Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl−/HCO3− exchange versus Na+-CO32− symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein–ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites  in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32− binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO Mutations in hAE1 cause distal renal tubular acidosis and red cell morphological abnormalities whereas mutations in hNBCe1 result in proximal renal tubular acidosis (pRTA) (The solute carrier family 4 (SLC4) transporters mediate the transport of HCOpressure , 6. The eostasis , 8. In lnditions . The AE1 to date , althougs (pRTA) , 8. Desp+ symporter  structures of hAE1 and hNBCe1 were resolved with X-ray diffraction  and cryoymporter , the bac SLC26Dg , and the SLC26Dg ). The id SLC26Dg  mapping −, HCO3−, and CO32− (cyan isomesh) and the nitrogen atoms of the methylammonium cations used as surrogate ions for the cotransported Na+ (yellow-orange isomesh). Although the acetate and methylammonium ions are larger than the anions normally transported by the SLC4 family proteins, they represent molecular probes with negative and positive charges with already developed parameters as part of SILCS. An enhanced density for these fragments in the protein center indicates that this region could accommodate as well the physiological ions, which are smaller and easier to fit in the putative binding sites. Both hAE1 and hNBCe1 feature a wide well-hydrated OF permeation cavity, lined with several positively charged (blue sticks) and negatively charged (red sticks) residues. These residues control the access of cationic and anionic fragments in the permeation cavity. Acetate can freely traverse the cavity to the protein center in both proteins and presents with a high density in the area of residues K539, R730, and K851 in hAE1 and residues K558, K559, and K924 in hNBCe1  and MD simulations to map protein regions with high affinity for various small solutes (fragments) with different structure and chemical properties, which are present in high concentrations in the simulation solutions. The flooding of the protein with the fragments and the corresponding fragment density maps (FragMaps) produced by SILCS can reveal previously unknown binding pockets and allosteric sites within the 3D protein matrix . The SILn hNBCe1 . Permeat−, CO32−, HCO3−, and Na+). To explore further the dynamics of Na+, Cl−, and HCO3− ions in the OF permeation cavity of hAE1 in conditions closer to the physiological environment, we performed two 1.2 μs long all-atom MD simulations starting from an empty (apo-) hAE1 protein embedded in a lipid bilayer solvated in either 150 mM NaCl, approximating the 0.9% NaCl in physiological saline, or an equimolar solution of 75 mM NaHCO3 + 75 mM NaCl, with elevated HCO3− concentration relative to Cl−  are displayed in 3− routinely persists for more than 100 ns in the protein center (average residence time 151.17 ns), while the presence of Cl− in this area is more short-lived  for fas1 and S2 , flankede cavity . Howevere cavity . Entry o of hAE1 , left. S of hAE1 .Figure 232−, HCO3−, or Cl−), a single cation (Na+), and different 1:1 cation:anion combinations  in the three SLC4 systems with binding stoichiometry corresponding to the previously published functional measurements  cells , confirming the functional significance of these protein regions.Experimental confirmation of the functional significance of the residues outlined in Computational modeling provides indispensable complementary information to many experimental structural biology and bioinformatics techniques , 31, 32.As a first step toward the identification of putative binding sites in hAE1 and hNBCe1, we applied the recently developed SILCS protocol for identification of accessible protein regions capable of binding of anionic and cationic molecular fragments . The SIL+, Cl−, and HCO3−) in the permeation cavity of apo-hAE1 at near-physiological concentrations. These results were complemented by a set of 250 ns MD simulations with different ion loads in the central site S1 in hAE1 and hNBCe1  which provided additional refinement of the ion binding sites in the permeation cavity and at the center of the protein where the ions have to bind before translocation. Multiple binding events were observed during the MD simulations in line with previously outlined solute behavior in open and nonoccluded structures of ion channels and secondary transporters  are routinely applied nowadays in the field of protein-aided membrane transport  or the protein center (location of sites S1 and S2 in hAE1), 3− or one Cl− , hNBCe1 loaded with one Na+ and one CO32− (for illustration of stable ion pair binding at site S1) and hNBCe1 loaded with one CO32− (for illustration of binding at site S2 after the CO32− ion exited from site S1).Both hAE1 and hNBCe1 feature a large well-hydrated OF permeation cavity framed by a number of charged residues, with the positively charged residues outnumbering the negatively charged ones  and S2. i.e., transfer and modifier sites) at the intracellular and extracellular side of hAE1  without impacting protein expression levels in the membrane . Anion coordination by residue F464 is achieved via its protein backbone atoms. Single point substitution at position F464 with smaller side chains, such as cysteine therefore would be expected to have a less profound effect on hAE1 transport , as long as helical packing and electrostatics in the region are not heavily affected.The entry binding site S2 of hAE1 is occupied by an anion in most of our MD simulations. Site S2 is comprised of residues P419, F423, F464-G466, I528, V531, F532, E535, K539, T727, V729, R730, and K851 , which ahis site . These rhis site  scores 8his site . In accohis site C withoutmembrane  confirmidecrease C. Anion via the proton of HCO3−), which is one of the acidic residues in the binding pocket of hAE1  and for longer periods of time  to site S2 in the protein center. The trigonal planar geometry of HCO3− allows for a good overlap of the carbonate group with the guanidinium group of R730 and additional coordination with K539 with a formation of a H-bond between HCO3− and E535  because of the competition between HCO3− and Cl− for site S2. From a functional point of view, this enhanced binding pattern between glutamate and HCO3−  which is found in ~4-fold lower concentration in the extracellular fluid than Cl− (Site S2 shows direct involvement in bicarbonate coordination of residue E535 ( of hAE1 . Accordiand Ref. ). Our 1.and E535  in site nd HCO3−  may manithan Cl− .− driven base flux compared with WT (C), reinforcing the notion that these residues are important participants in the hAE1 transport function. In addition, K539 and K851 are the anchor points for the covalently bound 4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS) molecule, which blocks hAE1 transport and locks the transporter in the OF state , previous studies . The R730C mutation causes hereditary stomatocytosis and has a well-established association with impaired anion transport and increased cation leak in hAE1 . Cystei in hAE1 . In our  present , an obseversus model pKa of 4.5). To probe whether protonation in the identified site S1 has an impact on ion retention in the binding pocket, we performed a set of 250 ns MD simulations of hAE1 with protonated E681, loaded with either Cl− or HCO3− in site S1  although it falls outside of the 5 Å cut-off for protein residues, presented in −/HCO3− exchange . This implies that successful function of hAE1 requires a glutamate residue at position 681, because even the conservative E681D mutation leads to significantly lowered AE1 exchange activity  of site S2 than Cl−, and the majority of MD steps feature a CO32− bound in this area  in the areas of high anion excess density from the hNBCe1 SILCS maps , in anal2CO3  and hNBC2CO3  and S15 2CO3  and S3 w+-driven base flux comparable to WT hNBCe1 is recorded for the cysteine substitutions of K559 and K562 (C) from site S2, which are positioned further away from the protein center than K558 and D555, and their long flexible side chains move freely and often stray away from site S2 . Previous studies on the lysine residues of site S2 in hNBCe1 are also inconclusive about the mechanistic significance of these residues: K559 has been identified as important for reversible DIDS blockade, whereas K558, K559, and K562 are not essential for irreversible DIDS blockade (+-induced base flux (C). A D555E substitution in hNBCe1 induces a Cl− flux (3− binding to residue E535 in hAE1), the acidic residues at this position in the SLC4 family. The sodium-dependent chloride bicarbonate exchanger (SLC4A8) also features a glutamate residue in the area of site S2 (+ + CO32−versus Cl−) but also between the two substrates of hAE1 (Cl−versus HCO3−).Naand K562 C from si site S2 . Substitblockade . Howeverase flux C. A D555Cl− flux , which iC), confirming the transport relevance of these residues. Site S1 residues that have seemingly little impact on transport are the residues from the V798-V802 stretch which participate in ion coordination via their backbone NH (CO32− coordination) or CO groups (Na+ coordination) . The polar residue T801 is involved in CO32− coordination also through its hydroxyl group. This coordination may be recaptured by the thiol group of cysteine in the T801C single point mutant, leading to the observed negligible deviation from the Na+-driven base flux values of WT hNBCe1 . From the residues whose mutation has more considerable effect  on hNBCe1 transport, residue T485 has shown clear functional significance, because the conservative serine substitution . Considering the immediate exit of Na+ placed without the stabilizing CO32− ion in the binding site of hAE1 (+), the presence of an arginine residue in the binding site of hNBCe1 most likely would impede Na+ binding leading to the observed transport deficiency.The central site S1 of hNBCe1 is comprised of S483, T485-P487, F544, I548, D754, I757, T758, V798-V802, and K924 , all of Fig. 5ination) . Substitination)  however mutation ) at thisransport . Anotherransport , shows h+ ion in site S1 of hNBCe1 is coordinated by the carboxyl group of D754, the hydroxyl group of T758, and the backbone carbonyl oxygen of A799 in addition to the CO32− ion (+ binding residue in site S1 of hNBCe1 both from SILCS mapping (C). Site S1 of the non-protonated WT hNBCe1 systems exhibits stable substrate coordination, which persists for more than 250 ns only when Na+ and CO32− are present together  feature an aspartate residue at this position, whereas the Na+-independent anion exchangers (SLC4A1-3) and the H+-NH3 transporter SLC4A11 have a conserved glutamate residue. Na+ binding is very sensitive to protonation at residue D754 . The pr site S2 . Based oike R730 .