Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host’s ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection. The pathogen causes tularemia, a zoonotic disease for which there is currently no licensed vaccine available and that generally is treated by antibiotics  is a small, conservative protein consisting of 76 amino acids that performs an essential role in post-translational modification of proteins. The process, known as ubiquitination, involves three distinct steps. First, an ATP-dependent Ub-activating enzyme (E1) activates the C-terminus of the Ub molecule. Next, E1 is replaced by a Ub-conjugating enzyme (E2), which is then followed by a Ub-ligase (E3). E3 ligase is responsible for the specificity of the Ub-substrate bond, which occurs when Ub binds to lysine residues on the substrate via its C-terminus , 9.F. tularensis live vaccine strain (LVS) has been found to interfere with K63 polyubiquitination, thereby modulating and inhibiting TRAF6 and TRAF3 complexes that control pattern recognition receptor response  are proteases that cleave ubiquitin chains, thereby playing a role in maintaining ubiquitin homeostasis and modifying Ub-protein conjugates. The activity of the host ubiquitin system and, in particular, DUBs during an infectious process can be modulated by various pathogens . F. tularesponse . F. tulan system . Study onfection .Exosomes are small, endosomal membrane microvesicles released by all eukaryotic cells. Most commonly, their sizes range from roughly 30 nm to 150 nm. Their content and function depend on the given situation and the type of cells by which they are produced, and at the same time reflect their metabolic state . Their bF. tularensis subsp. holarctica FSC200. We revealed that F. tularensis interferes with the host’s ubiquitination system. We observed increased activity of deubiquitinating enzymes in THP-1 cells. Using proteomic analysis of selectively isolated enzymatically active DUBs, we confirmed decreased amounts of active USP10 and UCH-L5, as well as increased amount of active USP25, in cells after infection. We also detected enrichment of these three enzymes in THP-1-derived exosomes after F. tularensis infection. Our findings shed light on the molecular mechanisms of this pathogenic bacterium and contribute to better understanding the role of DUBs during infection. The alteration of the activities of particular DUBs may play an important role in Francisella’s ability to resist and escape the host defense mechanisms, and therefore this study could contribute to the development of an effective tularemia treatment.In the work presented here, we focused on DUB enzyme activity during infection of human THP-1 macrophages by 22.12. The cell lines presented in this study were obtained from Sigma-Aldrich (88081201). To induce THP-1 cells differentiation to macrophages, 100 nM phorbol-12-myristate 13-acetate  was used.Human THP-1 monocytes (ATCC TIB-202) were cultivated in RPMI 1640 medium without glucose  supplemented with 10% fetal bovine serum , 2 mM glutamine , and 100 U/mL penicillin–streptomycin  at 37°C in atmosphere of 5% COFrancisella tularensis subsp. holarctica FSC200 was grown in brain heart infusion medium  supplemented with 0.1% cysteine  at 37°C (200 rpm) or on McLeod agar plates at 37°C in normal atmosphere. F. tularensis subsp. holarctica FSC200 is classified as a pathogen of Biosafety Level 2 (BSL-2) according to the Regulation of the Government of the Czech Republic and thus all experiments were performed under the BSL-2 conditions.Gram-negative bacterium 2.28 cells/dish . Following 3 days of cell differentiation by 100 nM phorbol 12-myristate 13-acetate (PMA), the morphology of the differenciated THP-1 macrophages was validated using microscopy (data not shown). THP-1 macrophages were infected by F. tularensis subsp. holarctica FSC200 strain at 500:1 multiplicity of infection. Cells were incubated for 10 or 60 min at 37°C. Uninfected cells were used as a negative control. At the proper time point, THP-1 cells were washed once with ice-cold phosphate-buffered saline . Infected and uninfected cells were scraped into ice-cold PBS, centrifuged for 5 min at 500 × g, and cell pellets were used for further analysis.Submerged cultivated THP-1 monocytes were plated onto dishes at concentration of 102.3The cell culture supernatant from uninfected and infected macrophages at time points 10 and 60 min were analyzed by LDH Cytotoxicity Assay . The measurement of cytotoxicity mediated by infection was in accordance with the manufacturer’s protocol.2.42, 50 mM Tris pH 7.5, 1% glycerol, 2 mM 2-mercaptoethanol,1 mM phenylmethanesulfonyl fluoride , 2 U/mL Turbo™ DNase , and 20 U/mL RNase A/T1 mix ). The cell lysates were centrifuged for 15 min at 21,000 × g at 4°C to remove cell debris. The protein concentration of the supernatant was determined using the Pierce™ BCA Protein Assay Kit  or Qubit Protein Assay Kit  according to the manufacturer’s protocol.THP-1 macrophage pellets were lysed by French pressure cell press in lysis buffer . The deubiquitinase activity was measured utilizing a fluorescent deubiquitinase substrate according to the manufacturer’s protocol.2.6Samples containing equal amounts (1 mg) of cell lysates were subjected to enzymatic reaction with the ubiquitin propargylamid  or vinylmethyl ester  HA-tagged probe at a probe:protein ratio of 1:250 for 30 min at 37°C. After the enzymatic reaction, the samples were immunoprecipitated using 100 µL of anti-HA resin  for 2 h at 4°C. The resin was washed once with 3 volumes of lysis buffer; then once with 3 volumes of 300 mM NaCl, 50 mM Tris pH 7.5, 2 mM 2-mercaptoethanol; and finally three times with 3 volumes of 150 mM NaCl, 50 mM Tris pH 7.5, and 2 mM 2-mercaptoethanol. Elution was performed twice with 100 µL of 50 mM NaOH. The pH of the eluate was adjusted with 1 M Tris to pH 7.5.2.72.7.1Following the manufacturer’s protocol, 100 µg of proteins isolated from cell lysates were prepared for TMT labeling using the TMTsixplex Isobaric Mass Tagging kit . Proteins were digested overnight at 37°C with trypsin  in the ratio trypsin:proteins = 1:30 (w/w). Upon acidification of samples with 1% trifluoroacetic acid (TFA), the sodium deoxycholate  precipitate was formed and then removed by centrifugation for 1 min 3,000 × g. An equal volume of ethyl acetate was added to the supernatant and the mixture was vortexed and centrifuged for 1 min at 3,000 × g. The upper organic phase was removed, and the extraction process was repeated four times to remove DOC. The samples were vacuum-dried and redissolved in 71 µL of 100 mM triethyl ammonium bicarbonate and isobaric labeled by TMTsixplex Isobaric Mass Tagging Reagents . The pooled samples were desalted using Empore C18-SD extraction disk cartridges , eluted by 60% acetonitrile (ACN)/0.1% TFA, vacuum-dried, then stored at −20°C. The isobaric-labeled peptides were fractionated by a home made gradient capillary chromatography wherein reverse phase C18 under basic pH and hydrophilic interaction liquid chromatography conditions were used. Each fraction was vacuum-dried and stored at −20°C.2.7.2The eluted proteins were precipitated by 0.18 volume of 100% trichloroacetic acid , incubated for 4 h on ice, then centrifuged for 30 min at 21,000 × g and 4°C. The supernatant was discarded; the pellet was washed with ice-cold 90% acetone and then centrifuged for 15 min at 21,000 × g and 4°C. The acetone was removed and the pellet was dried. Next, the pellet was solubilized in 4% DOC in 25 mM ammonium bicarbonate . The proteins were reduced with 4 mM dithiothreitol  in 25 mM ABC and directly alkylated with 16 mM iodoacetamide  in 25 mM ABC. The excess of IAA reagent was quenched by the addition of DTT to a final concentration of 4 mM DTT in 25 mM ABC. Digestion was performed overnight at 37°C using SOLu-trypsin in a ratio of 1:30 (w/w). Next, 20 µL 1% TFA  was used for acidification the sample mixture. The DOC precipitate was removed by centrifugation and ethyl acetate extraction as described above. The samples were desalted using Empore C18-SD extraction disk cartridges .2.7.3The samples were dissolved in 20 µL of 2% ACN/0.1% TFA (v/v), and 1 µL of the sample was analyzed using a gradient nanoLC system with UV detection  containing a µ-Precolumn  and analytical NanoEase column . The optimized sample loadings were then used for LC-MS analysis on the UltiMate 3000 RSLC-nano HPLC system (Dionex) with a trap column (75 µm × 20 mm) packed with 3 µm Acclaim PepMap100 C18 particles and a separation column (75 µm × 150 mm) packed with 2 µm Acclaim PepMap RSLC C18 particles, all coupled to the QExactive system  in positive ion mode. Instrumental data acquisition was done using Xcalibur software v. 4.2.47 and Tune control software v. 2.11.0.3006. The instrument settings for TMT quantitation analysis were set as follows: full MS scan (400–1650 m/z) at 70,000 full width at half maximum (FWHM) with maximum filling time 100 ms and automatic gain control (AGC) target 3E6; top 10 precursors in MS/MS at 35,000 FWHM with isolation window 1.2 m/z, fixed first mass at 110 m/z, maximum filling time 250 ms, and AGC target 2E5. The settings for label-free quantitation analysis were these: full MS scan (350–1650 m/z) at 70,000 FWHM with maximum filling time 100 ms and AGC target 1E6; top 12 precursors in MS/MS at 17,500 FWHM with isolation window 1.6 m/z, fixed first mass at 140 m/z, maximum filling time 100 ms, and AGC target 1E5.2.7.4Francisella tularensis reference proteome UP000006302; enzyme – trypsin; maximum missed cleavage sites – 1; min. peptide length – 7; precursor mass tolerance – 10 ppm; fragment mass tolerance – 0.02 Da; weight of b- and y-ions – 1; static modifications – carbamidomethyl/+57.021 (C); dynamic modifications – oxidation/+15.995 Da (M); dynamic modifications (protein terminus) – acetyl/+42.011 Da (N-terminus), met-loss/−131.040 Da (M), met-loss+acetyl/−89.030 Da (M). The search results thus obtained (msf file) were processed in the consensus workflow containing PSM Grouper, Peptide Validator, Protein and Peptide Filter (two peptides with strict target FDR 0.01), Protein Scorer, Protein FDR