P7C3 is a neurogenic compound that exhibits neuroprotective properties in neural cells. However, its target proteins and effects in glioma are unknown.in vitro and in vivo was analyzed based on cell viability, apoptosis and proliferation, migration and invasion, and xenograft model. Glial cells were marked by antibodies via immunohistochemical staining.The candidate P7C3 target proteins were analyzed using a human protein microarray containing 23136 human proteins. A streptavidin agarose affinity assay was used to verify the direct interaction between P7C3 and phosphoglycerate kinase 1 (PGK1). Mass spectrometry was used to identify the binding sites of PGK1 for P7C3 binding. Seahorse XF96 extracellular flux analyzer was used to measure the cell oxygen consumption rate and extracellular acidification rate. Glycolytic metabolites were measured using the related kits. Protein level was detected by western blotting and immunohistochemical staining. Autophagy was analyzed using a transmission electron microscope and western blotting. The malignancy of tumor progression in vitro and in vivo. Furthermore, P7C3 did not damage normal glial cells under concentration, which exhibit an inhibitory effect on gliomas.The human protein microarray identified 577 candidate P7C3 target proteins. The global profile of P7C3 target proteins indicated that P7C3 regulates glycolysis. Metabolic experiments confirmed that P7C3 regulates aerobic glycolysis in glioma cells. The underlying mechanism of P7C3 was found to be direct targeting PGK1 at lysine residues and asparagine residues, and the specific P7C3-PGK1 interaction led to decreased protein level and total intracellular kinase activity of PGK1. The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases indicated that the mRNA level of PGK1 is significantly increased in high-grade glioma, and the abnormally high mRNA level of PGK1 is associated with a poor prognosis in patients with glioma, suggesting that PGK1 is a promising target for glioma therapy. The inhibition of PGK1 and the subsequent suppression of aerobic glycolysis caused by P7C3 inhibited the malignant growth of glioma via directly targeting PGK1. Furthermore, we identified the P7C3 target proteins for the first time which is expected to provide scientific clues for future studies.This study revealed that P7C3 suppresses glioma by regulating aerobic glycolysis  P7C3 was firstly reported as a compound that exerts pro-neurogenic activity by protecting newborn neurons and enhancing neurogenesis in adult mice . Follow-in vitro and in vivo, while it did not damage normal neural cells at a concentration that exhibited an inhibitory effect on glioma. Taken together, this study is the first to report that the neurogenic compound P7C3 exhibits an inhibitory effect on malignant growth of glioma in vitro and in vivo, possibly due to its effect on the glycolytic pathway, specifically on PGK1, as the direct target of P7C3 in glioma cells. Most importantly, we were first to identify the target human proteins of P7C3. We believe that these findings may provide important scientific clues for future studies.In our study, we performed a systematic screening of the one-to-one P7C3 interacting proteins by using a human proteome microarray containing 23136 human purified proteins. We identified 577 candidate P7C3 targeting proteins. We found that P7C3 targeting human proteins is mainly involved in energy metabolic pathways, including central carbon metabolism, glutathione metabolism, pyruvate metabolism, and, most importantly, glycolysis, which is one of the most important hallmarks of cancer cells , 12. We 21H18Br2N2O) was purchased from MedChemExpress . For in vitro experiments, P7C3 was dissolved in dimethyl sulfoxide (DMSO)  as a stock solution to obtain a concentration of 20 mM. For in vivo experiments, DMSO was used as a co-solvent to aid in the dissolution of P7C3 in normal saline (20 mg/mL), and a dose of 15 mg/kg body was used. Biotin (C10H16N2O3S) was purchased from MedChemExpress . P7C3-Biotin (P7C3-Bio) was synthesized by Chemdow Biotechnology . Cy3-Streptavidin was purchased from Sigma-Aldrich . Streptavidin Mag Sepharose™ was purchased from Sigma-Aldrich . Recombinant human PGK1  was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 , HK2 , GCK , ALDOA , PGK1, PGK2 , GST , GAPDH , Beclin-1 , LC3A/B , Iba1 , SOX10 , GFAP , and β-Tubulin .P7C3 (Chttps://www.proteinatlas.org/) was used to analyze PGK1 and PGK2 mRNA levels in human cell lines. Transcriptomics datasets from TCGA  and Chinese Glioma Genome Atlas  were used for analyzing the profile of PGK1 and PGK2 mRNA level in patients with glioma. The data shown in this study, based on the above genome atlases, were downloaded from the Gliovis data portal for visualization and analysis of brain tumor expression datasets: (http://gliovis.bioinfo.cnio.es/) (The Human Protein Atlas (nio.es/) .2. Normal human astrocytes (HA) were purchased from Cell Science and cultured in astrocyte medium  incubated at 37°C humidified atmosphere containing 5% CO2.Human glioma cell lines, U87MG, U251G, and T98G were purchased from the Chinese Academy of Sciences cell bank , U118MG were purchased from Obio Technology Co., LTD . Glioma cell lines were culture in Dulbecco’s Modified Eagle Medium (DMEM) , containing 10% fetal bovine serum , incubated at 37°C in a humidified atmosphere containing 5% COZ-Score > 2.8, in a P7C3-Bio-treated microarray, and < 2.8, in a biotin-treated microarray, was identified as the candidate positive protein. The Z-Score ratio between P7C3-Bio treated microarray and biotin treated microarray was defined as IMean_Ratio, and the candidate positive protein with IMean-Ratio>1.4 was identified as the final P7C3 target protein.Identification of P7C3 targeting proteins was performed by Wayen Biotechnology  using a HuProt™ 20K Proteome microarray containing 23,136 purified human proteins. Firstly, blocking buffer  was used to block the microarrays at room temperature (RT) for 1 h. Then, the microarrays were incubated with 10 μM P7C3-Bio or 10 μM biotin at RT for 1 h. Next, the microarrays were washed with TBST three times and incubated with Cy3-Streptavidin  at RT for 1 h. Finally, the microarrays were washed again and spun to dry. Observations and data analysis were performed by GenePix 4000 microarray scanner  and GenePix Pro-6.0 software . The protein spot with the signal strength indicator http://string-db.org/). The Network of protein-protein interaction was built by Cytoscape v2.8.1 (http://www.cytoscape.org). Densely connected regions were calculated by using a graph theoretic clustering algorithm called molecular complex detection (MCODE)  pathway, biological process, and cellular component, was performed using DAVIA 6.7 . Protein (MCODE) . Motif e (MCODE) .Seahorse XF96 Extracellular Flux Analyzer was used for measuring cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). U87MG or U118MG cells were plated in XF96 cell culture plates , and incubated for 8 h at a normal cell incubator. Then, cells were treated with a new medium containing 0.3% DMSO (0 μM P7C3), 30 μM P7C3, or 50 μM P7C3 for 24 hours. Then, cells were equilibrated with bicarbonate-free buffered DMEM for 1 h without CO2 immediately before the extracellular flux (XF) assay. The glycolytic rate assay was performed in XF Base Media without phenol red, containing 5 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, and rotenone/antimycin A (0.5 μM) and 2-deoxy-glycose (50 mM) were added in proper order. The mitochondrial stress test was performed in XF Base Media containing 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, and oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (1 μM), and rotenone/antimycin A (0.5 μM) were added in proper order.4 cells/well/100 μL/were seeded in a 96-well plate in treatment medium which containing 0.3% DMSO (0 μM P7C3), 30 μM P7C3, or 50 μM P7C3 for 18 h. The medium was then replaced with a new growth medium, and the cells were cultured for another 6 hours before experiments. Then, the cells were treated as desired, and 10 μL/well 2-Deoxy-d-glucose was added, and the cells were incubated at 37°C for 30 min. To wash and lyse cells, 50 μL/well 2-Deoxy-d-glucose uptake assay working solution was added, and the cells were incubated at RT for 90 min. The optical density (OD) ratio at 570/610 nm was measured using a spectrophotometer .Glucose Uptake Assay Kit  was used for glucose uptake measuring. First, 5 × 106 cells from each sample) were collected for the subsequent measuring procedure according to the product manual. Relative absorbance was measured under a spectrophotometer .Pyruvate assay kit , lactate assay kit , and ATP assay kit  were used to measure the intracellular levels of pyruvate, lactate, and ATP, respectively. Glioma cells were treated with 0.3% DMSO, 30 μM P7C3, or 50 μM P7C3 for 24 hours. After that, an equal number of cells  of each sample was mixed with 50 μL of reaction mix buffer, and the absorbance was measured immediately at OD340 at two points, with an interval of 30 minutes. The OD340 difference between the two time points was used to calculate the PGK1 activity.The total intracellular kinase activity of PGK1 in glioma cells was measured using phosphoglycerate kinase activity assay kit  according to the manufacture’s protocol. Briefly, cells were treated with P7C3 for 24 h, and 4 × 105 cells were stereotactically injected into 6-weeks-old male BALB/cA nude mice  (12 mice per group). Transplanted cells in vivo grow for 7 days. Then, the mice were divided into the control group and treatment group based on tumor size. The mice in the control group were given daily intraperitoneal injections of normal saline, while the treatment group was given a daily intraperitoneal injection of 15 mg/kg/body P7C3. On day 14 after beginning P7C3 treatment, tumor size in vivo was measured again. Tumor size was measured using an IVIS imaging system . The photon flux was used to evaluate the tumor size. Subsequently, three mice from each group were used to obtain 4% PFA perfusion and paraffin section, and three mice in each group were used for collecting the total protein of xenograft tumor. The remaining six mice in each group were used for Kaplan–Meier survival analysis of the tumor-bearing mice. Animal experiments in this study were performed in accordance with the relevant guidelines and were approved by the Ethics Committee of the Southern Medical University and The Seventh Medical Center of General Hospital of PLA.U87MG-Luc cells used in the intracranial glioma xenograft model were purchased from Obio Technology Co., LTD . Briefly, 3 × 102O2 solution was used to eliminate endogenous peroxidase activity. The slides were then incubated with the primary antibodies at 4°C overnight. The antibodies were detected with Polink-2 plus polymer horseradish peroxidase detection system . Images including tumors and surrounding mouse brain tissue were taken under a Leica microscope . Images were evaluated using Image-Pro Plus software .Tumor tissues, obtained from the intracranial glioma xenograft model on day 21 after U87MG cells transplantation, were used for immunohistochemistry staining assay. The slide was dried at 60°C for 1 h, deparaffinized with xylene, and rehydrated with gradient alcohol. Tris-EDTA buffer was used for antigen retrieval, and a 3% H2O2 solution was used to eliminate endogenous peroxidase activity. The slides were then incubated with a terminal deoxynucleotidl transferase reaction mix for 1 h at 37°C. Next, the slides were washed with phosphate-buffered saline (PBS), and incubate with streptavidin-biotin-peroxidase for 30 min, stained with 3,3′-diaminobenzidine tetrahydrochloride, and counterstain slides with hematoxylin. Slides were observed and photographed under a Leica microscope .The in-situ cell death detection kit-POD  (TUNEL) was used for detecting apoptotic cells in tumor tissues obtained from the intracranial glioma xenograft model. The slides were dried at 60°C for 1 h, deparaffinized with xylene, and rehydrated with gradient alcohol. Tris-EDTA buffer was used to conduct antigen retrieval and 3%-H4 cells were seeded in a 96-well plate and culture in a growth medium for 8 h. The medium was replaced with a treating medium containing P7C3 at concentrations of 0, 10, 20, 30, 40, 50, 75, 100, 150, and 200 μM. The cells were then cultured for another 24 h. Next, the medium was removed, and Cell Counting Kit-8 reagent was added, and the cells were further incubated at 37°C for 1 h. Finally, the OD450 value was measured using a spectrophotometer .The effect of P7C3 on cell viability was analyzed using Enhanced Cell Counting Kit-8 . Briefly, 1 × 104) cells collected and resuspended in 195 μL Annexin V-FITC binding buffer. Next, 5 μL Annexin V-FITC and 10 μL PI were added to the cells, mixed well, and the mixture incubated at RT for 20 mins away from light. The results were determined using flow cytometry .An Annexin V-FITC