+ may be able to traverse the cavity and bind at residue D754 . The presence of CO32− in site S2 draws Na+ ions from the extracellular solution, which form short lived ion pairs with the bound CO32− , the analogous residues to K558 and K562 are substituted with acidic residues leading to a cluster of three negatively charged amino acids in the area of site S2  in the SLC4 family. We also highlight potential new avenues for future investigations, including specific mechanistic roles of the newly identified sites S1 and S2, role of individual residues in sites S1 and S2 in substrate selectivity and ion binding, role of protonation of the acidic residues at the protein center in substrate selectivity, binding and kinetics of transport, and role of the side-chain of residue R730 and adjacent nonpolar residues at the protein center in ion binding and gating. Better understanding of the substrate binding sites in SLC4 and their involvement in the overall transport process opens the door to design of specific and non-specific activators and inhibitors of SLC4 transport, which can be potentially applied in the treatment of SLC4-related diseases.In conclusion, we combine several complementary computational techniques with functional mutagenesis measurements to probe the putative binding pockets in hAE1 and hNBCe1 and to identify substrate binding sites and specific functionally relevant ion–protein binding motifs. Our MD simulations are in good agreement with our functional mutagenesis data and previous functional studies in the SLC4 family and provide novel insights into the transport mode determinants  and hNBCe1 (PDB ID 6caa) were taken from Refs. , 12, and−/HCO3− and Na+/CO32− ion binding sites in hAE1 and hNBCe1. SILCS is implemented in the Gromacs MD simulation package  simulations  that the target proteins favor. One of the major advantages of the SILCS methodology is that by incorporating MD simulations to GCMC computations it takes into account protein flexibility in identifying the possible ligand-targeted regions which improves conformational sampling of the proteins in addition to the configurational sampling of the probe species. FragMaps are free energy-based 3D-maps (GFE Maps) representing the occupancy possibilities (binding affinity) of small probe molecules/ions that target the specific volumes of the relevant proteins. The SILCS GCMC/MD protocol utilizes benzene, propane, methanole, formamide, imidazole, methylammonium, methanole, acetaldehyde, and acetate species. In the current work, we make use of a Methylammonium N Map which represents the regions that are likely to be targeted by positively charged donor fragments  and an Acetate O Map, which represents the putative binding sites for negatively charged donor fragments . Other maps  were also generated but are not discussed in the current work. The simulation protocol employed here is described below.SILCS Software  , 77, 78  package , 80, 81 ulations  to yield−1 nm−2 harmonic force constant on the Cα atoms of the proteins. In this step, temperature and pressure were maintained at 300 K and 1 bar by means of Nose-Hoover and semiisotropic Parrinello-Rahman controlling schemes. LINCS algorithm was used to constraint the hydrogen bonds. The approximate number of atoms of each simulated systems was ~90,000.Equilibrated frames from 250 ns MD simulations (see below) were used as the starting structures for the SILCS feeds. Gromacs MD (version 2018) , 80, 81 ex values alternate in each three-cycles for a better sampling  according to the formula below (FragMaps were generated from the 100 steps GCMC/MD simulations where 100 frames were collected from each step (each 10 ps from the production stages). A grid spacing of 1 Å is used in the calculation of FragMaps from the simulation data. First, occupancy maps were generated from each simulation step, and then, selected atom types from the solutes were binned in a 1 Åla below :GFExyzT=t values . In the t values . In the 3− and Cl−  , where the ions have to bind to translocate, is unlikely. Thus, the hAE1 and hNBCe1 transport most probably involves a single CO32−, HCO3−, or Cl− ion  , 96. The species , 27. The Cl− ion .versus symport)  . One or symport)  were plasymport) , 97  . Both hAsymport) . A negat in hAE1 . The basan anion  as evidean anion , right aO3− ions . FollowiO3− ions  in the pg pocket . The samg pocket  were useg pocket , 72 for The systems were then equilibrated following the six-step constrained equilibration procedure implemented in CHARMM-GUI  and wereThe analysis of all MD trajectories was done with Visual Molecular Dynamics (VMD) 1.9.3 . In-hous− and HCO3− permeation within the hAE1 OF cavity, long MD runs (1.2 μs) were performed with the Anton 2 supercomputer of apo-hAE1 (with either deprotonated or protonated E681) with 150 mM NaCl solution or in a 75 mM NaCl +75 mM NaHCO3 mixture (with sizes and lipid compositions as described above) under applied voltage of 0.055 kcal mol−1 Å−1 e−1 , 10% fetal bovine serum, and 1% penicillin-streptomycin in a 5% CO2 and cultured onto polyethylenimine-coated coverslips for the functional studies and 60-mm dishes for the sulpho-NHS-SS-biotin experiments. The cells were transfected with lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol except that after a 2 h exposure, the transfection solution was removed, and the cells were studied 24 h later. The cells were transfected with various human AE1 and NBCe1-A constructs or an empty pcDNA3.1 vector.The HEK293 cells were grown at 37 °C in media containing Dulbecco's Modified Eagle Medium with l-glutamine (200 mg li) was measured 24 h following transient transfection with the various constructs in HEK293 cells grown on polyethylenimine-coated coverslips. The coverslips were placed on the stage of a microscope fluorometer in a custom designed chamber and then loaded at room temperature for ~20 min with the fluorescent pHi probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM) (Life Technologies). The dye loading solution contained 140 mM KCl, 5 mM TMACl, and 5 mM Hepes. In the experiments, the fluorescence excitation ratio  was obtained from ~200 cells and averaged. The coverslips were continuously perfused at 2 ml min−1 (37 °C) with the bathing solutions. The intracellular fluorescence excitation ratio was calibrated at the end of each experiment using 26 μM nigericin (Sigma-Aldrich) and 5 μM valinomycin (Sigma-Aldrich) to equilibrate pHi, external pH, and K+. The intrinsic cell buffer capacity (βi) was measured over a range of intracellular H+ (Hin+) values in Hepes-buffered solutions using NH4+ addition/removal protocols and calculated as Δ[NH4+]/Δ[Hin+]. The bicarbonate buffer capacity βHCO3 was calculated as 2.3 × [HCO3−]in. The total buffer capacity was therefore βi + βHCO3. The rate of change of pHi (dpHi/dt) was measured in the initial 10 to 15 s after a bath solution switch and converted to the rate of change of [Hin+] (d[Hin+]/dt), and the H+ flux (mM s−1) was calculated as (βi + βHCO3) × (d[Hin+]/dt).Internal pH -amiloride (EIPA). AE1-mediated Cl− flux was induced by switching to the following Na+-free Cl−-containing solution: 115 mM tetramethylammonium chloride, 2.5 mM K2HPO4, 1 mM CaCl2, 1 mM MgCl2, 24 mM tetramethylammonium bicarbonate, 5% CO2, pH 7.4, and 30 μM EIPA. (2) Na+-driven base transport assay: The cells were initially bathed in the following Na+-free, Cl−-containing, bicarbonate-free solution: 140 mM tetramethylammonium chloride, 2.5 mM K2HPO4, 1 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH 7.4, and 30 μM EIPA. NBCe1-A–mediated Na+-driven base flux was induced by switching to the following Na+- and Cl−-containing solution: 115 mM NaCl, 2.5 mM K2HPO4, 1 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3, 5% CO2, pH 7.4, and 30 μM EIPA. The statistical analysis of the flux measurements is presented in (1) Anion exchange transport assay: The cells were initially bathed in the following Nag (4 °C), and then, the supernatant (containing >90% of the plasma membrane protein fraction) was collected and then incubated on a rotating shaker at 4 °C for 4 h with streptavidin-agarose resin (50 μl) (Thermo Fisher Scientific). The resin was pelleted by brief centrifugation and washed with the lysis buffer at 60 °C for 5 min with 2 × SDS buffer containing 2% 2-mercaptoethanol  to elute bound proteins. For the lysate detection, the cells were lysed in lysis buffer  Igepal [Sigma-Aldrich], 10 mM Tris HCl, 5 mM EDTA [Sigma-Aldrich] pH 7.5), and AE1 was pulled down using the AE1 2-M anti-human monoclonal antibody (Alpha Diagnostics) (1:1000 dilution), and NBCe1-A was pulled down with the previously described rabbit polyclonal anti-human NBCe1-A antibody (1:1000 dilution).Sulfo-NHS-SS-biotin was used to label plasma membrane proteins. Streptavidin-agarose resin was used to pull down these proteins. The following protocol was utilized; the cells were washed with phosphate buffered saline at room temperature, pH 8.0, 24 h following transfection and were then incubated at 4 °C for 30 min (pH 8.0) with 1.1 mM sulfo-NHS-SS-biotin (Thermo Fisher Scientific). 50 mM Tris buffer at 4 °C  was used to stop the reaction. The cells were collected and washed with phosphate buffered saline, then lysed on ice in a solution containing 150 mM NaCl, 0.5% sodium deoxycholate (Thermo Fisher Scientific), 1% (vol/vol) octylphenoxy poly(ethyleneoxy)ethanol  (Sigma-Aldrich), 10 mM Tris HCl, 5 mM EDTA (Sigma-Aldrich), pH 7.5, with protease inhibitors (Roche Life Sciences). The insoluble material was pelleted over 10-min centrifugation at 20,000The protein samples were initially resolved on 7.5% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The expression levels of the pulled down biotinylated proteins and whole cell lysates were assessed by probing the blots with either the mouse monoclonal AE1 2-M antibody  or the rabbit polyclonal NBCe1-A antibody  in Tris buffered saline with polyethylene glycol sorbitan monolaurate (TBSTM) buffer (0.1% (vol/vol) Tween 20; 137 mM NaCl, 20 mM Tris, pH 7.5, containing 5% (wt/vol) nonfat milk). After 1 h incubation at room temperature, the blots were washed with TBST and then probed with Peroxidase AffiniPure Donkey Anti-Mouse IgG (H + L)  at 1:10,000 dilution or Peroxidase AffiniPure Mouse Anti-Rabbit IgG (H + L)  at 1:10,000 dilution in TBSTM buffer and incubated at room temperature for 1 h. The blots were washed with TBST, and signals were detected with Enhanced Chemiluminescent (ECL) Western Blotting Detection Reagent .All necessary data is provided in the manuscript. The custom scripts used for analysis of the MD trajectories will be provided upon request by the corresponding author Sergei Noskov.