Validator, Protein Grouping, Protein in Peptide Annotation, Modification Sites, Protein Annotation and Protein Marker nodes. In the TMT6 labeling experiment, Reporter Ions Quantifier node was used in the processing workflow and the Reporter Ions Quantifier node in the consensus workflow (with pairwise computed protein ratio and background-based t-testing). For the label-free quantitation analysis, Minora Feature Detector node was used in the processing workflow and Feature Mapper and Precursor Ions Quantifier (with pairwise computed protein ratio and background-based t-testing) nodes were applied in the consensus workflow.The raw spectra files thus obtained were processed in Proteome Discoverer software  to identify proteins and to quantify relative protein abundance in the samples. The data processing workflow contained a spectrum selector, non-fragment filter, top N peaks filter (LFQ only), precursor detector, SequestHT search engine, Percolator validator, Spectrum Confidence Filter, SequestHT search engine and Percolator validator  nodes. The parameters for the first SequestHT database search were: protein database – UniProt human reference proteome UP000005640 and UniProt The raw MS data as well as processed identification results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD043492 (DOI: 10.6019/PXD043492).2.8Eluates and cell or exosome lysates were separated by SDS-PAGE electrophoresis in gradient 4–12% NuPAGE gels  and transferred onto PVDF membrane  using Western blot. Membranes were blocked for 1 h in Blotting-Grade Blocker  and subsequently incubated with primary antibodies for 1 h at room temperature. The TBS  buffer supplemented with 0.1% Tween 20  was used in all steps. Rabbit anti-UCH-L5 , rabbit anti-USP10 , rabbit anti-USP25 antibody , and mouse anti-tubulin antibodies , respectively, were used for UCH-L5, USP10, USP25, and tubulin detection. Rabbit anti-CD9  was used for detection of the CD9 exosome marker.After incubation with appropriate horseradish peroxidase-labeled secondary antibodies and washing membranes in TBS supplemented with 0.1% Tween 20, proteins were detected using enhanced chemiluminescence reagents . Images were visualized on medical X-Ray blue film .2.94 · 7 H2O), 0.2% silver nitrate in water for 1 min. Then the membranes were rinsed with water and air-dried.Activated membranes were washed in distilled water and stained in staining solution prepared from 2% sodium citrate, 0.8% ferrous sulfate  was used for reverse transcription reaction . The cDNA obtained was used as a template in subsequent polymerase chain reaction (PCR) reactions. Transcription of UCH-L5, USP10, and USP25 genes was analyzed by PCR using appropriate primers , appropriate KiCqStart SYBR Green primers . The samples were then ultracentrifuged at 100,000 × g for 70 min at 4°C. The supernatants were removed, the pelleted exosomes were washed with ice-cold PBS, and additional centrifugation was carried out at 100,000 × g for 70 min at 4°C. The exosomes were finally resuspended in PBS containing an EDTA-free protease inhibitor cocktail .2.12For electron microscopy, the exosomes were visualized by negative staining. Parlodion-carbon-coated grids were floated on the top of a 10 µL drop of sample for 5 min. The grids were then stained by 2% uranyl acetate, for 2 × 30 s and dried. The grids were visualized with a JEOL JEM-2100 Plus electron microscope operated at 200 kV.2.138 particles/mL. Five 60-second videos were recorded for each sample, with the camera level set to 16. The following settings were used for analysis: detection threshold 5, automatic blur size, automatic maximum track length, and automatic maximum expected particle size.The size and concentration of exosomes were assessed by nanoparticle tracking analysis (NTA) using a NanoSight NS300 analyzer  equipped with a green laser (532 nm), sCMOS camera, automatic injection pump system, and NTA 3.4 software. Before NTA measurements, isolated vesicles were diluted in 0.1 µm filtered PBS to achieve a concentration of approximately 1.0 × 1033.1Francisella can manipulate the host deubiquitination system, we analyzed the expression level of selected DUBs in THP-1 macrophages after Francisella infection. In advance, the cell cytotoxicity at 10 and 60 min after infection was measured and no toxicity was found  can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.VV, PR, KH, PS, and PP conceived and designed the experiments. VV, KH, JH, RH, PS, and PP performed the experiments. VV, PR, KH, JH, RH, PS, and PP analyzed the data. VV, PP, PS, KH, PR, and JS wrote the paper. All authors contributed to the article and approved the submitted version.


The structures of the new compounds were determined by analyzing their detailed spectroscopic data, while the absolute configurations were further determined through electronic circular dichroism calculations. Snatzke’s method was additionally used to determine the absolute configurations of the diol moiety in 1. In a bioassay, compounds 7 and 10 performed greater inhibitory activities on interleukin-6 and interleukin-1β than the positive control (dexamethasone) at the concentration of 25 μM. Meanwhile, compounds 5 and 6 showed potent effects with stronger inhibition than dexamethasone on IL-1β at the same concentration.Four new sorbicillinoids, named trichodermolide E ( The mangrove is a complex ecosystem occurring in tropical and subtropical intertidal estuarine zones . The speAspergillus, Penicillium, Trichoderma, Phialocephala, etc. + , see Trichodermolide E (2): yellow oil (6.7 mg). λmax (log ε): 213 (2.92), 286 (2.65), see 1H NMR , 13C NMR  and other NMR data, see m/z 293.1034 [M−H]− , see Trichosorbicillin J (3): red oil (10.2 mg). λmax (log ε): 306 (2.27); ECD (MeOH) λmax (Δε): 214 (−5.03), 247 (+3.41), 286 (−1.42), 333 (+7.75), see 1H NMR , 13C NMR  and other NMR data, see m/z 499.2326 [M−H]− , see Bisorbicillinolide B (5): yellow oil (8.4 mg). λmax (log ε): 213 (2.62), 237 (2.42), 287 (252), 338 (2.65), 360 (2.63); ECD (MeOH) λmax (Δε): 214 (−1.37), 239 (+4.97), 289 (−12.18), 306 (−9.47), 342 (+11.88), see 1H NMR , 13C NMR  and other NMR data, see − , see Demethylsorbiquinol (Spartan’14 (v1.1.0) [Gaussian 09 [SpecDis (v1.64) [Conformational analyses were carried out via random searching with an MMFF force field using (v1.1.0) . Subsequssian 09 . Then, T (v1.64) . The the2(OAc)4 for 1 were subjected to ECD measurement at a compound concentration of 1.0 mg/mL in DMSO for each. After mixing, the first ECD was recorded immediately, and the time evolution was surveyed until stationary (about 10 min after mixing). The inherent ECD was subtracted. In the induced ECD spectra, the observed signs of the diagnostic bands at around 310 and 400 nm were correlated to the absolute configuration of the 17,18-diol moiety.A modified version of Snatzke’s method was performed according to the published literature ,18. Mixt−△△CT values of the same gene in different samples, which were then converted into inhibition ratios. The real-time PCR method was performed to measure the expression of the LPS-induced inflammatory factors IL-1β and IL-6 in RAW264.7 cells. Cells were randomly divided into control, stimulation, and administration groups, with 3 wells in each group. The control groups were cultured with a complete culture medium, the stimulation groups were stimulated with LPS , and the administration groups were stimulated with the mixture of LPS  and test compounds . After culturing in an incubator for 4 h, reverse transcription was performed. Real-time PCR was performed to quantitatively analyze the products. The difference in CT values between the target gene and the internal reference gene GADPH (ΔCT) was used to perform the quantitative detection of genes in the samples. Results were given with the 2Trichoderma reesei BGRg-3, derived from the mangrove plant Avicennia marina. Compound 1 exhibited a special skeleton that was rare in the sorbicillinoid family. Its 1D and 2D NMR data gave the relative configuration, and its absolute configuration was deduced based on ECD calculations and Snatzke’s method. Compound 2 was a derivative of sorbicillinol. Based on the comparison with similar compounds, constitutional formulas of compounds 3 and 5 were confirmed. Their absolute configurations were also determined through ECD calculations. In the bioactive evaluation, known compounds 7 and 10 exhibited the highest inhibition of IL-6 and IL-1β among the tested compounds, even more effective than dexamethasone. Compounds 5 and 6 also presented potent inhibition of IL-1β and moderate inhibition of IL-6. In addition, compound 6 showed the advantage of little cytotoxicity, which makes it possible to develop a nontoxic anti-inflammatory drug. Compound 8 exhibited remarkable cytotoxicity against RAW264.7 cells at the test concentration level; therefore further research on its cytotoxicity against other cells is expected.Our work discovered four new sorbicillin-based derivatives from the secondary metabolites of