Apoptosis Detection Kit  was used for apoptosis assay. Glioma cells and normal human astrocytes were treated with P7C3 at a concentration of 0 μM or 35 μM  for 24 h. Then, cells were harvested and washed by PBS buffer. Cells  was used for the EdU incorporation assay. Briefly, cells were culture in a 12-well plate overnight, and then P7C3 was added at a final concentration of 35 μM for 24 h. Next, the medium was replaced with 20 μM EdU solution, and the cells were further incubated for 2 h at 37°C. Subsequently, 4% paraformaldehyde solution was used to fix cells for 10 min, followed by permeabilization of the cells for 15 min at RT using 0.5% Triton X-100 solution. The cells were washed three times with PBS and then incubated with click reaction solution for 30 minutes at RT away from light. Finally, the cells were rinsed and stained with 4′,6-diamidino-2-phenylindole. The results were analyzed using a Leica DMI3000B fluorescence microscope .5 cells in 200 μL DMEM containing 1% fetal bovine serum and 0 μM or 35 μM P7C3 were seeded in the upper chamber, and 600 μL DMEM which containing 20% fetal bovine serum and 0 μM or 35 μM P7C3 was added to the lower chamber. Cells were cultured in the chamber for 24 h. Then, the medium was removed, the cells were fixed with methanol, and the cells were stained with 0.1% crystal violet. Invasion cells were photographed and counted for statistical analysis.The transwell system  was used for the cell invasion assay. The wells were firstly coated with 12.5% Matrigel  at 37°C for 1 h, and then rinsed with DMEM before use. Then, 1 × 105) were seeded in 6-well plates at 90% confluency. Scratches of a predetermined length were vertically introduced into the monolayer using a 200 μL pipette tip. Then, the plate was rinsed to remove the detached cells. Next, a culture medium containing 0 μM or 35 μM P7C3, and 5 mM hydroxyurea  for distinguishing the contributions of cell proliferation to wound closure from those of migration, was added. The wounds were photographed first, and the 6-well plate was placed back into the incubator for cultivation for 16 h. Then, the wounds were photographed again. Migration cells were counted for statistical analysis.Cells  according to the manufacturer’s protocol. PrimeScript RT Reagent Kit  was used to construct cDNA. qRT-PCR analysis was performed by Platinum SYBR Green qPCR SuperMix-UDG w/ROX kit  according to the protocol. Quantitative PCR reactions were repeated at least three times. The expression level was normalized to β-actin. The primer sequence information was as follows: for PGK1 the forwards primer is 5’- ATCTGCCACAGAAGGCTGGT-3’, and the reverse primer is 5’- ACTTTAGCTCCGCCCAGGAT-3’; for β-actin, the forwards primer is 5’- GGCACCCAGCACAATGAAGA-3’, and the reverse primer is 5’- CATTTGCGGTGGACGATGGA-3’.Total protein in cells was extracted with radioimmunoprecipitation assay lysis buffer  supplemented with protease inhibitor . Protein concentration was measured using a bicinchoninic acid protein assay kit . Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. QuickBlock™ Blocking Buffer for Western Blot  was used for antigen blocking at RT for 15 min. Then, the membranes were incubated with primary antibodies (1:1 000 diluted in QuickBlock™ Blocking Buffer) overnight at 4°C. Membranes were then washed thrice with cool TBST Buffer , followed by incubation in horseradish peroxidase-linked antibody  at RT for 1 h. Membranes were washed thrice with cooled TBST Buffer thrice and then reacted with Immobilon Western Chemiluminescent horseradish peroxidase Substrate  for 10 s. The protein bands in the membrane were detected using ChemiDoc™ XRS+ with Image Lab™ Software . Quantification analysis of the related protein was performed densitometrically using ImageJ software.To identify P7C3 binding to the intracellular target protein of glioma cells, two experimental methods were adopted. Firstly, glioma cells cultured in the dish were first treated with 50 μM P7C3-Bio for 24 h, and then the cell lysates were extracted using radioimmunoprecipitation assay buffer. Secondly, cell lysates extracted from normally cultured glioma cells using radioimmunoprecipitation assay buffer, and then incubated with 10 μM P7C3-Bio at 37°C for 1 h. Subsequently, a mixture based on these two methods was incubated with streptavidin