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Results showed that angelicin could regulate the M1/M2 ratio and function and alleviate the development of PTOA in the meanwhile. Bone marrow monocytes were isolated and induced by macrophage colony-stimulating factor (M-CSF), lipopolysaccharide (LPS) and interferon (IFN)-γ for M1 polarization and interleukin (IL)-4/IL-13 for M2 polarization. Subsequently, repolarization intervention was performed. The results indicate that angelicin can repolarize M1 toward M2 macrophages by upregulating the expression of CD9. Besides, angelicin can also protect and maintain M2 polarization in the presence of LPS/IFN-γ, and subsequently downregulate the expression of inflammatory mediators such as IL-1β and TNF-α. Mechanistically, angelicin can activate the p-STAT3/STAT3 pathway by conducting CD9/gp130 to repolarize toward M2 macrophages. These results suggest angelicin can alleviate the progression of OA by regulating M1/M2 polarization via the STAT3/p-STAT3 pathway. Therefore, angelicin may have a promising application and potential therapeutic value in OA clinical treatment.Post-trauma osteoarthritis (PTOA) is the most common articular disease characterized by degeneration and destruction of articular cartilage . Inflammatory response of local joint tissue induced by trauma is the most critical factor accelerating osteoarthritis (OA) progression ( Osteoarthritis (OA) is the most common and severe joint disease worldwide and creates a substantial socioeconomic burden, affecting 10% of men and 18% of women over 60 years of age . OA was Previous studies have shown that OA is a low-grade chronic inflammatory disease, causing damage to multiple articular and periarticular tissues. Emerging evidence suggests local inflammation, synovitis, in particular, plays a crucial role in the pathogenic process of OA . MacrophBreaking the original balance of macrophage polarization has been proved to contribute to accelerate the pathological process of various diseases. Exorbitant activation of the M1 type phenotype can cause tissue damage and metabolism disorders . IncreasAngelica Sinensis, used in traditional Chinese medicine, is widely recognized as an effective herb to heal multiple diseases. Angelica is gaining ground all over the world, including Europe, North American and Southeast Asia. In folk medicine, angelica is believed as an anti-inflammatory, anti-aging medicine with wide use . AngelicIn this study, for the very first time, we revealed that angelicin is an effective pharmaceutical therapy for regulating M1/M2 macrophage polarization. Angelicin plays a role in suppressing M2 polarization, especially when M2 macrophages repolarized toward M1 and facilitating M2 polarization through the STAT3 pathway. These findings have implied that angelicin may have potential therapeutic value in the future to alleviate the inflammation of articular and periarticular tissue and then restrain the progression of OA.Angelicin with purity > 95% was purchased from Med Chem Express  and dissolved in DMSO. Alpha Minimum Eagle’s Medium , penicillin , streptomycin  and fetal bovine serum (FBS) were purchased from Hyclone . Soluble mouse recombinant M-CSF was purchased obtained from R&D system . Interleukin (IL)-4, IL-13, LPS, and IFN-γ were purchased from PeproTech Inc. . The Cell Counting Kit-8 (CCK-8) was purchased from Med Chem Express . Anti-CD9, anti-gp130, anti-STAT3, anti-phospho-STAT3 (Ser727), anti-CD206, anti-iNOS, anti-Arg-1, anti-β-actin antibodies were purchased from Bioworld Technology .3 per well and were treated with a range of 10 μmol/L-100 µmol/L for 24 or 48 h. Next, CCK-8 kit solution was added, and then the system described above was incubated for 2 h away from light. Absorbance was detected at 450 nm and recorded by a microplate absorbance reader .The cytotoxicity evaluation of angelicin in bone marrow monocytes was examined using CCK-8 kit. BMMs isolated from mice femur were seeded in 96-well plates at a density of 3 × 102. BMMs were incubated for 72 h in α-MEM supplemented with 30 ng/ml M-CSF, 10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin. Then, non-adherent cells were removed with PBS. After reaching the confluence of 80%, BMMs were seeded in 6-well plate with the complete medium in an incubator at 37 C with 5% CO2.BMMs were isolated from the femurs of 6-week-old C57BL/6 mice under aseptic condition. α-MEM with 10% FBS was applied for flushing the bone marrow capacity. The mixtures described above were filtered with 200 mesh nylon filter. BMMs were seeded into 60mm cell culture dish containing a complete medium composed of 90% α-MEM and 10% FBS in an incubator set at 37 C with 5% CO5 cells per well and cultured in a complete medium containing 30 ng/ml M-CSF for 24 h, with 20 ng/ml IL-4 and 20 ng/ml IL-13 or 20 ng/ml LPS and 100 ng/ml IFN-γ for 36 h. Then cells were lysed using Trizol reagent (Invitrogen) 1 ml per well. Complementary DNA (cDNA) was synthesized from 1μg RNA from each sample using a reverse transcriptase kit . For real-time PCR, 1ug RNA was mixed with primers and SYBR green super mix. The gene expression levels were calculated with 2 –∆∆CT values by CFX Manager™ Software v3.1(Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control gene in this experiment. The primer sequences are listed in Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate marker gene expression in M1/M2 macrophages in different groups. BMMs were seeded in 6-well plates at a density of 1 × 10Cell samples were harvest at 36 h in 6-well plates after the intervention described above. Cells were lysed in RIPA lysis buffer with phenylmethanesulfonyl fluoride (PMSF). Protein samples were loaded to SDS-PAGE with 20 ul per lane and then transferred to PVDF membranes. Next, membranes were blocked with 5% bovine serum albumin (BSA) blocking agent for 1.5 h and washed with TBS-T buffer for three times, 15 min for each. Primary antibodies against iNOS , Arg-1 , CD9 , gp130 , STAT3 (1:500), p-STAT3 (1:500), β-actin  were incubated with the membranes in the BSA solution overnight at 4 C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies  for 1.5 h and washed three times with TBST buffer. Lastly, the Chemi Doc XRS + Imaging System  was performed to capture images of the protein bands, which were analyzed with the ImageJ v2.0. β-actin was used as an internal control in this experiment.The secretion of IL-1β and TNF-α of polarized macrophages in cell supernatant was measured by ELISA Kits  according to the instructions. All measurements were repeated five times.C57BL/6 mice were obtained from the animal center of the Third Medical University . All experiments in this research were performed according to the Third Military Medical University Sciences Guide for Laboratory Animals. Animal suffering weas reduced by all means with all our forces. All animals were maintained at 25 C under 12 h’ light/dark cycle with 50% humidity. Eighteen 6–8 weeks of female C57/BL6 mice were separated randomly into three equal groups: Control, LPS and LPS + ANG groups. The Control group were performed intraperitoneal injection with Saline. LPS group was performed intraperitoneal injection with LPS . LPS + ANG group were performed intraperitoneal injection with LPS  and angelicin . Injections were performed intraperitoneal every other day continuously for two weeks. 14 days later, all mice were sacrificed, and the femur was isolated under aseptic condition. Femurs were fixed with 4% paraformaldehyde for 48 h and decalcified in EDTA (pH = 7.2) for 14 days for further testing.To confirm the protective effect of angelicin, DMM models were established as previously described . Accordi+/F4/80+and iNOS+/F4/80+ cell proportion of macrophages. After complete euthanization of mice, macrophages were isolated from femurs and resuspended in ice-cold PBS. Cells seeded in the 6-well plate were digested into single cells by trypsin. Half of the collected cells were incubated with PE-conjugated CD206 and AF 780-conjugated iNOS antibodies  for 45 min, respectively. Cells were further incubated with BV421-conjugated F4/80 antibody  for 45 min at 4°C. iNOS+F4/80+ macrophage was recognized as M1 phenotype and CD206+F4/80+ macrophage was recognized as M2 phenotype.Flow cytometry was applied for examining CD206For apoptosis detection, Annexin-V Apoptosis Detection Kit  was used to detect apoptosis of BMMs. Cells were harvested with 0.25% trypsin and washed with PBS. Then cells were resuspended with 100 μL Annexin-V and propidium iodide (PI) staining buffer diluted in PBS for 20 min on the ice from light. The cells were analyzed on CytoFLEX Flow Cytometer , and data were analyzed in FlowJo  followed by instructions.To further investigate the polarization and repolarization of macrophages, isolated BMMs were stimulated with LPS/IFN-γ for M1 polarization and IL-4/IL-13 for M2 polarization alone or combined with 30 μmol/L angelicin. After treatment, the samples were rinsed with PBS for three times. Then the samples were fixed by 4% paraformaldehyde and permeabilized by Triton X-100 diluted in PBS for 10 min. Samples were blocked with 5% BSA at 37°C for 30 min and washed with PBS, and incubated with iNOS (1:150) and CD206 (1:150) primary antibodies overnight at 4°C. Subsequently, samples were washed by PBS for three times and incubated with AF647-conjugated secondary antibody (1:400) for 1 h and stained with DAPI for 5 min at room temperature. After staining, the fluorescence microscope  was used to observe the plates and slides. Fluorescence intensity was measured and quantified using ImageJ v2.0.The keen joints were isolated on the day of execution (8 weeks after surgery) and fixed in 4% paraformaldehyde for 48 h and decalcified in EDTA (pH = 7.2) for 14 days. After dehydration, embedding and cutting, the slides were stained with Safranin-O/Fast Green and hematoxylin and eosin (H&E). An experienced histological investigator blinded to evaluate medial femoral condyle and medial tibial plateau according to the International Osteoarthritis Research Society International (OASRI) scoring system as described previously .p < 0.05 and **p < 0.01 being regarded as significant.All values are representative of at least three parallel experiments of resembling results except independently demonstrated. Data are expressed as mean ± SD and comply with the normal distribution. Independent groups are compared by one-way ANOVA followed by Student-Newman-keul post hoc tests to determine the significant difference between results, with *Angelicin is a kind of furocoumarin, a naturally occurring tricyclic aromatic compound. The chemical formula of angelicin is showed in the figure . To detein vitro, primary BMMs were isolated for further investigation. BMMs were stimulated with LPS/IFN-γ for M1 polarization and IL-4/IL-13 for M2 polarization. During the stimulation of M1 macrophage, angelicin treatment has shown the effect of angelicin on this polarization procedure. However, the repolarization procedure was performed that M2 macrophages were treated with LPS/IFN-γ alone or combined with angelicin was conducted to observe repolarization (+ (represent as M2 macrophages) cell number and iNOS+ (represent as M1 macrophages) cell number was shown on the left , while the angelicin treatment group was lower compared with the DMM group   .It is widely reported that OA is the most common and disabling type of arthritis, causing heavy health burdens with severe complications for the affected individuals . At presPrevious studies suggest that angelicin was potentially advantageous in preventing inflammatory diseases. Angelicin, an ancient herb widely used worldwide, was recently reported to have various effects on regulating inflammatory response . In thisM1/M2 macrophages have been formulated decades ago to be a group of cells with high plasticity and polarization under the conditions of multiple cytokines . MacrophAngelicin have been reported for their special cellular biological functions these years. Recent research has shown that angelicin can inhibit the malignant behavior of human cervical cancer. Combined administration of psoralen and angelicin can significantly regulate anti-inflammation and osteogenesis effect . Meanwhiin vivo and in vitro. Our results had shown that angelicin could inhibit M1 polarization and promote M2 polarization in synovial tissues. Angelicin is protective of maintaining the M2 phenotype in the presence of IFN-γ via CD9/gp130/STAT3 pathway to alleviate synovitis in OA. Angelicin can significantly increase the expression of STAT3 and phosphorylation of STAT3 and then facilitate the expression of M2-related genes (In the study, we evaluated the effect of angelicin in regulating the polarization of macrophages both ed genes . In animOverall, our studies suggest that angelicin could regulate M1/M2 ratio via CD9/gp130/STAT3 pathway to reduce the inflammatory response and then protect cartilage in OA. These results further raise the possibility that angelicin could be a potential therapeutic agent against OA.