The comparison between both insecticides’ reveals that only the Envidor® 240SC induces concentration-dependent DNA damage, as well as structural changes in the germarium. We determined that the DDR induced by Movento® 240SC depends on the activation of the tefuATM, grpChk1 and lokChk2 kinases by significantly increasing the percentage of expression of γH2AV in regions 2b and 3 of the germarium, and that mei−29DATR and dp53p53 kinases only respond at the highest concentration of 37.3 mg/L of Movento® 240SC. With the Envidor® 240SC insecticide, we determined that the DDR depends on the activation of the mei−29D/Chk1grpATR and tefu/Chk2lokATM kinases, and dp53p53 by significantly increasing the percentage of expression of γH2AV in the germarium.DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento Both insecticides were diluted with deionized water to the final concentrations of  of Movento® 240SC and  of Envidor® 240SC. Two hundred microliters of each concentration were mixed with yeast to form a paste that was deposited in the bottom of Drosophila Genesee Scientific  food vials. The concentrations of both insecticides that induced DNA damage in ovarian cells were reported in a previous study and used in three independent experiments [The keto-enol insecticides Moventoeriments .Drosophila melanogaster strains: Oregon R, tefuATM, mei−29DATR, grpChk1/lokChk2, grpChk1 and dp53p53, were collected for 3 days, grouped into 4 groups with 10 females each, to be incubated and fed in vials containing a yeast mixture with a concentration of 11.2, 22.4 y 37.3 mg/L of Movento® 240SC and of 12.3, 24.6, 41.1 mg/L of Envidor® 240SC, independently, for 72 h at 25 °C. As a control group, food without pesticide (0.0 mg/L) was used, under the same conditions as the experimental groups  were obtained, which were disaggregated into ovarioles . Ovarioles were fixed in 800 µL of fixative solution , for 20 min. Subsequently, they were washed with 1 mL of 1X PBST (0.1% Tween-20 1X PBS), 3 times for 10 min.After 72 h of exposure to the keto-enol insecticides MoventoD. melanogaster strains after 72 h of exposure to the three concentrations of the keto-enol insecticides Movento® 240SC and Envidor® 240SC, and control groups, were incubated for 1 h in 1 mL of 1% PBST + BSA (10 mL PBST + 0.1 g BSA). After this time, they were incubated overnight in 500 μL of primary antibody diluted in 1 mL of (1% PBST + BSA) at 4 °C on a rocking nutator. Afterwards, they were washed three times with PBST and incubated in 500 μL of secondary antibody diluted 1:500 in (1% PBST + BSA) for 2 h, and for 10 min with 5 μL of DAPI. Subsequently, they were washed with 1X PBST three times for 15 min and mounted on a slide with 35 μL of ProLong Gold [The ovarioles of the wild-type and DDR mutant ong Gold .The following antibodies were used for staining the nuclei: primary (rabbit α-γH2AvD (p5137), 1:1000) ), secondary (Alexa fluor-488 goat α-rabbit IgG (H+L), 1:500) ) and DAPI  ). The primary antibody γH2AvD was used as a marker for DNA double-strand breaks in the Drosophila germarium as described in .tefuATM, mei−29DATR, grpChk1/lokChk2, grpChk1 and dp53p53) of each experimental and control group were taken with a Zeiss LSM880 confocal laser scanning microscope, using a 63 ×/0.65 NA oil immersion objective, using ZEN 2.1 software . The images were saved as .czi files with a key unknown to the reader for processing with FIJI software . The intensity of the emitted fluorescence of γH2AV was reported as the average of the total percentage of γH2AV expressed in the germarium modified from [Images of the germarium  of females of the strain ; statistical analysis was performed using the GraphPad Prism version 9 program.The values obtained for the percentage of expression of γH2AV in the germarium of DDR mutant and wild-type females were reported as means ± standard deviation of three independent experiments for each experimental and control group. The data were analyzed with two-way analysis of variance (ANOVA) and a multiple comparison post hoc test (Tukey) to determine significant differences between the experimental and control groups of each strain (Drosophila melanogaster (Oregon R) germarium, after 72 h of oral exposure to the concentrations of 11.2, 22.4, and 37.3 mg/L of the keto-enol insecticide Movento® 240SC, compared to the average value of basal damage in the DNA of the germarium of the control group (0.0 mg/L of pesticide) (p < 0.0001)  (p < 0.0001)  germarium (p < 0.0001) (D. melanogaster (Oregon R) exposed to the three concentrations of the insecticide Envidor® 240SC (At a concentration of 12.3 mg/L of the insecticide Envidor 0.0001) D. Additi® 240SC and Envidor® 240SC shows a concentration–effect response for Envidor® 240SC (r2 = 0.92), but not for Movento® 240SC (r2 = 0.55)  induced by the keto-enol insecticides Movento = 0.55) E.tefATMu mutant females, after 72 h exposure to concentrations of 11.2, 22.4 and 37.3 mg/L of Movento® 240SC (1)), through the significant increase in the percentage of expression of γH2AV compared to the values of the control groups (tefuATM 0.0 mg/L of pesticide) (p < 0.0001)  (tefuATM strain (r2 = 0.82) ( 0.0001) A. Additi = 0.82) B.mei−29DATR mutant females, after 72 h of exposure to concentrations of 11.2, 22.4 and 37.3 mg/L of Movento® 240SC, there was a significant increase in DSBs in DNA in regions 2b and 3 of the germarium (mei−29DATR 0.0 mg/L of pesticide) (p < 0.0001)  (® 240SC does not induce concentration-dependent DNA damage in mei−29DD. melanogaster ATR (r2 = 0.75) (In  0.0001) A. The li = 0.75) C.grpChk1/lokChk2 mutant females, after oral exposure to concentrations of 11.2 and 37.3 mg/L of Movento® 240SC, we determined a significant increase in DSBs in DNA, in all regions of the germarium (grpChk1/lokChk2 0.0 mg/L of pesticide) (p < 0.0001)  (p < 0.0001)   (In  0.0001) A. At a c 0.0001) A. Howeve = 0.20) D.grpChk1 mutant females exposed for 72 h at a concentration of 11.2 mg/L, there was no significant increase in DSBs in the DNA and in the percentage of expression of γH2AV, in the germarium (grpChk1 0.0 mg/L of pesticide) (p < 0.0001)  (p < 0.0001) (® 240SC we observed inhibition of cell proliferation evidenced by the absence of cell nuclei in region 1 and reduction in the size of the germarium (grpChk1 0.0 mg/L of pesticide)  (In  0.0001) A. Howeve 0.0001) A. Additi = 0.91) E.dp53p53 mutant females, after exposure to concentrations of 11.2 and 22.4 mg/L of Movento® 240SC, there was no significant increase in DNA double-strand breaks and in the percentage of expression of γH2AV, in the germarium (dp53p53  0.0 mg/L of pesticide)  (p < 0.005)   A. Howeve< 0.005) A. The li = 0.88) F.tefuATM mutant females after 72 h exposure to concentrations of 12.3, 24.6 and 41.1 mg/L of Envidor® 240SC (1)) and the percentage of expression of γH2AV compared to the control groups (tefuATM 0.0 mg/L of pesticide) (p < 0.0001)   ( 0.0001) A. Additi = 0.77) B.mei−29DATR mutant females, after 72 h of exposure to concentrations of 12.3, 24.6 and 41.1 mg/L of Envidor® 240SC, there was a significant increase in DSBs in DNA of the germarium (mei−29DATR 0.0 mg/L of pesticide) (p < 0.0001)   (In  0.0001) A. At a c = 0.29) C.grpChk1/lokChk2 females exposed to 12.3 and 41.1 mg/L concentrations of Envidor® 240SC for 72 h, there is a significant increase in DNA double-strand breaks in regions 1 and 3 of the germarium (grpChk1/lokChk2 0.0 mg/L of pesticide) (p < 0.0001)  (p < 0.0001)   (In Drosophila  0.0001) A. At a c 0.0001) A. Howeve = 0.11) D.grpChk1 mutant females, exposure to concentrations of 12.3, 24.6 and 41.1 mg/L of Envidor® 240SC significantly increases DSBs in DNA in all regions of the germarium (grpChk1 0.0 mg/L of pesticide) (p < 0.0001)  (In  0.0001) A. The li = 0.69) E.dp53p53 mutant females of D. melanogaster, exposure to concentrations of 12.3, 24.6 and 41.1 mg/L of Envidor® 240SC significantly increased DSBs in DNA in all regions of the germarium (dp53p53 0.0 mg/L of pesticide) (p < 0.0001) (dp53p53 0.0 mg/L of pesticide) (® 240SC in the dp53p53 mutant (r2 = 0.52)  A. Concen = 0.52) F.® 240SC and Envidor® 240SC and their active ingredients (Spirotetramat and Spirodiclofen) have been shown to induce DNA damage in non-target organisms [® 240SC and Envidor® 240SC induce a significant increase in damage in the ovary DNA of wild-type Drosophila melanogaster (Oregon R) [Drosophila melanogaster in the wild-type strain Oregon R and DDR mutants: tefuATM, mei−29DATR, grpChk1/lokChk2, grpChk1 and dp53p53 exposed for 72 h orally to elucidate DDR pathways.The commercial keto-enol insecticides Moventorganisms ,14. In aregon R) . HoweverD. melanogaster (Oregon R) after exposure to concentrations of 11.2, 22.4, 37.3 mg/L of Movento® 240SC and 24.6, 41.1 mg/L of Envidor® 240SC for 72 h show a significant increase in DNA double-strand breaks (DSBs) in regions 2b and 3 of the germarium. However, at a concentration of 12.3 mg/L of Envidor® 240SC, there were no significant changes in DSB production in the germarium of D. melanogaster (Oregon R) compared to the percentage of endogenous expression in region 2a of the control group. (0.0 mg/L) (p < 0.0001). However, exposure to the three concentrations of Envidor® 240SC produced structural changes in the germarium, compared to the germarium of the control group.The results of the expression of histone γH2AV in the germarium of the ovaries of wild-type Drosophila melanogaster is exclusively found in oocytes in the pachytene subphase of prophase I of meiosis I, in region 2a. These are repaired before differentiation into region 3 [® 240SC and Envidor® 240SC, indicate that both insecticides act as genotoxic agents through DSB induction. When comparing the induction of DNA damage in the germinal cells of the germarium in relation to both insecticides, we observed that only the insecticide Envidor® 240SC induces concentration-dependent DNA damage, as well as structural changes of the germarium in wild-type Oregon R females. It is reported that one of the mechanisms of genotoxicity and cytotoxicity of Spirodiclofen (active ingredient of Envidor® 240SC) is the induction of oxidative stress by increasing the production of reactive oxygen species (ROS), such as superoxide anion (O2−•), the hydroxyl radical (HO•), hydrogen peroxide (H2O2), with increased activity of endogenous antioxidants SOD and CAT, and induction of lipid peroxidation [® 240SC. In female zebrafish (Danio rerio), an induction of morphological alterations in the ovary, oxidative stress and lipid peroxidation were reported after exposure to the keto-enol insecticide Spirotetramat [It has been reported that the endogenous induction of DSBs for meiotic recombination and visualization through the expression of γH2AV in region 3 ,58. Therxidation . One of xidation , which mtetramat .