Especially, LuAG:Ce film attracts more attention due to the outstanding advantages, such as the efficient heat dissipation, the saving of rare earth, and so on. Here, LuAG:Ce film on sapphire was successfully prepared by the ultrasonic spray pyrolysis process. The phase, microstructure and photoluminescence of LuAG:Ce films were investigated. LuAG:Ce films had a thickness of around 5 μm, which were well crystallized at 1000 °C in air atmosphere to form the typical garnet structure. Under the protection of CO atmosphere, increasing the annealing temperature greatly enhanced the photoluminescence performance. After annealing at 1500 °C for 5 h in CO atmosphere, 3.0 mol.% Ce3+ doped LuAG:Ce film exhibited the highest emission and excitation intensity. The emission intensity of 3.0 mol.% Ce3+ doped LuAG:Ce film annealed at 1500 °C in CO atmosphere increased up to five times, when compared with the best LuAG:Ce film annealed at 1000 °C in air atmosphere. The effects of Ce3+ doping concentration on the photoluminescence were also examined. As the Ce3+ doping concentration increased from 0.2 mol.% to 7.0 mol.%, the color of LuAG:Ce films changed from yellowish green to greenish yellow. When coupling the 3.0 mol.% Ce3+ doped LuAG:Ce film with a 0.5 W 450 nm blue laser, the formed device successfully emitted white light.LuAG:Ce (Lu Therficiency ,12,13.−1 K−1) and high reliability (1.9% decrease after 1000 h at 85 °C and 85% humidity) [2O3-LuAG:Ce composite ceramic phosphor (CCP) by vacuum solid-state sintering method [2O3 greatly improves the CCP’s thermal conductivity (18.9 W m−1 K−1) and thermal stability (the luminescence loss of only 3.2% at 200 °C) [Phosphor ceramics are thought to be the favorable choices to replace phosphor-in-silicon color converters ,15. LuAGumidity) . Zhang eg method . They fo 200 °C) . Clearly−1 K−1) [3+ doping concentrations were prepared. Their phase compositions, microstructure and photoluminescence properties were investigated.To solve the problem, LuAG:Ce film on sapphire is a wonderful choice, which effectively combines the high thermal conductivity of sapphire and the high luminous efficiency of LuAG:Ce phosphor. The synthesis methods of film mainly include the sol-gel method , ultraso−1 K−1) . LuAG:Ce2O3, Al(NO3)3·9H2O, Ce(NO3)3·6H2O and HNO3. All chemical reagents were of analytical grade, which were purchased from Sinopharm Chemical Reagent Corporation  and used as received. The solution of Lu(NO3)3 was prepared by dissolving Lu2O3 with proper amount of nitric acid. The spray solution was composed of Lu(NO3)3, Ce(NO3)3 and Al(NO3)3. The molar ratio of rare earth to Al was 3:5, and the total concentration of rare earth ions was 0.01 mol/L. 3 wt.% PVP was added as the surfactant. To investigate the effect of Ce3+ doping concentration on the photoluminescence, the spray solutions with different Ce3+ concentrations (0.2–7.0 mol.%) were prepared. The sapphire substrates  were ultrasonically cleaned with deionized water, acetone and ethanol. The experimental setup for ultrasonic spray deposition was shown in The chemicals used were LuThe phase compositions of thin films were determined by X-ray diffraction , utilizing Cu-Kα radiation (1.54056 Å). The microstructure was observed by scanning electron microscopy . The photoluminescence (PL) and photoluminescence excitation (PLE) were analyzed at room temperature using an FP-8600 fluorospectrophotometer  equipped with a 60 mm diameter integrating sphere  and a 150 W Xe-lamp for excitation. The thickness of films was measured by a stylus profiler .3+ contents on the phase compositions of LuAG:Ce films were investigated. The results are shown in 3Al5O12 (PDF#18-0761). Upon annealing below 1000 °C in air atmosphere, the Ce3+ doping concentration has no significant influence on the phase compositions of films. Besides, it is known that Ce3+ can be easily oxidized to Ce4+, resulting in the decrease in luminescence intensity. In order to improve the luminous intensity, LuAG:Ce films were further annealed at 1500 °C for 5 h in CO atmosphere. Their XRD patterns are shown in 3Al5O12 (PDF#18-0761) phase, and the crystallinity of films is significantly improved, as evidenced by the sharp diffraction peaks. In addition, Ce3+ (1.143 Å) has a larger radius than Lu3+ (0.977 Å). Therefore, introducing Ce3+ into the lattice of Lu3Al5O12 increases the interplanar spacing. According to the Bragg equation, the strongest diffraction peak would shift to the lower 2θ side. 3+ doping concentration increases from 0.2 mol.% to 5.0 mol.%. Due to the large amount of Ce3+ was incorporated into the Lu3Al5O12 lattice, unknown phase was observed, as shown in The effects of annealing temperature, annealing atmosphere and Ce3+ doping concentrations are shown in 3+ doping concentration has significant influence on the microstructure and grain shape. For the Ce3+ doping concentration of 0.2 mol.%, 0.5 mol.% and 1.0 mol.%, the average grain size is 400 nm, 650 nm and 850 nm, respectively. Obviously, Ce3+ doping significantly improves the grain growth. The average grain size is even up to 1.0 μm when 2.0 mol.% Ce3+ was doped. The grain shape is also affected by the Ce3+ doping. As the Ce3+ doping concentration increases to above 1.5 mol.%, the grains become rounded. The 7.0 mol.% Ce3+ doped film exhibits a peculiar microstructure, whose crystal grains are rounded and isolated.The FE-SEM images of 1500 °C CO atmosphere-annealed LuAG:Ce films with different Ce3+ ions, which leads to the electronic transition from the ground state  to the excited 5d levels. The emission spectra consist of a broad band ranging from 490 nm to 650 nm, with the maximum at around 520 nm, which are ascribed to the electronic transitions of Ce3+ from the lowest 5d level to the 4f ground state. After annealing at 1000 °C for 5 h in air atmosphere, 1.5 mol.% Ce3+ doped LuAG:Ce film exhibits the highest emission and excitation intensity. Improving the crystallinity and inhibiting the oxidation of Ce3+ could greatly enhance the photoluminescence properties. Upon annealing at 1500 °C for 5 h in CO atmosphere, 3.0 mol.% Ce3+ doped LuAG:Ce film exhibits the highest emission and excitation intensity. Compared with the 1.5 mol.% Ce3+ doped LuAG:Ce film annealed at 1000 °C in air atmosphere, the emission intensity of 3.0 mol.% Ce3+ doped LuAG:Ce film annealed at 1500 °C in CO atmosphere increases up to five times. Further increasing the Ce3+ doping concentration above 3.0 mol.% declines the emission intensity of LuAG:Ce films, which may be due to the effect of concentration quenching.The annealing temperature and atmosphere significantly influence the photoluminescence properties of LuAG:Ce films. The photoluminescent spectra of LuAG:Ce films were measured at room temperature, as shown in 3+ doping concentration increases from 0.2 mol.% to 7.0 mol.%. After coupling the 3.0 mol.% Ce3+ doped LuAG:Ce film with a 0.5 W 450 nm blue laser, the formed device emits white light, as shown in The corresponding chromaticity coordinates of LuAG:Ce films were obtained. The coordinates were calculated from the luminescence spectral data shown in In order to further determine the photoluminescence properties of LuAG:Ce films, the color purity was investigated. The color purity can be calculated according to the following formula :(1)C=x−xi, yi) stands for the color coordinates of standard white light, while  are the color coordinates of the dominant wavelength. The calculated color purity of LuAG:Ce films is plotted in 3+ doped LuAG:Ce film.Here,  are the CIE coordinates of LuAG:Ce film. (x3+ doping concentrations are shown in 3+ doping concentration increases from 0.2 mol.% to 7.0 mol.%, the color of LuAG:Ce films changes from yellowish green to greenish yellow, which agrees well with the results of CIE color coordinates analysis. The thickness of LuAG:Ce film is also examined. 3+ doped LuAG:Ce film, which was annealed at 1500 °C for 5 h in CO atmosphere. As can be seen, 3.0 mol.% Ce3+ doped LuAG:Ce film has a thickness of around 5 μm.The appearance of 1500 °C CO atmosphere-annealed LuAG:Ce films with different Ce3+ greatly enhanced the photoluminescence properties. After annealing at 1500 °C for 5 h in CO atmosphere, 3.0 mol.% Ce3+ doped LuAG:Ce film exhibited the highest emission intensity, which increased up to five times when compared with the emission intensity of the best LuAG:Ce film annealed at 1000 °C in air atmosphere. Coupling the 3.0 mol.% Ce3+ doped LuAG:Ce film with a 0.5 W 450 nm blue laser, the formed device successfully emitted white light.LuAG:Ce films were successfully prepared by the ultrasonic spray pyrolysis process. The film thickness was around 5 μm. After annealing at 1000 °C in air atmosphere, LuAG:Ce films were well crystallized to the typical garnet structure. Improving the crystallinity and meanwhile inhibiting the oxidation of Ce


In this study, selective laser melting (SLM) has for the first time successfully been employed to tune the crystallographic structure of bulk MoS2 to a 2H/1T phase and to distribute Mo2S3 nanoparticles in-situ in MoS2/Mo2S3 nanocomposites used in electrochemical energy conversion/storage systems (EECSS). The remarkable results promote further research on and elucidate the applicability of laser-based powder bed processing of 2D nanomaterials for a wide range of functional structures within, e.g., EECSS, aerospace, and possibly high-temperature solid-state EECSS even in space.MoS Recently has a high thermal stable hexagonal boron nitride (h-BN) been introduced as a promising anode material, though its low electrical conductivity may limit its electrochemical performance1. Molybdenum disulfide (MoS2) belongs to a class of advanced next-generation materials called Transition Metal Dichalcogenides (TMDs) with a direct bandgap of ~ 1.8 eV and a melting point of 1185 ºC in nonoxidizing environments3. MoS2, with strong covalent S–Mo–S bonds and weak inter-layer van der Waals forces4, and its nanocomposites dominate electroactive layered structures due to their transitional structure from semiconductor 2H to metallic 1T, which is favorable in electrochemical applications6. Similar to Li+, the accommodation of Na+ between 1T-MoS2 results in considerable capacitance and interesting electrochemical properties7.Na-S energy conversion/storage devices have earlier been demonstrated as possible alternatives to mitigate thermal decomposition, short circuit, heat, fire, or explosion of Li-ion electrodes2-based nanocomposites have been widely studied using a variety of conventional methods12 which have different technical drawbacks such as complex expensive pre-processing , processing (deposition or lack of precise morphology