® 240SC in mutant tefuATM of D. melanogaster demonstrates that the production of DSB in the DNA of germarium cells activates DDR mediated by ATM kinase, and both ATMtefu and ATRmei−29D kinases are only activated in the presence of DSB at the highest concentration (37.3 mg/L). Some studies have indicated that ATR may have functional redundancy with ATM even by activating effectors initially phosphorylated by ATM [Esenia fetida) exposed to 1.25 and 2.5 mg/kg−1 of Spirotetramat for 14, 21 and 28 days, in which induction of oxidant stress and lipoperoxidation correlated with DNA fragmentation were determined [® 240SC in grpChk1 and grpChk1/lokChk2 mutant strains demonstrates the increase in DNA damage and the deficiency of DNA repair mechanisms in regions 1, 2a and 2b of the germarium, and consequently, induction of cell death evidenced by the absence of DAPI-stained cell nuclei. The significant increase in the expression of γH2AV in dp53p53 mutant females in relation to the Oregon R wild-type strain (p < 0.005) only at the highest concentration of 37.3 mg/L of Movento® 240SC, allowed us to infer that, in general, the mechanisms of DNA damage response and repair are mainly regulated by Chk2lok, but not dependent on p53dp53. Additionally, it supports our hypothesis that this concentration generates many ROS that activate multiple DNA damage response and repair mechanisms. In addition to activating p53, Chk2 can regulate the activation of transcription factors, such as FOXO1 [grpChk1/lokChk2 deficient mutant strain, where, in the absence of Chk2, FOXO1 cannot be activated and consequently regulate cell proliferation and survival, in addition perhaps to responding to oxidative stress induction. Negative regulation of FOXO1 expression has already been reported in female mice ovaries exposed to Movento, in which histological analysis revealed damage to cell membranes, nuclear fragmentation, and cell death of ovarian granulosa cells [Exposure to the same concentrations of Moventod by ATM ,62. Thisd by ATM . Our restermined . In thisas FOXO1 . FOXO1, as FOXO1 ,66 as weas FOXO1 . This cosa cells .® 240SC, after exposure to concentrations of 12.3, 24.6 and 41.1 mg/L, we determined a significant increase in the percentage of γH2AV expression in the germarium of tefuATM and mei−29DATR mutant females independently, which could perhaps indicate that Envidor® 240SC acts as a genotoxic agent through the induction of SSBs (single-strand breaks), which consequently lead to the formation of DSBs. ATR is activated when SSBs and stress occur during replication [tefuATM and mei−29DATR mutant strains, cell death is induced, evidenced by the absence of nuclei in region 3, perhaps derived from the induction of oxidative stress and ROS production that could be interfering with the DNA damage response and repair mechanisms, leading to cell death. A recent study reported that Spirodiclofen (the active ingredient of Envidor® 240SC) increases the frequency of micronuclei (MN), chromosomal aberrations (CA) and DNA fragmentation, in addition to decreasing the percentage of the mitotic index in meristem cells of Allium cepa, correlated with the induction of oxidative stress and lipid peroxidation. Spirodiclofen administration also caused structural damage, such as cell wall thickening, flattened cell nucleus, cell deformation and necrosis [grpChk1 mutant females compared to Oregon R wild-type females (p < 0.0001), supports our hypothesis that Envidor® 240SC primarily induces SSBs and that, in this context, lesion repair initially depends on ATR-mediated activation of Chk1. However, SSB formation ultimately leads to DSB formation exceeding Chk1 repair capacity, upon which Chk2 is activated to initiate repair mechanisms and/or cell death [grpChk1 and grpChk1/lokChk2 kinases, we identified a significant increase in DNA damage, and an absence of cell nuclei in all regions of the germarium, which could also be a consequence of the induction of oxidative stress. With the significant increase in DNA damage induced by the insecticide Envidor® 240SC in dp53p53 mutant females, we define its direct participation in activating DNA damage response and repair mechanisms [® 240SC, there are other effectors downstream of Chk2 that initiate cell death processes, such as BRCA1 and transcription factors such as FOXO1 and E2F1, that also regulate the proliferation and induction of cell death [In relation to the commercial insecticide Envidorlication . In resplication ,71; faillication , simultalication . Additionecrosis ,75. The ll death ,77. Thischanisms  but not ll death ,79.® 240SC and Envidor® 240SC . Movento® 240SC depends on the activation of ATMtefu, Chk1grp and Chk2lok kinases, with ATRmei−29D and p53dp53 kinases only responding at the highest concentration of 37.3 mg/L. With the Envidor® 240SC insecticide, we determined that the DDR depends on the activation of the ATRmei−29D/Chk1grp and ATMtefu/Chk2lok kinases, and that the repair mechanisms are dependent on p53dp53. The data obtained support our hypothesis that both keto-enol insecticides represent a potential risk for the female reproductive system, affecting the proliferation, maturation, and development of germ cells, which could consequently affect the quality of the oocytes and the fertility rate. However, further studies are still required to fully elucidate the DNA response and repair mechanisms induced by this class of insecticides.Based on our results, we show that the commercial keto-enol insecticides Movento


Such a two-dimensional/three-dimensional heterostructure enables suppressed light-induced halide segregation, reduced non-radiative interfacial recombination, and facilitated charge extraction. The wide-bandgap perovskite solar cells demonstrate a champion power conversion efficiency of 19.6% and an open-circuit voltage of 1.32 V. By integrating with the thermal-stable FAPb0.5Sn0.5I3 narrow-bandgap perovskites, our all-perovskite tandem solar cells exhibit a stabilized PCE of 28.1% and retain 90% of the initial performance after 855 hours of continuous 1-sun illumination.Light-induced halide segregation constrains the photovoltaic performance and stability of wide-bandgap perovskite solar cells and tandem cells. The implementation of an intermixed two-dimensional/three-dimensional heterostructure via solution post-treatment is a typical strategy to improve the efficiency and stability of perovskite solar cells. However, owing to the composition-dependent sensitivity of surface reconstruction, the conventional solution post-treatment is suboptimal for methylammonium-free and cesium/bromide-enriched wide-bandgap PSCs. To address this, we develop a generic three-dimensional to two-dimensional perovskite conversion approach to realize a preferential growth of wider dimensionality (n ≥ 2) atop wide-bandgap perovskite layers (1.78 eV). This technique involves depositing a well-defined MAPbI The conventional solution post-treatment is suboptimal for methylammonium-free and cesium/bromide-enriched wide-bandgap perovskite solar cells. Here, the authors develop a 3D-to-2D perovskite conversion approach for a preferential growth, achieving stabilized efficiency of 28.1% for tandem cells. In the past few years, the power conversion efficiency (PCE) of all-perovskite tandem solar cells has already surpassed that of single-junction perovskite solar cells (PSCs), positioning themselves as a promising next-generation photovoltaic (PV) technology8. Despite the significant advancements in PCE, instability issues still hinder the commercialization of all-perovskite tandem solar cells9.All-perovskite tandem solar cells, which consist of a wide-bandgap  perovskite top cell paired with a narrow-bandgap  perovskite bottom cell, offer the potential for higher efficiency than the SQ limit of single-junction solar cell while maintaining the benefits of low-cost solution processing12, thereby limiting the operational lifetime of tandem PSCs. Various strategies have been employed to address this challenge, such as passivating the interface14, relaxing lattice strain16, modifying compositions22, and developing new transport layers23. Such efforts have, to some extent, mitigated light-induced halide segregation. Nevertheless, understanding the fundamental mechanism of phase segregation in full device structure is still a matter of ongoing investigation.The WBG subcells, which alloy a high content of bromide (~40%), suffer from severe light-induced halide segregation under continuous illumination26. Here, we reveal that charge accumulation accelerates the halide segregation in WBG PSCs under illumination, originating from the mismatch of energy levels and high trap density at contacting heterointerface with the electron transport layer . This prompts us to design the perovskite/C60 interface with facilitated charge extraction and durable interfacial contact.In single-junction PSCs with bandgaps ~1.5 eV, carrier charges trapped in perovskite films were found to induce irreversible degradation29. The solution-based post-treatment involves the dissolution of the 3D perovskite, leading to a more PbI2-rich surface suitable for the ammonium ligands to intercalate30. However, when applied to the MA-free and Cs/Br-enriched WBG perovskites, recognized for their better photostability and thermal stability31, this technique yields inconsistent results due to intricate surface chemistry with increased activation energy for surface reconstruction growth.The construction of an intermixed 2D/3D heterostructure, accomplished by spin-coating long-chain ammonium ligands dissolved in isopropyl alcohol (IPA) onto the surface of a 3D perovskite, has been widely utilized to optimize contact heterointerfaces in PSCs3 thin layer by a hybrid evaporation/solution method, followed by its complete transformation into a 2D structure via a long-chain ammonium ligand. This approach overcomes the composition-dependent surface sensitivity of conventional solution-processing strategies and enables the preferential growth of wider dimensionality (n ≥ 2) on the MA-free WBG perovskite layer. The resulting 2D/3D heterostructure effectively accelerates the charge extraction at the perovskite/C60 interface and thereby suppresses light-induced halide segregation. We achieved a champion PCE of 19.6% and an impressive Voc of 1.32 V in 1.78-eV WBG PSCs. Meanwhile, the devices exhibited superior operational stability – maintaining 95% of their initial PCE after 1000 h operation at the maximum power point (MPP). These enabled us to realize an impressive PCE of 28.1% in monolithic all-perovskite tandem solar cells.Herein, we present a generic 3D-to-2D perovskite conversion approach: depositing a well-defined MAPbIx-SAM/perovskite/C60/ALD-SnO2/Cu (SAM: self-assembled monolayer). The photo-generated carriers under OC condition are expected to be unextractable and would accumulate at a contacting selective heterointerface, in contrast to the MPP condition where carriers are extracted from the absorber during operation34. For devices with different bandgaps, we found that devices under MPP tracking showed significantly less degradation than those under OC condition  conditions for 1.55-, 1.68-, and 1.78 eV-bandgap devices Fig. . The p-i0.2FA0.8Pb(I0.6Br0.4)3 device under OC and short-circuit (SC) conditions. Under SC conditions, there is a minimal built-in electric field within the devices, thus allowing for rapid carrier extraction. We used a 532 nm laser with an intensity equivalent to 15 suns, in order to accelerate the degradation process. The PL spectra under the OC condition exhibited a growing red shift from 700 nm to over 750 nm, corresponding to the formation of an I-rich phase after 10 min of continuous illumination  evolution of Csion Fig. . In contion Fig. . When thurs Fig. . Combini0.2FA0.8Pb(I0.6Br0.4)3 and MA0.3FA0.7Pb(I0.6Br0.4)3  and fill factor (FF) rather than the open-circuit voltage (Voc)  curve are significantly higher than those from the J–V tests after the same aging time  perovskite layer on CsFA-based WBG perovskite film via a hybrid evaporation/spin-coating deposition method  and spin-coated MAI (2 mg mL−1). Then, 2 mg mL−1 PEAI was spin-coated on top to transform the thin MAPbI3 perovskite layer into a 2D layer  in films, providing further confirmation of the VAQ-2D structure. , which can be ascribed to the non-radiative recombination losses at the perovskite/C60 junction. In contrast, the VAQ-2D stack exhibited a much smaller reduction (only 20 meV) in QFLS, suggesting a substantially suppressed non-radiative recombination at the VAQ-2D perovskite/ETL junction. These results are consistent with the steady-state PL measurements  of perovskite films with different stacks Fig. . This al60  of VAQ-2D film can block hole transport and thus reduce electron-hole recombination at the interface  and UV-vis measurements to investigate the energy level alignment between WBG perovskite and C60 Fig. , 24. The60 interface, we conducted TRPL measurements using an excitation wavelength of 405 nm for both bare films and full device stacks  in PL lifetimes  lifetime, as extracted from the slower decay (τ2) at low excitation fluences, was increased from 14 ns to 31 ns after VAQ-2D treatment, which can be attributed to the suppressed non-radiative recombination. All fitting results of TRPL spectra are summarized in Supplementary Table 60 interface.To further explore the charge extraction at the perovskite/Ccks Fig. . The VAQmes Fig. . The τ1 We then carried out steady-state PL measurements with an excitation of 532 nm continuous-wave laser to elucidate the photostability of devices. Figure 60 side. The VAQ-2D device showed stronger PL emission than those of the control and standard-2D films  mapping 3 composition in our previous work15, the VAQ-2D strategy significantly improves the efficiency while the standard-2D treatment has no optimal effect. The champion VAQ-2D device for DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3 achieved a PCE of 19.6%, with a Voc of 1.302 V, a Jsc of 18.1 mA cm−2 and a high FF of 83.3% under reverse scan. We also achieved a champion PCE of 24.6% for a Cs0.05FA0.95PbI3 device (bandgap of 1.51 eV) in the p-i-n structure, while the PCE of control and the standard-2D device is 22.5% and 22.2%, respectively 3 device retained 90% of its performance after about 1020 h of light illumination, while the control device exhibited a T90 of 385 h. For DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3, after 1060 hours of continuous operation under 1-sun illumination, the VAQ-2D-treated device still retained 95% of its initial PCE, with a linear extrapolation to a T90 > 1700 hours. The stability of standard-2D devices, as shown in Supplementary Fig. 0.2FA0.8Pb(I0.6Br0.4)3 and DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3 maintained 88% and 93% of their initial PCE after 500 hours of MPP tracking under continuous light illumination compared to the control devices, which degraded roughly to 54% of their initial PCE over the same timespan . We fabricated 30 tandem devices (area ~0.049 cm2), showing an average PCE of 27.7 ± 0.3% and a relatively narrow PCE distribution , showing a stabilized PCE of 28.1%. The integrated Jsc values of the front and back subcells from EQE curves are 16.2 and 16.2 mA cm−2, respectively, agreeing well with the Jsc determined from J–V measurements. We also achieved a champion PCE of 27.3% for large-area tandem (1.05 cm2 aperture area), with an average PCE of 26.7 ± 0.3%  combined with high PCE represents a crucial step toward the practical application of all-perovskite tandem solar cells  of 2D fragments in 2D/3D heterostructure, which effectively accelerated the charge extraction and reduced nonradiative recombination at the perovskite/C60 heterointerface. The optimized WBG single-junction cells, with a high PCE of 19.6% and an impressive Voc of 1.32 V, exhibited promising operational stability, maintaining 95% of their performance after 1100 h of MPP operation under full 1 sun illumination. The improved WBG perovskites further enabled the fabrication of monolithic all-perovskite tandem solar cells with a stabilized PCE of 28.1% and a high Voc of 2.135 V, retaining >90% of their initial efficiencies after 855 hours of operation at the MPP. This study notably reveals the degradation mechanism of WBG perovskite devices and proposes an unprecedented technical approach to simultaneously improving efficiency and stability. The strategy offers a crucial step toward the full commercialization of all PSCs.In this work, we reveal that halide segregation in WBG PSCs is correlated to charge accumulation and that accelerating charge extraction improves photostability. We developed a 3D-to-2D perovskite conversion approach to tailor the dimensionality (2 (99.99%), PbBr2 (99.99%), PbCl2 (99.99%), 4PACz (>98.0%) and MeO-2PACz (>98.0%) were purchased from TCI Chemicals. SnI2 (99.999%) was purchased from Alfa Aesar. SnF2 (99.9%), formamidine sulfinic acid , CsI (99.999%), DMF, (99.8% anhydrous), DMSO (99.9% anhydrous), IPA (99.8% anhydrous), ethyl acetate (99.8% anhydrous) and ITO NCs (<100 nm particle size (DLS) were purchased from Sigma-Aldrich. The organic halide salts  were purchased from GreatCell Solar Materials . Guanidinium thiocyanate (99%) and BCP (>99% sublimed) were purchased from Xi’an Polymer Light Technology Corp. PEDOT:PSS aqueous solution (Al 4083) was purchased from Heraeus Clevios (Germany). The C60 was purchased from Nano-C. NiO nanocrystals were synthesized according to previous reports.All materials were used as received without further purification. PbI0.2FA0.8Pb(I0.6Br0.4)3 and DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3 perovskite: the precursor solution (1.2 M) was prepared from four precursors dissolved in mixed solvents of DMF and DMSO with a volume ratio of 4:1. Cs0.2FA0.8Pb(I0.6Br0.4)3: The molar ratios for CsI/FAI/PbI2/PbBr2 were 0.8:0.2:0.4:0.6, respectively. DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3: The molar ratios for DMAI/CsI/ FAI/PbI2/PbBr2 was 0.1:0.4:0.5:0.625:0.375, respectively. 5 mol% MAPbCl3 (relative to Pb) was additionally added. The precursor solution was stirred at 50 °C for 2 h and then filtered through 0.22 μm PTFE membrane before use.WBG Cs0.5Sn0.5I3 perovskite: the precursor solution (2.0 M) was prepared in mixed solvents of DMF and DMSO with a volume ratio of 2:1. The perovskite precursor solution was prepared in an N2-filled glovebox . The FAPb0.5Sn0.5I3 perovskite precursor solution was prepared by dissolved FAI, PbI2, SnI2, SnF2, and GuaSCN at a molar ratio of 1:0.5:0.5:0.05: 0.015 in a mixed solvent of DMF and DMSO with the volume ratio of 2:1. The precursor solution was stirred at room temperature for over 2 hours and then filtered through a 0.20-μm PTFE filter before use.NBG FAPb−1 in deionized water) solution was spin-coated on cleaned ITO flexible substrates at 4,000 r.p.m. for 20 s, followed by annealing at 100 °C for 10 min in air. Subsequently, the solutions of 2PACz and MeO-2PACz with the same concentration (1 mmol L−1 in IPA) were mixed with 3:1 volume ratios and then were spin-coated on the NiO film at 4,000 r.p.m. for 20 s, followed by annealing at 100 °C for 5 min in air. Next, the substrates were transferred into a nitrogen-filled glove box and the WBG perovskite films were deposited with a two-step spin-coating procedure. The first step was 2000 r.p.m. for 10 s with an acceleration of 200 r.p.m. s−1. The second step was 6,000 r.p.m. for 40 s with an acceleration of 2000 r.p.m. s–1. For Cs0.2FA0.8Pb(I0.6Br0.4)3 films, chlorobenzene (200 µl) was dropped on the spinning substrate during the second spin-coating step at 20 s before the end of the procedure, followed by annealing at 100 °C for 15 min. For DMA0.1Cs0.4FA0.5Pb(I0.72Br0.24Cl0.04)3 films, no antisolvent was used; instead, a gas quenching method was introduced by using a blow dryer H300 made by Xiaomi. The diameter of the air outlet is 9.5 cm. The temperature of hot air is 57 °C and the air flow rate is 20 m/s. The blow dryer was fixed 15 cm above the sample and hot N2 gas was blown on the wet film surface to extract excess DMSO and DMF solvents at 20 s before the end of the second spin-coating procedure. After cooling to room temperature, the substrates were transferred to the evaporation system. PbI2 (10 nm) was sequentially deposited by thermal evaporation (Beijing Technol Science Co.) at the rates of 0.1 Å s−1. The thickness of PbI2 layer was read from the evaporation equipment . Then, the MAI solution in IPA (2 mg mL−1) was drop-casted on perovskite film at 5000 rpm for 20 s, followed by drying at 100 °C for 3 min. Then the cooled films were deposited by PEAI (2 mg/ml) IPA solution at 5000 rpm for 20 s, followed by drying at 100 °C for 3 min. Finally, C60 (20 nm), BCP (7 nm), and Cu (150 nm) were sequentially deposited by thermal evaporation (Beijing Technol Science Co.) at the rates of 0.2 Å s−1, 0.2 Å s−1, and 1.0 Å s−1, respectively.The pre-patterned ITO substrates were cleaned in an ultrasonic cleaner with acetone and isopropanol (IPA) for 15 min, respectively. Then, the ITO flexible substrates were dried by nitrogen flow and fixed onto the glass substrates by double-sided adhesive at four edges to facilitate the fabrication of flexible devices. Before the fabrication of devices, the ITO flexible substrates were treated with UV-ozone for 15 min. NiO nanocrystals were synthesized according to the previous report. NiO nanocrystal  system (Veeco Savannah S200) to deposit 20 nm SnO2 at 75 °C using precursors of tetrakis(dimethylamino) tin (IV)  and deionized water. Then 1 wt.