control), and complementary post-processing stages 13. Despite the mitigation of existing drawbacks, scientists have enhanced processing possibilities which have led to novel 3D printing techniques20. The use of these techniques is still limited due to drawbacks such as restricted resolution, weak mechanical properties, low printing speed, or high cost of equipment23.MoS2 in a controlled manner24 to enable green fabrication of 3D shapes from 2D nanomaterials26, and improve the charge capacitance properties of Mo2S3 nanostructures27. The thinning mechanism of MoS2, with a high laser absorption coefficient due to the weak van der Waals forces between poorly coupled MoS2 layers, can be attributed to localized temperature-dependent sublimation of upper layers of bulk 2H-MoS2 lattice, arising through the absorption of high-energy laser28. At the next steps, laser irradiation on thinned 2H-MoS2 can lead to further photoinduced exfoliation to form few-layered nanosheets25. On the other hand, the more laser reflection due to the high reflection ratio of Mo powder28, the more thinning of 2H-MoS2 existing nearby Mo powders25.Laser-based thinning can effectively exfoliate MoS2 is the thermodynamically stable trigonal phase consisting of prismatic coordination of Mo atoms by six surrounding sulfur atoms, whereas metallic 1T-MoS2, metastable octahedral coordination of Mo atoms in MoS2, can be a result of the first-order phase transition due to the change in the density of states29. It has already been shown that the exfoliated form consists of a 1T-MoS2 phase with lattice distortions according to the exfoliation technique. The annealing of metastable 1T-MoS2 at temperatures lower than 300 ºC can lead to restacking of the layers and restoration of 2H-MoS230. Hence, generally, integrated hybrid 1T/2H-MoS2 exists in which the 2H phase can stabilize the metastable 1T phase, avoiding restocking and restoration30. It has though been proven that 2H-MoS2 at temperatures higher than 600 ºC can be transformed into 1T-MoS2 via nucleation of an intermediate and subsequently 2H/1T boundaries migration over time31.2H-MoS2 can impose a highly localized temperature resulting in the thinning of MoS2 as well as the creation of many α-phase nucleation sites for 2H to 1T phase transformation31. Mo2S3 nanostructures can due to surface phase transformation also be formed through thermal annealing or sputtering of MoS2 in an Ar-isolated atmosphere and at high temperatures above 1300 ºC25. Imposing sulfur vacancies into the structure of MoS2 in the exposure of high-energy laser beam in the presence of melted Mo at a high temperature can also result in the formation of Mo2S3 nanostructures at the edges of laser-thinned 1T-MoS2 and sulfur vacancies26. Consequently, the rapid cooling rate of the SLM process as well as the presentation of Mo2S3 nanostructures can significantly prevent the restoration of 1T/2H-MoS2.Accordingly, high-energy laser processing of MoS23, this technology can be used for rapid prototyping of future energy storage materials32. Despite the specific characteristics of MoS2 in electrochemical applications, the weak van der Waals forces make direct SLM processing of MoS2 structures difficult. The addition of Mo powder, as a metallic additive, has been shown to both facilitate the printability of MoS2 based structures and result in the formation of stable electrocatalytic Mo2S3 nanostructures via Mo–Mo zigzag chains, which enhance the electron transfer within and between the S–Mo–S layers27.Compared to other 3D printing methods, SLM relies on versatile high-energy Nd:YAG laser equipment which enables printing of parts of improved mechanical properties and better resolution. Since the SLM technique can overcome many of the demerits of both conventional and other 3D printing techniques2 from bulk 2H to metallic 1T through one-step laser-based exfoliation but also on the simultaneous formation and uniform distribution of Mo2S3 nanoparticles in the nanocomposite structure. The unprecedented electroactivity of the one-step SLM-processed MoS2/Mo2S3 nanocomposite (SLM-Mo(x)S(x+1)) is demonstrated, paving the way for the fabrication of the next generation EECSS. Our recent findings demonstrate that the SLM technique is able to directly utilize raw materials, exfoliate MoS2 in-situ during fabrication of intricate parts, such as electroactive components of EECSS, and directly deliver parts of different functionalities without any special additives or binders.This study reveals the extraordinary influence of the SLM technology not only on the crystallographic phase transformation of MoS2 particles can be ascertained from Fig. 50) of 25–35 µm and 2.5–7.5 µm, respectively S(x+1) indicated a similar microstructure which can be ascribed to the optimized LSS (laser power of 62.5 W and a laser scan speed of 85 mm s−1) resulting in a relatively stable melt-pool with a nearly constant thermal gradient during the SLM process  illustrate the exfoliation of bulk 2H-MoS2 into thin 1T-MoS2 nanosheets and the distribution of Mo2S3 nanoparticles in the matrix 7. Illustrated in Fig. −1  patterns verified the purity of Mo and MoSess Fig. a. The lo(x)S(x+1) sample compared to both pure Mo and MoS2 electrodes   at different current densities revealed nearly 1.0 V in output voltage even at a current density of 15 mA cm−2 for the SLM- Mo(x)S(x+1) electrodes  and a decreased iR voltage drop. The enhanced capacitance induced by increasing the number of GCD cycles can be attributed to facilitated intercalation of Na+ cations, activation of the electrode texture, and improved electrical conductivity S(x+1) surface37.Cyclic voltammograms (CV) show considerably enhanced current densities for the SLM-Modes Fig. a. Due to−1) Fig. b7. Quasides Fig. c. Herein2, separated by Mo2S3 nanoparticles, resulted in a considerably lower Rct (75.36 Ω) and a quasi-semicircle shape of the Nyquist plot of SLM-Mo(x)S(x+1) compared to pure Mo. The higher slope of the diffusion impedance in the low-frequency region for SLM-Mo(x)S(x+1) compared to pure 2H-MoS2, confirms facilitated diffusion and accumulation of Na+ charges into the defect-rich 1T-MoS2 nanolayers within the porous surface S(x+1) electrode before and after 2000 cycles GCD in Fig. + intercalation into the deeper active sites and spaces of electrode materials, respectively, by increasing the number of GCD cycles.As shown in Fig. ace Fig. e38. Comp(x)S(1+x) electrode revealed capacitance retention of 53% even at a scan rate of 50 mV s−1 (44.16 mF cm−2) compared to a low scan rate of 10 mV s−1 (83.1 mF cm−2). This is considerably higher than their counterpart electrodes processed by different 3D printing techniques S(x+1) electrodes offered higher power (53.34 mW cm−2) and energy density (1.66 mWh cm−2) compared with ink-jet printed MXene-39, and graphene-based electrodes40, as well as 1T-MoS2/graphene-based electrodes14.Areal rate capability studies of the SLM-Mo2/Mo2S3 electrodes with a remarkable capacitive behavior compared to other 3D-printed counterparts provides a new possibility to manufacture the next generation of 3D-printed electrochemical devices. In this study, we elucidate that a high-energy Nd:YAG laser can simultaneously exfoliate and tune the structure of MoS2, distribute electroactive Mo2S3 nanoparticles into the structure of the nanocomposite, and therefore considerably enhance the power density and energy density of 3D-printed structures. The preliminary results promote further research on laser-based processing of 2D nanomaterials for a wide range of functional structures e.g., EECSS, high-temperature solid-state energy conversion/storage systems, aerospace parts, and green energy storage device fabrication, even in space.The successful fabrication of SLM-processed MoS2, 99.99%, Sigma Aldrich) was used as feedstock for SLM processing of the electroactive nanocomposite. A mixture of Mo and MoS2 (20 wt.%) was prepared by mixing for 6 h at 20 rpm to obtain a homogenous mixture  and ultrapure water, with a conductivity of 18.2 MΩcm and a pH of 6.5 ± 0.1 at 25 °C, was used to prepare the 0.5 M Na2SO4 (pH 5.5 ± 0.1) electrolyte for the electrochemical investigations.Gas atomized powder (GAP) of commercial pure molybdenum  was used as the feedstock to fabricate both the current collector and the electroactive nanocomposite. Pure molybdenum disulfide powder  was carried out as a function of energy density to obtain the optimized printing parameters, where the energy density is defined in Eq.  by using optimized SLM parameters (laser power − 62.5 W and scan speed − 85 mm s−1). Figure 2. Shown in Fig. A laser scattering particle size distribution analyzer  was used to evaluate the particle size distribution of the Mo and MoS2SO4 electrolyte as well as to clean the surface of the 3D-printed electrodes. A standard pH meter  was used to measure the pH of the Na2SO4 electrolyte. A PARSTAT MC multichannel potentiated , an Ag/AgCl reference electrode , and a Pt wire counter electrode  were used for the electrochemical studies. The surface area of the working electrode (anode) was 0.33 cm2. Cyclic voltammetry (CV) was carried out using scan rates between 10 and 100 mV s−1 in a potential window between −1.1 and 0.2 V. Electrochemical potentiostatic impedance (EIS) measurements were carried out in the frequency range between 10–1 Hz and 100 kHz . Galvanostatic charge–discharge (GCD) measurements were carried out at different current densities . 2000 cycles of GCD at a current density of 8 mA cm−2 were performed on the electrodes to evaluate their stability and capacity retention.A stereomicroscope  was used to study the effect of different single laser scanning parameters on the stability of the melt pool to optimize the 3D printing parameters Fig. e. An ult2, and Mo2S3, respectively. Raman measurements were carried out using a Horiba LabRAM HR800 micro-Raman system equipped with a cooled multichannel CCD detection system in the back-scattering configuration with a spectral resolution of 1 cm−1. A Nd:YAG laser (λ = 532 nm) was used for excitation with a laser spot size diameter of approximately 5 μm. A high-resolution scanning electron microscope  was employed to study the surface morphology of 3D-printed samples in both back-scattered (BE) and secondary (SE) electron modes (X-ray diffraction (XRD) studies were carried out by using a Smartlab  diffractometer equipped with a rotating 9 kW Cu anode X-ray tube with Cu-Kα1 radiation (λ = 1.5406 Å), and a silicon strip detector D/teX Ultra. Based on the Rigaku database, ICDD card numbers 9008543, 1531960, and 1531960 were selected to identify the XRD patterns of Mo, MoSon modes .Supplementary Video 1.Supplementary Information.