% ITO NCs solution (dispersed in isopropanol) was spin-coated on the substrates at 5000 rpm for 15 s and annealed at 100 °C for 5 min in ambient air. Then we immediately transferred the substrates to an N2-filled glovebox for the deposition of FAPb0.5Sn0.5I3 NBG perovskite films with identical procedures used for the single-junction devices. Finally, 20-nm C60, ~15-nm ALD-SnO2, and 150-nm Cu films were sequentially deposited on the surface of NBG perovskite. Details on the deposition of ALD-SnO2 layers can be found in our previous work50.The WBG films were prepared as mentioned above. The substrates were transferred to the evaporation system after the fabrication of all-inorganic perovskite films, and 20-nm-thick CJ–V) characteristics were measured using a Keithley 2400 sourcemeter under the illumination of the solar simulator  at the light intensity of 100 mW cm−2 as checked with NREL calibrated reference solar cells . Unless otherwise stated, the J–V curves were all measured in a nitrogen-filled glovebox with a scanning rate of 200 mV s−1 (voltage steps of 20 mV and a delay time of 100 ms). The active area was determined by the aperture shade mask (0.049 cm2) placed in front of the solar cells. EQE measurements were performed in ambient air using a QE system (EnliTech) with monochromatic light focused on device pixels and a chopper frequency of 20 Hz. For tandem solar cells, the J–V characteristics were carried out under the illumination of a two-lamp high spectral match solar simulator . The spectrum from the simulator was finely tuned to ensure that spectral mismatch is within 100 ± 3% for each 50-nm interval between wavelength range 400–1000 nm. The solar simulator was set at the light intensity of 100 mW cm−2 as checked with a calibrated crystalline silicon reference solar cell with a quartz window (KG-0). EQE measurements were performed in ambient air, and the bias illumination from highly bright LEDs with emission peaks of 850 and 460 nm was used for the measurements of the front and back subcells, respectively. No bias voltage was applied during the EQE measurements of tandems.For single-junction solar cells, the current density-voltage (−2 using a home-build LabVIEW-based MPP tracking system and a Perturb and observe method in ambient conditions (humidity of 30–50%). The solar cells were encapsulated with a cover glass and UV epoxy  which was cured under a UV-LED lamp (peak emission at 365 nm) for 3 min. No UV filter was applied during operation. The illumination intensity was regularly calibrated to check the degradation of the LED lamp.The operating stability tests were carried out under simulated AM 1.5 G illumination  with an intensity of 100 mW cmλ = 1.54 Å) radiation. SEM images were obtained using a TESCAN microscope with an accelerating voltage of 2 kV.XRD patterns were acquired using a Bruker D8 AVANTAGE Diffractometer with Cu Kα  and detected by an EMCCD camera  after passing through 550 nm , long-pass filters. We used a pulsed supercontinuum white laser  with AOTFs to obtain a monochromatic light of 532 nm . A fast avalanche photodiode  coupled with a time-correlated single photon counting module  was used for the time-resolved PL decay measurements. The PL decay curves were fitted with biexponential components to obtain a fast and a slow decay lifetime.PL and TRPL measurements were conducted on a home-built wide-field microscope based on Olymplus IX73. A 532 nm CW diode laser was used as the excitation light for the sample measurements with the laser power density on the film surface of 100 mW cm−10 mbar). UPS employed the He I 21.22 eV as the excitation source with an energy resolution of 50 meV. The work function was derived from the secondary electron cutoff and the ionization potential from the frontier edge of the occupied density states to the vacuum level. Transient photovoltage decays were measured on a homemade system. A 540 nm green light-emitting diode was used to modulate the Voc with a constant light bias, and the repetition rate was set to 2000 Hz. The voltage-decay lifetime was fitted by a single exponential decay. Measurements were carried out in a glovebox using a Keithley 2400 source meter. The LED device characterizations were carried out with a Keithley 2400 source meter and a 100 mm integrating sphere coupled with a spectrometer .UPS was performed in an ultrahigh vacuum (UHV) surface analysis system equipped with a fast entry load-lock, a transfer chamber, and an analysis chamber (base pressure ~10Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73–98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The Vmax, Km, and kcat of purified (r)OnTRAP5b were found to be 0.484 μmol × min−1 × mg−1, 2.112 mM, and 0.27 s−1 with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions  and inhibitors . Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from  TStaphylococcus aureus when challenged in vivo [S. aureus [TRAP5 is a regulator of bone resorption and osteoblast metabolic function, exerting its effects through phosphatase activity and reactive oxygen species (ROS) production ,23. In h in vivo . In addi. aureus . When taS. agalactiae and A. hydrophila in vivo and in vitro. Additionally, we evaluated the phosphatase activity of prokaryotic recombinant protein (r)OnTRAP5b and its response to various metal ions and inhibitors. Furthermore, we investigated the immune-related molecules, ROS, and phagocytosis of head kidney macrophages (MΦ) in the presence of (r)OnTRAP5b. Moreover, we demonstrated that OnTRAP5b significantly influenced bacterial loads in different tissues using overexpression and RNAi technology. These findings provide insights into the functional role of OnTRAP5b in teleost fish.Currently, there is limited knowledge regarding the role of TRAP5 in teleost fish in response to immune challenges and bacterial infections. Nile tilapia is a commercially important fish species that is susceptible to pathogenic bacterial infections during breeding. Therefore, it is essential to investigate the innate immune system of Nile tilapia. The Nile tilapia transcriptome database contains two types, TRAP5a, and TRAP5b. TRAP5a has three distinct isoforms, while the mRNA sequence of TRAP5b is specific. In this study, we cloned the TRAP5b homolog in Nile tilapia, designated as OnTRAP5b, and analyzed its tissue expression patterns. We also examined the changes in OnTRAP5b expression following stimulation with Maylandia zebra and the lowest (61.73%) with Ictalurus punctatus. OnTRAP5b shared only 41.74 to 49.24% of the similarities of other species TRAP5a  and ACP5b (TRAP5b). ACP5a occurs as three distinct isoforms, namely TRAP5 isoform X1, TRAP5 isoform X2, and TRAP5 isoform X3. ACP5b has no isoforms, and the mRNA sequence of ACP5b is unique. Three distinct isoforms are completely similar except for the length difference of the N-terminal sequence. Sequence alignment of TRAP5 isoform X1 and TRAP5b exhibits a similarity of 42.27%.The open reading frame (ORF) of OnTRAP5b was found to be 975 bp in length, which is predicted to encode a protein consisting of 324 amino acids. The predicted signal peptide of OnTRAP5b contained 22 amino acid residues. The mature peptide of OnTRAP5b was estimated to have a molecular weight of 33.448 kDa. The NCBI conserved domains program prediction indicated that the metallophosphatase (MPP) domain of OnTRAP5b is located between the 28th and 311th amino acid residues A. Multips TRAP5a B. The MPs TRAP5a C. PhylogM. zebra D. The th sapiens E. In NCBIn this study, qRT-PCR was used to evaluate the tissue distribution and relative expression of OnTRAP5b in healthy tilapia. The results indicated that OnTRAP5b mRNA was most highly expressed in the liver (1797.83 ± 69.86-fold higher), followed by the stomach (708.06 ± 37.44-fold higher), intestine (437.94 ± 37.44-fold higher), terminal kidney (278.91 ± 11.73-fold higher), head kidney (218.95 ± 60.77-fold higher), spleen (108.95 ± 8.02-fold higher), heart (89.63 ± 21.59-fold higher), peripheral blood (72.88 ± 10.08-fold higher), gills (49.40 ± 9.12-fold higher), brain (32.00 ± 11.18-fold higher), and skin (8.74 ± 1.05-fold higher). Conversely, OnTRAP5b expression was lowest in muscle (set as 1) .S. agalactiae or A. hydrophila, the expression of OnTRAP5b was significantly elevated in the liver, spleen, and head kidney compared to the control group at 3, 6, 12, 24, 48, and 72 h post-infection (h p.i.)  was observed at 24 h p.i. in the liver  (Upon stimulation with (h p.i.) . FollowiS. agalactiae and A. hydrophila. The highest expression of OnTRAP5b in MΦ occurred at 24 h p.i. with S. agalactiae challenge, resulting in a 2.75-fold increase  (A. hydrophila (4.49-fold increase) OnTRAP5b and (r)MBP (The recombinant protein (r)OnTRAP5b and (r)MBP were expressed in d (r)MBP .pNPP (5 mM) in buffer at 37 °C for 30 min (pNPP with (r)OnTRAP5b at 50–300 μg/mL, was significantly higher than that of (r)MBP (0.324–0.390). While the absorbance value of the PBS control group was 0.321–0.325. In order to investigate the effect of temperature and pH on (r)OnTRAP5b phosphatase activity, the temperature was varied from 10–80 °C and the pH from 3–8. The optimal reaction temperature for (r)OnTRAP5b was found to be 50 °C OnTRAP5b using pNPP as substrate were determined to be 0.484 μmol × min−1 × mg−1, 2.112 mM, and 0.27 s−1, respectively.In order to assess the phosphatase activity of (r)OnTRAP5b, varying concentrations of the recombinant protein (r)OnTRAP5b (50–300 μg/mL) and (r)MBP (50–300 μg/mL) were incubated with substrate r 30 min A. The mebe 50 °C B, and thbe 50 °C C. The VmThe thermal stability analysis of (r)OnTRAP5b demonstrated its relative stability when subjected to incubation at 30 or 40 °C for 120 min D. The reThe stability of (r)OnTRAP5b was evaluated by incubating it overnight at 4 °C in buffer solution with pH values ranging from 3.0 to 8.0, and the residual phosphatase activity was determined at the optimal pH and temperature of 37 °C. (r)OnTRAP5b was found to be relatively stable between pH 4.0–7.0, with more than 70% residual phosphatase activity E.+, Na+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Fe3+ were found to significantly affect the phosphatase activity of (r)OnTRAP5b. The control for this study was the phosphatase activity of (r)OnTRAP5b without metal ions, which was set as 100%. The phosphatase activity measured with 1–50 mM of different metal ions was considered relative enzyme activity. K+, Na+, Ca2+, and Mg2+ were found to enhance the (r)OnTRAP5b phosphatase activity in a concentration-dependent manner within a certain range OnTRAP5b was investigated. The metal ions KThe experiments depicted in IL-1β, IL-6, IL-10, TNF-α, IL-8, and IL-12 in MΦ. The expression of IL-1β was increased compared to the control group at 3 (1.13-fold ± 0.25-fold higher) and 12 h (1.12 ± 0.18-fold higher), while down-regulated at 6 h (0.54 ± 0.04-fold) and not change at 24 h (0.94 ± 0.28-fold). The expression level of IL-1β was not statistically significant when compared to the control group at the three-time points of 3, 12, and 24 h. (IL-6 (~52.53-fold) (TNF-α (~8.14-fold) (IL-8 (~9.64-fold) (IL-12 (~14.91-fold) (IL-10 was significantly increased at 3 (~3.84-fold), 6 (~1.95-fold), 12 (~6.03-fold), and 24 h (~9.21-fold) OnTRAP5b (100 μg/mL) using qRT-PCR. The results demonstrated a significant increase in mRNA expression of nd 24 h. A The exp53-fold) B, TNF-α 14-fold) D, IL-8 (64-fold) E, and IL91-fold) F peaked 21-fold) C. In comS. agalactiae and A. hydrophila for flow cytometry analysis to determine whether treatment with (r)OnTRAP5b could enhance phagocytosis. The results showed that (r)OnTRAP5b could considerably increase MΦ phagocytosis OnTRAP5b at the dose of 100 μg/mL and 500 μg/mL OnTRAP5b, the concentrations of (r)OnTRAP5b and (r)MBP ranging from 100–500 μg/mL were investigated and were found to have no significant effect on the viability of MΦ compared to the control group . The proocytosis C. The ph00 μg/mL D. TreatmOverexpression of OnTRAP5b resulted in increased expression of OnTRAP5b in the liver (~1.42-fold), spleen (~1.67-fold), and head kidney (~1.67-fold) in the pcOnTRAP5b-treated group compared to the control group, as determined by qRT-PCR . ConversTo investigate the role of OnTRAP5b in bacterial infection, in vivo knockdown of OnTRAP5b was achieved using siRNA. At 12 h post-dsRNA injection, qRT-PCR analysis revealed a significant reduction in OnTRAP5b expression in the liver , spleen TRAP5 is a member of the purple phosphatase family of metalloenzymes, which catalyze the hydrolysis of various phosphate esters and anhydrides under acidic reaction conditions. TRAP5 plays a crucial role in the immune defense system and has been extensively studied in mammals . HoweverSciaenops ocellatus (SoACP5) and the OnTRAP5b sequence revealed that the similarity between OnTRAP5b and SoACP5 was only 42.69%, and phylogenetic analysis demonstrated that OnTRAP5b belongs to the fish group, while SoACP5 belongs to the TRAP5a branch.Alignment with other species revealed that the amino acid sequence of OnTRAP5b is highly conserved, exhibiting an MPP domain, eight active sites, and seven metal binding sites. However, OnTRAP5b differs from mammalian TRAP5b in that it contains an N-glycosylation site, whereas mammals possess two N-glycosylation sites ,28,29,30pNPP is commonly used as a substrate for ACP [Trypanosoma cruzi using pNPP as substrate was determined to be 5.0 [pNPP since pNPP could be used for the quick analysis of the protein phosphatase activity under any non-standard conditions. Previous studies have also investigated the substrate specificity of acid phosphatase and the compounds used, including pNPP, phosphoserine (P-Ser), phosphothreonine (P-Thr), phosphotyrosine (P-Tyr), adenosine triphosphate, adenosine diphosphate, glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, phenyl phosphate, pyridoxal-6-phosphate, glycerophosphate and so on [pNPP [Artocarpus communis maintained 50% of its activity at 55 °C [Thermus thermophilus ACP was found to be active within the range of 40–90 °C, with an optimum temperature of 70 °C [pNPP catalysis by OnTRAP5b was determined to be 5.0, with an optimal temperature of 50 °C. (r)OnTRAP5b was shown to be stable in the pH range of 4.0–7.0 based on acid-base stability analysis and stable in the temperature range of 30–40 °C based on temperature stability analysis. Previous research on SoACP5 revealed that the optimum pH for the catalytic activity of SoACP5 toward the substrate pNPP was 5.5, with an optimal temperature of 55 °C [+ concentration. (r)OnTRAP5b activity exhibited Michaelis-Menten behavior, with Vmax, Km, and kcat values of 0.484 μmol × min−1 × mg−1, 2.112 mM, and 0.27 s−1, respectively. In previous studies, T. cruzi tartrate-resistant acid phosphatase type 5 exhibited a Vmax for pNPP hydrolysis of 7.7 nmol × μg−1 × h−1 and apparent Km for pNPP of 169.3 μM [Km of acid phosphatase in Ctenopharyngodon idella (grass carp) was 3.56 mM, and the Vmax was 0.153 μmol × ml−1 × min−1 [Km and Vmax of acid phosphatase from Macrotyloma uiflorum seeds for pNPP were found to be 0.934 mM and 1.333 mM× min−1, respectively [Most acid phosphatases (ACP) exhibit activity in an acidic environment and demonstrate a broad range of heat stability. The synthetic organic chemical  for ACP ,32. The o be 5.0 . In thisnd so on ,34. Diffon [pNPP ,34. The at 55 °C . Thermusof 70 °C . In thisof 55 °C . These f169.3 μM . The Km  × min−1 . Km and +, Na+, Mg2+, and Ca2+ were found to promote the phosphatase activity of OnTRAP5b, while Mn2+, Cu2+, Zn2+, and Fe3+ downregulate phosphatase activity of (r)OnTRAP5b within a certain concentration range, similar to findings in grass carp [T. cruzi [2+ and Fe3+ inhibited the activity of SoACP5 and OnTRAP5b, while Ca2+, Mg2+, and K+ promoted their activity [The OnTRAP5b sequence was analyzed and found to possess a binuclear metal core. The spatial structure of a protein can be modified by combination with different metal ions, resulting in varying phosphatase activity. Kass carp . AdditioT. cruzi . EDTA waactivity . These rS. agalactiae and A. hydrophila, significant upregulation of OnTRAP5b expression was observed in vivo in the liver, spleen, and head kidney, as well as in vitro in MΦ and hepatocytes. Similarly, TRAP5 expression in the spleen and head kidney of S. ocellatus was dramatically up-regulated upon stimulation with Edwardia tarda [TRAP5 is a crucial factor for maintaining health, growth, metabolism, and homeostasis and is highly abundant in various tissues, including bone, spleen, liver, thymus, serum, and colon ,39,40,41ia tarda . These fIL-1β, IL-6, IL-10, TNF-α, IL-8, and IL-12) in MΦ. MΦ are essential components of the teleost professional phagocytic system, serving as one of the first lines of host defense against infections. Previous reports have demonstrated that TRAP5 can generate ROS both in vitro and in vivo. Overexpression of TRAP5 in mammalian macrophages resulted in increased intracellular ROS production with a neutral pH [S. aureus after overexpression [Previous research has established that TRAP5 is involved in non-specific immune responses, such as macrophage activity and inflammatory response ,42. In tutral pH . Furtherpression . OverallS. agalactiae and A. hydrophila. The phosphatase activity of (r)OnTRAP5b was affected by various metals and inhibitors. (r)OnTRAP5b was found to enhance phagocytosis, the release of ROS, and the expression of inflammatory molecules in MΦ. Overexpression or knockdown experiments demonstrated that OnTRAP5b played a crucial role in defending against pathogenic infections in vivo. These findings suggest that OnTRAP5b may be a key component of the innate immune system in Nile tilapia.In summary, the successful cloning of OnTRAP5b from Nile tilapia was achieved. OnTRAP5b was found to be expressed in a wide range of tissues. OnTRAP5b mRNA expression significantly increased in vivo and in vitro upon challenge with Nile tilapia were purchased from Guangdong Tilapia Breeding Farm . The fish were kept in the automatic filtering aquaculture system for two weeks prior to the experiments at 28 ± 3 °C to acclimate them to the experiment environment.