The performance of the electrolyte is analyzed based on the sintering temperature (550 to 950 °C) as well as the composition. The trend of XRD results reveals the presence of impurities in the sample, and from Rietveld Refinement, the purest sample is achieved at a sintering temperature of 950 °C and when x = 0.6. The electrolytes obey Vegard′s Law as the addition of Al3+ and Ti4+ provide linear relation with cell volume, which signifies a random distribution. The different composition has a different optimum sintering temperature at which the highest conductivity is achieved when the sample is sintered at 650 °C and x = 0.4. Field emission scanning electron microscope (FESEM) analysis showed that higher sintering temperature promotes the increment of grain boundaries and size. Based on energy dispersive X-ray spectroscopy (EDX) analysis, x = 0.4 produced the closest atomic percentage ratio to the theoretical value. Electrode polarization is found to be at maximum when x = 0.4, which is determined from dielectric analysis. The electrolytes follow non-Debye behavior as it shows a variety of relaxation times.The aim of this study is to synthesize Li Most researchers and engineers are rapidly changing their direction towards energy storage solutions, e.g., lithium-ion batteries and supercapacitors, due to growing awareness of the environmental impacts of fossil fuels and the resilience of energy grids worldwide. Lithium-ion batteries are used in various applications, such as automotive, aviation, electrical appliances, and smart devices ,2. Ceram3) [7La3Zr2O12 [3PS4 and Li4SnS4 [6PS5Br [2(PO4)3 system [2S–P2S5) [Typically, CSE is a great Li-ion conductor compared to polymer and polymer/composite electrolytes, due to the presence of channels in which alkaline ions can migrate easily . CSE als3) ; garnet-a3Zr2O12 ; sulfide Li4SnS4 ; argyrod [6PS5Br ; and NAS3 system . Other t2S–P2S5) —are some2(PO4)3 can be altered to various possible structures where M can be tin (Sn), titanium (Ti), germanium (Ge), hafnium (Hf) or zirconium (Zr) [2P3O12 has great electrical and thermal stability and withstands shock and pressure. However, LiSn2P3O12 usually has a low conductivity value. This obstacle can be solved by substitution method using trivalent cation like gallium (Ga), indium (In), yttrium (Y), aluminum (Al), vanadium (V), iron (Fe), chromium (Cr) or scandium (Sc). In this work, dual metal element substitution method has been implemented using Al3+ and Ti4+. Hence the new formula after dual metal element substitution is Li1+xAlxTixSn2−2xP3O12, where x is varied from 0.2 to 0.8. The substitution process creates positive charge deficiency, which is compensated by Li− ion [1+xAlxTixSn2−2x(PO4)3sample will be produced using several sintering temperatures from 550 to 950 °C. The influence of sintering temperature and composition on the electrical and structural properties of the samples will be the main focus. Based on our knowledge, there is no report on Li1+xAlxTixSn2−2xP3O12 electrolyte.The performance of NASICON-type is strongly dependent on the materials and composition used in the framework. This is because the parent compound, LiMium (Zr) . The PO4ium (Zr) . Other rium (Zr) ,17,18 re Li− ion . In this2O) 99%, tin (IV) oxide (SnO2) 98%, ammonium dihydrogen phosphate (NH4H2PO4) 98%, titanium (IV) oxide (TiO2) 99.8% and aluminum oxide (Al2O3) 99.99%. These materials were procured from Sigma Aldrich .Lithium oxide  was used to conduct structural studies by refinement method.Any changes on the surface of the electrolyte were analyzed using JEOL 7600F FESEM . The electrolytes were examined under vacuum condition with accelerating voltage of 5 kV, and magnification of 20 kx. EDX was conducted using Oxford INCA X-Max 51-XMX 0021  integrated with FESEM (JEOL 7600F).A Bruker AXS D8 Advance X-ray Diffraction spectrometer ,  was employed to study the phase and structural properties of the electrolytein d stands for the thickness of the electrolyte, bulkR is the bulk resistance and A is the interfacial contact area between the electrode and electrolyte. The dielectric constant may be used to portray the capacitive behavior of the electrolyte and to confirm the pattern of conductivity. The charge stored in the electrolyte is called the dielectric constant (ε′), while the energy dissipation is called the dielectric loss (ε″). Data from impedance analysis was used to obtain the dielectric parameters via the following equations [Z′ and Z″ are the real (x-axis) and imaginary (y-axis) parts of the impedance. oC is the vacuum capacitance, and ω stands for angular frequency. The ionic-conducting behavior of a material may also be examined via electrical modulus analysis. The real and imaginary parts of modulus are M′ and M″, respectively, where M′ and M″ can be expressed as:Solartron SI 1260 Impedance Analyzer  was used to analyze the conductivity of each electrolyte in the frequency range of 1 Hz to 32 MHz. The value of conductivity (quations :(2)ε′=Z″M″, relaxation time (rext) was determined using the following equation:peakω is the angular frequency of the relaxation peak.From the peak in the plot of 1+xAlxTixSn2−2x(PO4)3 with x = 0.2, 0.4, 0.6 and 0.8 that are sintered at five different temperatures. XRD analysis is conducted to identify the formation of the compound either single phase or multi-phase as well as the existence of impurity in the compound. It is noticeable in 2(PO4)3 and align with the International Center for Diffraction Data (ICDD) reference pattern (01-087-2078), which is reported in our previous work [ous work .3 phase is also observed in most samples. The impurity peaks are more obvious for the samples with x = 0.8. For the rest, the impurity peak to the signal ratio is quite low. It is attributed to a high amount of substituent, which causes Al3+ ions to react with PO3− ions due to the strength of their charge interaction. Al3+ loses 3 electrons while PO3− receives 3 electrons, thus producing high AlPO3 impurities [4.The samples sintered at 850 °C show almost pure samples with low impurity for all stoichiometries. However, the impurity peak of the AlPOpurities . This ispurities . These f1+xAlxTixSn2−2x(PO4)3 sintered at 950 °C. All parameters obtained from refinement analysis are tabulated in 4, while some are TiO2 and SnO2. There is no trend of impurity amount with the increased amount of substituent. The purest compound with impurity-free is when x = 0.6 is sintered at 950 °C. When more substituents are added into the system, the cell parameter and the cell volume become smaller than LSP. The refinement results show that the problem with this system or compound is the element vacancies in the system.The refinements are done for samples from 650 °C and above due to the crystallinity issues. All samples that are sintered at 550 °C portray amorphous structures. From Rietveld Refinement analysis, all samples have the lowest impurity amount at a sintering temperature of 950 °C; thus, 3+ (0.57 Å) and larger Ti4+ (0.75 Å) compared to Sn4+ (0.65 Å). Linear relation can be seen between the cell volume and x. This phenomenon indicates that these samples obey Vegard’s Law [x/2Li1-xTi2(PO4)3 NASICON-based electrolyte is expected to be random. Thus, Al3+ and Ti4+ in Li1+xAlxTixSn2−2x(PO4)3 system are in random distribution.rd’s Law . Kahlaourd’s Law  reportedConductivity measurement is done on all samples and sintering temperatures. The conductivity values are summarized in 1+xAlxTixSn2−2x(PO4)3 sample has the highest conductivity at sintering temperature of 650 °C, Nyquist plots of all samples at sintering temperature of 650 °C are chosen for a better comparison purpose. 1+xAlxTixSn2−2x(PO4)3 where x = 0.2, 0.4, 0.6, 0.8 and is sintered at 650 °C. The value of bulkR for this kind of plot is taken from the meeting point of the titled line and the semicircle. The semicircle at higher frequency region is due to the conduction of ions in the bulk of the electrolyte while the tilted line at low frequency indicates polarization effects [−6 S cm−1 when x = 0.4 (−6 S cm−1 for LiTi2(PO4)3 system. The inclusion of x = 0.4 of Al3+ has optimized the conductivity to 2.5 × 10−6 S cm−1 for Li1+xAlxSn1.2+xP3O12-based solid electrolyte, which is reported by Lu et al. [c side and the cell volume increases as x value increases. When the cell volume increases, this may cause the ion to move more easily in the cell, which in turn enhances the conductivity values. However, too large a volume can lead to conductivity decrement as ions require more energy to move to neighboring sites.As the Li effects . It is o x = 0.4 . Rao et u et al. . From Ta1+xAlxTi2−x(PO4)3 system has low ionic conductivity value. Furthermore, among all compositions, the sample with x = 0.4 has the most consistent particle arrangement and size distribution, while x = 0.2, 0.6 and 0.8 has larger grain structures. The pathway of ions and electrons to conduct is easier in compounds with consistent particle size distribution [FESEM micrographs of samples sintered at 650 °C are selected as the highest conductivity is obtained at this sintering temperature, as can be seen in ribution .Results 1.4Al0.4Ti0.4Sn1.2P3O12 sintered at low and high temperatures. Electrolyte sintered at 750 °C shows an increase in grain size, while the grain size is further increased for 950 °C and clear grain boundaries can be seen. The increment in grain size is usually due either to re-crystallization or the existing defects in the crystal [1.3Al0.3Ti1.7(PO4)3 system. The authors also stated that sintering temperature will affect the grain size and the resistance of the electrolyte. This explains the pattern of the conductivity value in  crystal ,33. Thes crystal  reported1+xAlxTixSn2−2xP3O12 is obtained at sintering temperature of 650 °C; thus, dielectric study at this sintering temperature is chosen to verify the pattern of conductivity. Dielectric analysis is a crucial method for identifying ionic transport and phase transition mechanism in a system. The pattern of Highest conductivity of Lif > 3) while high at low frequency region [f is more than 3. At rapid rate of electric field, charge carriers experience unstable flow, including collisions among charge carries, which disable proper formation of charge double-layer [1+xAlxTixSn2−2x(PO4)3, the number of charge carriers is the largest when x = 0.4. The trend of conductivity in As observed in y region . Rao et y region  reportedle-layer . The val1+xAlxTixSn2−2x(PO4)3at 650 °C is shown in 1.4Al0.4Ti0.4Sn1.2(PO4)3 has more free ions or charge carriers compared to other compositions. More energy loss is observed as more ionic collision occurs. The pattern of dielectric constant and loss in this study is similar to other NASICON-based solid electrolyte works [3−xMnxO4 ceramic is due to the presence of dielectric anomaly peak. According to the work by Hyatt et al. [The loss of energy of Lite works ,38. Theste works  reportedt et al.  and Luo t et al. , the hum1+xAlxTixSn2−2x(PO4)3 is further studied using electrical modulus, which analyzes the response of Li+ ions in the presence of electric field. Modulus is used to examine ionic conductivities in correlation with the ionic process and conductivity relaxation. It can be observed that the pattern of M′ in f = 7 for all compositions. The presence of this peak is common in a conductor of ion.The electrical properties of LiM′ is low and almost approaching zero from Log f = 0 to Log f = 3. Tripathi et al. [Relaxation process is usually located at the high frequency region, while conduction process is at low frequency region . The vali et al.  reportedM″. The peak of M″ shifted towards higher frequency from x = 0.2 to x = 0.4 and shifted back towards lower frequency regions for x = 0.6 and 0.8. Nikam and Deshpande [1.3Al0.3Ti1.7(PO4) system.eshpande  stated teshpande  with therext of each electrolyte is tabulated in rext is 98 × 10−8 s for x = 0.2 and reduces to 1.57 × 10−8 s when x = 0.4. The trend of rext supports the trend of conductivity. The presence of relaxation is attributed to conduction of free ions in alternating electric fields. It is noticeable that there are several humps in the frequency range between Log f = 4 to 6 in all composition. This is due to the presence of grain and grain boundaries. Polarization of ceramic is highly influenced by the existence of grain boundaries [The undaries . Supriyaundaries  stated t1+xAlxTixSn2−2x(PO4)3 system. Li1+xAlxTixSn2−2x(PO4)3 systems have been successfully prepared using mechanical milling method at 550, 650, 750, 850 and 950 °C. Enhancement of crystallinity with increasing sintering temperature