®Ⅱ 1st Strand cDNA Synthesis SuperMix according to the manufacturer’s instructions . Quantitative real-time PCR (qRT-PCR) assay did conduct with Hieff® qPCR SYBR Green Master Mix .Total RNA extraction was isolated from tissues using the Trizol reagent  according to the manufacturer’s instructions. The RNA sample concentration was determined using NanoDrop 2000 spectrophotometer . The first strand of cDNA was synthesized using the HifairO. niloticus TRAP5b (OnTRAP5b) ORF sequence was cloned and confirmed by DNA sequencing (GenBank accession XM_003457513.5). OnTRAP5b specific primers ). The location of the OnTRAP5b signal peptide site was performed using the SignalP 4.1 server ). Protein domain analysis was performed using Conserved domains (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 8 November 2021)). Multiple sequence alignments of TRAP5 amino acid sequences were performed using the DNAMAN 7.0 software. The phylogenetic relationship of OnTRAP5b was constructed using MEGA 6.0 software with 1000 bootstrap replications. The typical structures were predicted by SWISS-MODEL online software (https://swissmodel.expasy.org/interactive (accessed on 8 November 2021)) and modified by PyMOL 2.5 software.The complete length of the β-actin was amplified using qβ-actin-F and qβ-actin-R as internal control primers  were collected for qRT-PCR analysis of OnTRAP5b mRNA expression in healthy Nile tilapia. Total RNA and cDNAs were synthesized as described above. The qRT-PCR carried out on the CFX96 Touch Real-Time PCR  was applied to evaluate tissue distribution of OnTRAP5b transcription levels. The primers qOnTRAP5b-F1/R1  were app primers . The 2–ΔOnTRAP5b .S. agalactiae was cultured in brain heart infusion (BHI), and A. hydrophila was cultured in Luria-Bertani (LB) at 28 °C before being resuspended in sterilized phosphate-buffered saline (PBS). The experimental groups were injected with 100 μL (1 × 107 Colony-Forming Units (CFU)/mL) of S. agalactiae or 100 μL of A. hydrophila intraperitoneally, while the control group was injected with 100 μL of PBS intraperitoneally [−ΔΔCt method, and β-actin was used as an internal control [toneally . At 3, 6 control .O. niloticus (200 ± 10 g) were isolated according to a previous study [® 1077 , followed by centrifugation at 500× g for 40 min at 4 °C. The gradient interface band of cells was collected and washed three times with RPMI-1640 medium, 1% P/S, and centrifuged at 500× g for 10 min at 4 °C. The cells were seeded into 96-well plates  and incubated at 25 °C for 4 h. Then, the adherent MΦ were collected and resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS)  to the concentration of 1 × 107 cells/mL. The experimental group received 100 μL of formalin-inactivated S. agalactiae (1 × 108 CFU/mL) or A. hydrophila (1 × 108 CFU/mL). The control group was added with an equal volume of PBS. The cells were cultured at 25 °C, stimulated for 3, 6, 12, 24, 48, and 72 h, and then lysed in 1 mL of Trizol for RNA extraction.Head kidney MΦ from healthy us study . In brieg, 4 °C for 5 min and then resuspended in 3 mL of red blood cell lysis buffer . Finally, the hepatocytes were washed in RPMI-1640 medium and suspended to the concentration of 1 × 107 cells/mL in RPMI-1640 medium containing 10% FBS  [S. agalactiae (1 × 108 CFU/mL) or A. hydrophila (1 × 108 CFU/mL) and incubated in 96-well plates. The cell sample collection time was the same as that described above.In order to isolate Nile tilapia hepatocytes, 15 mL trypsin with 0.2% Ethylene Diamine Tetraacetic Acid (EDTA)  was added to each 5 g of the liver of three fish for enzymatic digestion, which was treated at 25 °C for 30 min. Following that, 2 mL of FBS was used to stop the reaction . The celCA, USA) . The hepEscherichia coli cells Top 10. OnTRAP5b was excised from pMD18T-OnTRAP5b by digestion with EcoR 32I and BamH I restriction enzymes . OnTRAP5b was inserted into pMAL-c5X  to produce pMAL-c5X-OnTRAP5b with an MBP-tag at N-terminus for protein purification, using the primer pair OnTRAP5b-F2/R2  and transformed into competent 5b-F2/R2 . To obta5b-F2/R2 , the OnTquencing .E. coli K12 strains TB1 competent cells  and cultured onto solid LB plates with ampicillin (100 μg/mL) at 37 °C. The culture medium containing fusion plasmid was inoculated into LB medium containing glucose and ampicillin and incubated at 37 °C to O.D.600 to 0.6–0.8. Then isopropyl-β-D-thiogalactopyranoside  was added and induced at 16 °C for 16 h. The cells were harvested by centrifugation, and the supernatant was discarded and resuspended the cells in column buffer . After freezing at 4 °C, the cells were lysed by sonication until the amount of released protein reached a maximum, and cell debris was removed with centrifugation. The target protein was attached to the MBP-tag affinity column after purifying the protein using an amylose resin column. In order to elute the target protein, a column buffer containing maltose (10 mM) was utilized. The purified protein was stored at −80 °C until further use.The plasmids pMAL-c5X-OnTRAP5b or backbone pMAL-c5X were transformed into p-nitrophenyl phosphate (pNPP). Purified (r)OnTRAP5b (50–300 μg/mL) was incubated at 37 °C for 30 min in substrate buffer consisting of 50 mM Hac-NaAc (pH 5.5) and 5 mM pNPP. The reaction was stopped by adding 10 μL 1 M NaOH, and the absorbance at 405 nm was measured using a microplate reader of multi-wavelength  [Km) and maximum reaction rate (Vmax), were determined using Origin 8.0 software. These values were then combined with the molecular weight of the enzyme to calculate the catalytic number (kcat) value of the (r)OnTRAP5b, which is the number of individual reactions catalyzed by the active site per time unit when the (r)OnTRAP5b is working at Vmax. This approach is consistent with the previous studies in the field [pNPP. 100 μg/mL (r)OnTRAP5b and (r)MBP were kept overnight at 4 °C in buffer (pH 3.0–8.0), and residual phosphatase activity was evaluated at the optimal pH and 37 °C. The highest phosphatase activity was set as 100%, and the absorbance values measured at other temperatures and pH were converted into relative phosphatase activity.The phosphatase activity of (r)OnTRAP5b was measured by spectrophotometrically quantifying the rate of hydrolysis of 5 mM MA, USA) ,49. In the field . Heat st2, CaCl2, MnCl2, CuCl2, ZnCl2, and FeCl3) on phosphatase activity, (r)OnTRAP5b was incubated at 37 °C for 30 min with a reaction mixture containing buffer  with pH of 5.0 and 5 mM pNPP, and the reaction was stopped by adding 10 μL 1 M NaOH. The assay was performed independently three times. The phosphatase activity without metal ions was considered to be 100%, and the phosphatase activity determined under the remaining conditions was converted to relative phosphatase activity. To determine the effect of phosphatase-specific inhibitors (sodium tartrate and sodium fluoride)  and EDTA were investigated as potential inhibitors of the (r)OnTRAP5b and (r)MBP. The phosphatase activity of (r)OnTRAP5b and (r)MBP was evaluated after 30 min of incubation at 37 °C with phosphatase-specific inhibitors or EDTA. The residual phosphatase activity was determined by adding 5 mM pNPP and incubating at 37 °C for 30 min. Then, 1 M NaOH (10 μL) was added to terminate the reaction, and the absorbance at 405 nm was measured using a microplate reader of multi-wavelength.To determine the effect of different metal ions , interleukin IL-6 (IL-6), interleukin IL-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin IL-8 (IL-8) and interleukin IL-12 (IL-12) were determined with Nile tilapia β-actin by the 2−ΔΔCt method [In order to investigate the effect of (r)OnTRAP5b on immune-related genes, three groups of MΦ were treated with (r)OnTRAP5b (100 μg/mL), (r)MBP (100 μg/mL) or PBS (as control). The MΦ were collected by centrifugation at 500× t method .6 cells/mL with cell culture medium and then treated with (r)OnTRAP5b  or (r)MBP  at 25 °C for 12 h. After treatment, the cells were collected, and washed with ice-cold PBS, added 1 mL of Calcein AM/PI test working solution was to resuspend the cells and incubated at room temperature in the dark for 30 min. After incubation, the cells were placed on ice for testing. The fluorescence intensity was analyzed by BD LSRFortessaTM .According to the manufacturer’s instructions, the Calcein AM/PI cell viability/cytotoxicity assay kit  was used to detect the viability of cells. The MΦ were adjusted to 1 × 106 cells/mL with cell culture medium and then treated with (r)OnTRAP5b , (r)MBP  or PBS (as control) at 25 °C for 6 h. After treatment, the cells were collected, and washed with ice-cold PBS, stained with 10 μM DCFH-DA in PBS, incubated in the dark at room temperature, and then cells were washed to remove the excess probe. The fluorescence intensity was analyzed by BD LSRFortessaTM .Following the manufacturer’s instructions, the 2,7-dichlorofluorescin diacetate (DCFH-DA) ROS assay kit  was used to detect the ROS levels in MΦ. The MΦ were adjusted to 1 × 106 cells/mL with cell culture medium and then treated with (r)OnTRAP5b or (r)MBP at a concentration of 100 and 500 μg/mL in cell culture at 25 °C for 6 h. After treatment, the MΦ were harvested and washed with ice-cold PBS by centrifugation at 100× g, 25 °C for 10 min. The cells were incubated with fluorescein isothiocyanate (FITC)-labeled S. agalactiae or FITC-A. hyfrophila. The cells and FITC-labeled S. agalactiae or FITC-A. hyfrophila were incubated in a dark environment for 4 h in a ratio of 1:20. After incubation the cells were centrifuged and washed with ice-cold PBS, and 0.25M D-sorbitol solution was added to remove FITC-labeled S. agalactiae or FITC-A. hyfrophila adhering to the cell surface. Finally, the collected and washed cells with ice-cold PBS, and then added 200 μL PBS to resuspend cells and analyzed by BD LSRFortessaTM . The phagocytosis rate was examined using the Flowjo V10 software.The MΦ were resuspended and adjusted to 1 × 10S. agalactiae infection. The fish (16 ± 4 g) were randomly divided into three groups (15 fish in each group) and injected intramuscularly with 100 μL of pcOnTRAP5b (150 μg/mL), pcDNA3.1 (150 μg/mL) or PBS (control group). qRT-PCR was performed using qOnTRAP5b-F1/R1 to evaluate the level of OnTRAP5b in the liver, spleen, and head kidney . At 6, 12, and 24 h p.i, liver, spleen, and head kidney tissues were collected and weighed, with three fish randomly in each group at each time point. The number of active bacteria was calculated by the plate counting method.Overexpression of OnTRAP5b in Nile tilapia was performed to explore its effects on d kidney . Then NiS. agalactiae infection. According to instructions, T7 RiboMAX™ Express RNAi System  kit was used to synthesize small interfering RNA (siRNA), which was utilized to silence the expression of OnTRAP5b in vivo. The primers dsOnTRAP5b-P1/P2 and dsOnTRAP5b-P3/P4, as well as dsGFP-P1/P2 and dsGFP-P3/P4 . Bacterial colonies were examined using the plate smearing method after the liver, spleen, and head kidney were collected at 6, 12, and 24 h p.i.This study evaluated the effect of OnTRAP5b on FP-P3/P4 , were deFP-P3/P4 . Then, tp) value < 0.05. One asterisk (*) indicated a p-value between 0.01 and 0.05, and two asterisks (**) a p-value was < 0.01.Excel 2019, GraphPad Prism 5, and Origin 8.0 were used to analyze statistical data. Data were presented as the mean ± standard deviation (SD), with statistical significance considered as a probability (