can be seen in XRD analysis. The purest Li1+xAlxTixSn2−2x(PO4)3 system can be obtained at sintering temperature of 950 °C and x = 0.6. Thus, in applications that require pure samples, this sintering temperature and composition can be used. The Li1+xAlxTixSn2−2x(PO4)3 system obeys Vegard’s Law: a linear pattern of cell volume can be observed as Al3+ and Ti4+ are added. The optimum sintering temperature to obtain highest conductivity for samples with x = 0.2, 0.4, 0.6 and 0.8 are 650 °C, 650 °C, 550 °C and 850 °C, respectively. Thus, it can be verified that sintering temperature has a great influence on conductivity. The Li1+xAlxTixSn2−2x(PO4)3 system has the highest conductivity at sintering temperature of 650 °C and x = 0.4. Growth in grain size and grain boundary can be seen at high sintering temperature. Maximum electrode polarization and dielectric constant are obtained when x = 0.4. The Li1+xAlxTixSn2−2x(PO4)3 system follows non-Debye behavior as it shows a variation of relaxation times. Thus, it can be concluded that different sintering temperatures and compositions produced Li1+xAlxTixSn2−2x(PO4)3 with various properties . Hence, the selection of sintering temperature as well as composition should be aligned with the desired application. The performance of the electrolyte can be further enhanced in order to be useful in energy devices. This ceramic electrolyte can be used in hybrid polymer–ceramic electrolytes where polymers like polyethylene oxides (PEO) and polyvinylidene fluoride (PVDF) are used. This can improve electrolyte flexibility and provide more channel for ions to be conducted. Thus, a number of improvements can be made in the future. Other than that, different synthesis approaches  might yield a different result.The main objective of this study has been achieved as different sintering temperature yields different grain size, grain boundary visibility, conductivity and impurity content for NASICON electrolyte with the Li


After treated with 2 µg/mL NOR (1/2 MIC), CBS content, H2S and NO contents decreased while H2O2 accumulation and the antioxidant-related genes mRNA level increased. Additionally, the endogenous H2S content in V. alginolyticus was increased by the exogenous NO, while H2O2 accumulation and the relative expression level of SOD (Superoxide dismutase gene) decreased under exogenous NO or H2S. And the content of endogenous NO and NOS in V. alginolyticus increased under the exogenous H2S as well. Taken together, these results showed that anti-oxidative ability in V. alginolyticus was respectively enhanced by the gas molecules of H2S and NO under NOR-induced stress, and there may be a crosstalk regulative mechanism between H2S and NO. These results lay a foundation for the research of regulation network of H2S and NO, and provide a hint to synthesize anti-vibrio drugs in the future.Antioxidant system is of great importance for organisms to regulate the level of excessive reactive oxygen species (ROS) under the environmental stresses including antibiotics stress. Effects of norfloxacin (NOR) on cystathionine-β-synthase (CBS), nitric oxide synthase (NOS) and antioxidant enzymes were investigated, and interaction between NO and H Antioxidant system, comprised of antioxidant enzymes and nonenzymatic antioxidants, is of great significance for bacteria to resist and reduce ROS under the adverse condition . Superoxnsmitter , and pla(MG1655) . Another content , also ac content . Althougeostasis .Vibrio alginolyticus is a gram-negative bacterium, and a marine pathogenic species which can cause diseases of many marine animals with heavy economic loss in aquaculture industry  and nitric oxide synthase (NOS), H2S and NO and hydrogen peroxide (H2O2), and the relative expression levels of antioxidant-related genes such as superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), were measured under different gas donors and scavengers treatment, which is expected to provide a hint to synthesize drugs to resist Vibrio spp. in the future.In this paper, in order to discover and obtain fundamental information about the role of HV. alginolyticus HY9901 strain used in this work was isolated and preserved in our laboratory, Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals in China , hypotaurine , sodium nitroprusside  and 2-phenyl-4, 4, 5, and 5-tetramethylimidazoline-1-oxyl 3-oxide  came from Sigma-Aldrich Co. LLC (USA), while NOR came from Guangzhou Technology Company Limited (China). ELISA kits for microorganism CBS and NOS, H2S and NO were provided by Shanghai Jianglai Industrial Ltd (China). Hydrogen Peroxide Assay Kit and Total Protein Quantification Assay Kit were from Nanjing Jiancheng Bioengineering Institute (China).Genes encoding antioxidant-related enzymes of SOD, GR and CAT in  results , referenNaSH, SNP, HT and PTIO were respectively weighed, and then dissolved and diluted with cold double-distilled water to yield stock solutions in a final concentration of 100.0 mM. Except for NaSH and SNP are prepared before using, all the other liquors were preserved in −20 °C for one week, after filtering through 0.22 µm microfiltration membrane.0.15 g of NOR was dissolved with 10 mL 12 mol/L cold HCl to yield a stock solution with a final concentration 15,000 µg/mL and preserved in −80 °C for one week.V. alginolyticus was respectively cultured in total volume of 0.5 mL at 28 °C for 48 h with NOR from 0.125 to 256 µg/mL  using two-fold serial dilution method. Briefly, the stock solution of NOR was added into 10 mL V. alginolyticus culture in logarithmic period with OD600 of 0.1, to yield 256 µg/mL NOR treatment, and subsequently 0.5 mL of V. alginolyticus culture with 256 µg/mL NOR was transferred into 0.5 mL of V. alginolyticus culture in logarithmic period without NOR, to yield 128 µg/mL NOR treatment. Similarly, repeat the above dilution till NOR concentration reach 0.125 µg/mL. As a control, a same volume of HCl was added into 10 mL V. alginolyticus culture, and diluted with the same method as described above. Growth of V. alginolyticus in different concentration groups was recorded to determine MIC value of NOR against the bacterium. Every treatment including control were triplicated (n = 3).V. alginolyticus was transferred to 50 mL fresh TSB medium and cultured at 28 °C for 10 h till OD600 value with 0.5, and subsequently treated with 0 (control) and 1/2 MIC concentration NOR, respectively, and continued to culture for 2 h. Then harvested cells and measured CBS and NOS, H2S and NO contents, and H2O2 accumulation as well as the relative expression levels of the antioxidant-related genes. Every treatment was triplicated (n = 3).+ reduced from Cu2+ by proteins in the alkaline condition can react with the bicinchoninic acid (BCA) reagent to form the purple complex which can be spectrophotometrically read at 562 nm, and to quantify the protein by comparing with the standard curve. The absorbance is proportional to the protein concentration, so concentration can be obtained following the formula: total protein = (ODsample- ODblank)/(ODstandard- ODblank) × standard sample (524 µg/mL) × sample dilution times.Total protein was measured according to the bicinchoninic acid method (with standard sample)  with Tot2S content, V. alginolyticus was treated with a final concentration of the following resolution respectively, (1) control; (2) 1.0 mM SNP; (3) 0.2 mM PTIO under NOR for 2 h. Every treatment was repeated thricely. Similar to the above, in order to determine the effect of H2S on NOS content and NO content, V. alginolyticus was treated with a final concentration of the following resolution respectively, (1) control; (2) 1.0 mM NaSH; (3) 0.2 mM HT for 2 h. Every treatment was triplicated (n = 3).To determine the effect of NO on CBS content and H2S on the antioxidant-related enzymes, V. alginolyticus was treated for 2 h with a final concentration of the following resolution respectively: (1) control; (2) 1.0 mM SNP; (3) 0.2 mM PTIO; (4) 1.0 mM NaSH; (5) 0.2 mM HT. Every treatment was triplicated (n = 3). H2O2 accumulation level and relative expression level of the antioxidant-related genes were subsequently determined.For studying the effect of NO and H2)2⋅H2O2)] and 50 µL substrate solution of 3, 3, 5, 5-tetramethylbenzidine (TMB) were added to plate wells. TMB substrate became blue after HRP enzyme catalyzed in 30 min at 37 °C, and this chromogenic reaction was terminated by the addition of 50 µL 2M sulphuric acid solution for the spectrophotometric measurement at 450 nm. The standard curve was set up on the same plate simultaneously, which was used for calculating CBS or NOS contents, and the result was further calibrated by the corresponding total protein concentration. The final results were showed with U/g protein.CBS and NOS contents were measured with ELISA method of the quantitative sandwich immunoassay technique (Sandwich ELISA) . The pri2S contents were measured with the sandwich ELISA method .HA method . Briefly2S measurement, NO was detected by the ELISA kit according to the manufacturer’s instructions . 16S rDNA gene of t-Test method and F test with GraphPad Prism 5. Results were considered statistically significant if P < 0.05 and presented as means with standard error (SEM).Differences between the control and treatments were determined by the statistical V.alginolyticus was 4 µg/mL. Hence subinhibitory concentration as 1/2 MIC concentration (2 µg/mL) of NOR was used to treat V. alginolyticus to assess the interaction of H2S and NO under NOR stress.NOR minimum inhibitory concentration (MIC) against P = 0.248), and H2S content was lower than that in the control group (P = 0.006) (P < 0.001) (P = 0.065) (2O2 accumulation (P = 0.002) and the relative expression level of SOD (P = 0.020) and CAT (P = 0.011) increased under NOR stress compared to the control    and 1C. < 0.001)  though i= 0.065) . Besides control  and 1G, = 0.324) .2S level increased in the treatment groups with exogenous NO by SNP treatment (P = 0.068), whereas CBS content did not change evidently in the groups treated with SNP compared to the control (P = 0.424) and H2S (P = 0.708) content decreased in the group treated with NO scavenger of treatment (PTIO), though P-value failed to achieve a statistical significance  improved NOS content in V. alginolyticus . Interestingly, NO content was up-regulated by both the exogenous H2S (P = 0.189) and H2S scavenger (P = 0.129) treatment compared to the control  compared to the control, but it increased slightly after treated with PTIO (P = 0.191)  (CAT decreased significantly in the treatment groups with different donors or scavengers (Data not shown).Under NOR stress, the relative expression level of = 0.191) . Also, t= 0.168) . It is nSOD, H2O2 accumulation in bacterial cells decreased significantly in the treatment group with SNP (P = 0.003), and also decreased in the treatment group with NaSH (P = 0.759), whereas it rose up when treated with PTIO (P = 0.004) or HT (P < 0.001) with significant difference  while it decreased after treated with PTIO (NO scavenger) (2S donor) and HT (H2S scavenger)  , and NO avenger) , indicatreatment , while Ncavenged . These s2S and NO) in smooth muscle, vasodilation in animal, and cobalt toxicity and heat tolerance in plant  in plants decreased under the exogenous H2S (2S decreased in rats under chronic restraint stress (2S and NO act synergistically on the antioxidant system is inconsistent in all the organisms and should be species-specific.Many studies focused on the synergistical regulation between the two gases (Hin plant , whereas and H2S  and 3, wolyticus . That isectively . Decreasnous H2S . And NO t stress , suggestt stress . Therefo2S and NO are potential to enhance the bactericidal effect, hence it is worthwhile to develop and synthesize these anti-vibrio drugs in the future, even though it needs more endeavors and cross studies in this fields. We believe that it was, and it will be a research hotspot in the future.Finally, the medicines that can disturb the metabolic pathway of HV. alginolyticus was respectively enhanced via the regulation of H2S and NO under NOR-induced stress, and there may be a crosstalk mechanism between H2S and NO to regulate the antioxidant system of V. alginolyticus treated with NOR, which lay a foundation for the research of regulation network of H2S and NO, and for study on the anti-drug mechanism. It also provides a novel target for the synthesis of anti-vibrio drugs.Anti-oxidative ability in 10.7717/peerj.12255/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12255/supp-2Supplemental Information 2Click here for additional data file.


Eumegistus illustris (Bramidae) was 2.94 ± 0.27 µg CTX1B eq. kg−1. Its toxin profile consisted of 52-epi-54-deoxyCTX1B, CTX1B, and 54-deoxyCTX1B, as assessed by LC–MS/MS. This is the first study demonstrating that deep-water fish are potential ciguatera vectors and highlighting the importance of a systematic monitoring of CTXs in all exploited fish species, especially in ciguatera hotspots, including deep-water fish, which constitute a significant portion of the commercial deep-sea fisheries in many Asian–Pacific countries.Ciguatera poisoning (CP) cases linked to the consumption of deep-water fish occurred in 2003 in the Gambier Islands (French Polynesia). In 2004, on the request of two local fishermen, the presence of ciguatoxins (CTXs) was examined in part of their fish catches, i.e., 22 specimens representing five deep-water fish species. Using the radioactive receptor binding assay (rRBA) and mouse bioassay (MBA), significant CTX levels were detected in seven deep-water specimens in Lutjanidae, Serranidae, and Bramidae families. Following additional purification steps on the remaining liposoluble fractions for 13 of these samples (kept at −20 °C), these latter were reanalyzed in 2018 with improved protocols of the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography tandem mass spectrometry (LC–MS/MS). Using the CBA-N2a, the highest CTX-like content found in a specimen of  Aphareus, Aprion, Etelis, and Pristipomoides represent the main targeted species and are also commercially important marine species in many tropical–subtropical fisheries as well PbTx-3 (15 Ci/mmol) were obtained from Latoxan  and Amersham Perkin Elmer Life Science, respectively. The final assay concentration of [3H]PbTx-3 was 0.85 nM. The rat brain synaptosomes were prepared following the protocol described by Dechraoui et al. [−1 giving no more than 10% of the total radioactivity. The radioactivity was determined using a Perkin Elmer Microbeta Trilux 1450 liquid scintillation counter in 2 mL Perkin Elmer Betaplate scintillation cocktail. Nonspecific binding was measured in the presence of saturating concentration of PbTx-3 (0.67 µM) and subtracted from the total binding to yield specific binding. For the pure CTX toxin, six distinct concentrations  of a CTX1B standard solution sourced from ILM ranging from 10−12 to 10−7 g mL−1 were tested in three independent experiments (n = 3). The concentration (Cx) of 2000 mg flesh eq. mL−1 corresponds to the maximum concentration of extract (MCE) that does not induce nonspecific binding on VGSCs from samples of S. powelli found negative by both rRBA and MBA. For deep-water fish samples, the three subsamples (5 g) per fish sample were tested in the same experiment at seven distinct concentrations ranging from 6 to 1200 mg mL−1  of flesh tissue equivalent for half of the samples  and at eight concentrations ranging from 6 to 2000 mg mL−1 for the remaining half samples , each concentration tested in duplicate. The dose-response curves were established by plotting net count per minute (cpm) values vs. pure toxins or fish concentrations tested based on the four parameters logistic regression model (4PL) using GraphPad Prism software version 9.0.2. . From these dose-response curves, the effective concentration, which causes 50% inhibition (IC50), was determined. For the pure CTX1B from the ILM, the mean IC50 value was 0.27 ± 0.01 ng mL−1 (n = 3). The limit of detection (LOD) and quantification (LOQ) for fish samples were estimated to 0.07 ± 0.02 and 0.13 ± 0.01 µg CTX1B eq. kg−1 of flesh, respectively, according to the following formula:Briefly, each subsample (5 g) was resuspended with 7 mL methanol (MeOH) in 50 mL tubes  and sonicated for 2 h. After one night at −20 °C, the crude extracts were centrifuged and further purified on Sep-Pak C18 cartridges  of 3 × 5 g flesh eq. per fish sample tested during the same rRBA experiment.S. powelli, specimens originating from the same fishing area were pooled  and 2 × 500 mL MeOH/H2O 60/40, shaken, and left at room temperature overnight. The resulting CH2Cl2 phase was dried under vacuum and further defatted by a second partition step using 2 × 200 mL cyclohexane and 100 mL MeOH/H2O 80/20. The liposoluble fraction (CH2Cl2 purified into MeOH/H2O 80/20) and the hydrosoluble fraction (MeOH/H2O 60/40) likely to contain CTXs and maitotoxins (MTXs), respectively, were then evaporated, dried under vacuum, and weighed. Upon completion of the MBA analyses, the remaining liposoluble extracts for two pooled samples and eight individual specimens  were stored as archived fractions at −20 °C until 2018 and were further used in CBA-N2a and LC–MS/MS analyses.For the MBA, which requires larger amounts of biological material, sample extraction followed the protocol published later by Laurent et al.  with some pooled . Brieflye pooled  was extrt et al. . The res50) is defined as the i.p. dose of fish flesh killing 50% of a group of two mice (18–21 g) over 24 h, and the lethal potency of CTX1B was 0.35 µg kg−1 [50 for a 20 g mouse, which is equivalent to 7 ng of CTX1B [−1) [−1 of fish flesh. The limits of detection/quantification LOD/LOQ of the MBA was defined as 0.005 MU g−1 of fish flesh corresponding to 0.035 µg CTX1B eq. kg−1 of fish flesh.The liposoluble extracts were tested for their toxicity using the MBA according to Legrand et al. . Groups  µg kg−1 ,107. Oneof CTX1B . Total fX1B [−1) . CTX-lik−1 of dry extract and stored at −20 °C. The CBA-N2a analyses were conducted using mouse neuroblastoma (N2a) cell line (CCL-131) purchased from the American Type Culture Collection  and according to the optimized protocol published later by Viallon et al. [−1 of dry extract (established as the maximum concentration of dry extract or MCE that does not induce nonspecific cytotoxic effects on N2a cells). The qualitative screening results of LF90/10 and LF100 extracts are expressed in N2a cell viability percentages (%) relative to their corresponding controls in OV− (COV−) and OV+ (COV+) conditions according to the following equations:For CBA-N2a analyses, an aliquot of the liposoluble fraction equivalent to 10 g of flesh was dried and resuspended in methanol for each of the 13 available deep-water fish samples . After pn et al. . The inin et al. ) by test50 value could be determined. A CTX1B standard solution sourced from ILM ranging from 0.15 to 19.05 pg mL−1  were tested in parallel with each fish sample. Among the suspect and positive specimens, final concentrations ranged from 74 to 9524 ng mL−1 for LF90/10 dry extracts  and from 372 to 47,619 ng mL−1 for LF90/10 dry extracts (#1–3 and #18) and for LF100 dry extract (#22). Each concentration was tested in triplicate in OV− and OV+ conditions. Toxicity of each sample was then evaluated with dose-response curves established by plotting net absorbance values vs. toxin standard or dry extract concentrations based on the sigmoidal model 4PL using GraphPad Prism software version 9.0.2. The raw EC50 values were expressed in mg of dry extracts mL−1 and converted into mg of flesh eq. mL−1 based on the “dry extract weight/fresh flesh weight” ratio characterizing each fish sample. Indeed, for the same quantity of 10 g extracted flesh, the quantity of dry extract obtained varied from 1.5 to 20.9 mg for the samples, as also observed in previous studies [50 values expressed in mg of flesh eq. mL−1.Second, the composite toxicity estimation was assessed in the LF90/10 and LF100 extracts only from fish specimens previously identified as positive or suspect in the qualitative screening step. To this end, eight distinct concentrations  of CTX1B or fish extracts were analyzed to generate a dose-response curve from which the EC studies . Thus, t50 value was 1.76 ± 0.07 pg mL−1 (n = 4). The LOD and LOQ of the LF90/10 fractions were determined according to the following formulas:−1 flesh, respectively, depending on the “dry extract weight/fresh flesh weight” ratio of fish samples. This variation of LOD and LOQ values according to fish specimens and/or species is well documented in previous studies [For pure CTX1B (ILM), the mean EC studies ,109.50 values of CTX1B and fish extracts determined in the same experiment according to Viallon et al. [The composite cytotoxicity in fish extracts was finally calculated by comparing the ECn et al. , using t−1 of fish flesh and reported as mean concentration ± SD per fish sample tested in one microplate in four independent assays (n = 4) run at different days.The toxin content was expressed in µg CTX1B eq. kg® cartridges  were conditioned with 3 mL of EtOAc/MeOH 85/15. Then, 2 mL of sample extracts were loaded onto the cartridge and eluted with 2 × 2 mL of the same solvent. The two fractions were combined (1 × 2 mL + 2 × 2 mL) and evaporated under nitrogen (N2) at 40 °C. The purified extract was dissolved in MeOH/H2O 70/30 (2 mL). For the second SPE purification, Bond Elut LRC C18 cartridges  were conditioned with 3 mL of MeOH/H2O 70/30, then the purified extract was loaded onto the cartridge. The C18 cartridges were then washed with 3 mL MeOH/H2O 75/25, and then CTXs were eluted with 2 × 3 mL of MeOH/H2O 90/10. The two eluting fractions (2 × 3 mL) were combined and evaporated under N2 at 40 °C. The purified extract was reconstituted with 500 µL of MeOH prior to LC–MS/MS analysis at a concentration ranging from 80 to 160 g flesh eq. mL−1. LC–MS/MS analyses were performed using a UHPLC system  coupled to a hybrid triple quadrupole-linear ion-trap API4000 QTRAP mass spectrometer  equipped with a TurboV® electrospray ionization source (ESI). The instrument control, data processing, and analysis were conducted using Analyst software 1.6.3 . The quantitative targeted analysis of CTXs was investigated according to the method previously described [m/z transitions are listed in −1. For fish samples, 5 µL of sample solution was injected corresponding to 400 to 800 mg flesh equivalents. The LOD and LOQ were determined with the ordinary least-squares regression data method [−1 of fish tissue, respectively.For LC–MS/MS analyses, an aliquot of the liposoluble extracts equivalent to 100–200 g of flesh according to fish specimen was prepared  and resuescribed . The sela method , at 0.00n = 3) and CBA-N2a (n = 4) could be evaluated based on the coefficient of variation (CV) calculated following the formula ((SD/mean) × 100%).No statistical comparison could be made with MBA or LC–MS/MS data, as only one value per specimen was obtained. Conversely, the repeatability of CTX contents obtained with rRBA (n = 3) differed significantly with CBA-N2a values (n = 4), the Student’s t-test (multiple unpaired) was performed using GraphPad Prism software version 9.0.2 .To determine whether rRBA values , CTX-like activities were detected in seven individuals and one pooled samples of deep-water fish using three biological methods , as well as the presence of three CTX analogs confirmed by LC–MS/MS in three of the toxic specimens. It is also worth mentioning that there are presently very few studies in the bibliography that document the comparison of four CTX detection/quantitation methods in fish, and our results showed good agreement between the methods used, with discrepancies observed only in samples contaminated with low levels of CTXs.Pristipomoides filamentosus and Etelis coruscans in many countries of the Asia–Pacific region [Fortunately for local populations, CP cases linked to the consumption of these highly prized deep-water fish species remain scarce . If morec region ,7,9.