agarose overnight at 4°C. To detect P7C3 binding to purified PGK1 protein, 25 μg recombinant human PGK1 solution was incubated with 10 μM P7C3-Bio at 37°C for 1 h, and then incubated with streptavidin agarose overnight at 4°C. Next, the streptavidin agarose was washed thrice with cool PBS buffer and boiled for subsequent western blot analysis.Glioma cells were treated with P7C3 for 24 h. The culture medium was removed, and 2.5% glutaraldehyde solution  was added to fix the cells for 1 h at RT. Then, cells were harvested, washed gently three times in PBS, and postfixed in 1% osmium tetroxide-PBS for another 1 h. After dehydration in graded ethanol series, cells were critical point dried and sputter-coated with 10% gold using JEOL JFD-320 Cold Ice Dryer and JFC-1600 Ion Sputterer. Observations were performed using a transmission electron microscope  at an accelerating voltage of 80 kV.The P7C3 binding to protein was analyzed using a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer . Briefly, P7C3 was dissolved at a final concentration of 20 μmol/L and incubated with 1 μg/μL recombinant human PGK1 at 37°C for 1 h. Samples were loaded to SDS-PAGE, and the protein bands were excised from SDS-PAGE gel. After digestion, analysis was performed using a mass spectrometer. The raw MS files were analyzed and searched against a protein database based on the species of the samples using Byonic. The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), P7C3 (+456.179)  (variable); the enzyme specificity was set to trypsin or chymotrypsin; the maximum missed cleavages were set to 3; the precursor ion mass tolerance was set to 20 ppm, and MS/MS tolerance was 0.02 Da. Only high confident identified peptides were chosen for downstream protein identification analysis.t test, Kaplan–Meier survival analysis with a log-rank test. Each group of experiments was repeated at least three times, standard deviation (SD) was calculated to indicate the variation within each experiment and data, and values represent mean ± SD. P < 0.05 is considered to be statistically significant.GraphPad Prism 8  was used to perform the statistical analysis and statistical chart making. Quantitative data were analyzed by unpaired To comprehensively understand the pharmacological function of P7C3, we explored the P7C3 target proteins using a HuProt™ human protein microarray. We firstly synthesis biotin-labeled P7C3, the molecular structure of which is shown in To thoroughly understand the biological function of P7C3 target proteins, enrichment analysis was performed, including Kyoto Encyclopedia of Genes and Genomes pathway, cellular component, and biological process. The top 20 enriched terms are listed. The top 20 enriched pathways were mainly involved in energy metabolism, including carbon metabolism, glutathione metabolism, glycolysis/gluconegene, central carbon metabolism, and pyruvate metabolism , 12, 18.Energy metabolism remodeling is an important feature in cancer cells, which is manifested as desirable cooperation between aerobic glycolysis (known as the “Warburg effect”) and tricarboxylic acid cycle , to provide efficient energy for the rapid growth of cancer cells in different tumor microenvironments . AccordiTo investigate the substantive effect of P7C3 on glycolysis, we measured the ability of glioma cells to consume glucose and a series of metabolites that are involved in glycolysis under normal oxygen conditions cultured cells. The results showed that P7C3 reduced glucose uptake, and the intracellular levels of pyruvate level, lactate level, and ATP in U87MG and U118MG cells , and the mRNA level of PGK1 was significantly higher than that of PGK2. This profile suggested that PGK1, but not PGK2, is the responsible driver gene in glioma , IYEYKKSLKK , and EEGEEVVRKI   and CQ (a lysosome inhibitor) to block the ubiquitin-mediated proteolytic pathway and autophagy-lysosome proteolytic pathway, respectively, and then detected the protein level of PGK1 by western blotting in U87MG and U118MG cells . Although we did not investigate the underlying mechanism, we confirmed that the declining protein level of PGK1 is not dependent on the mRNA level of PGK1. The posttranslational modification is an important mode of PGK1 activity. For example, the phosphorylation levels of PGK1 at Y324 and T243 are important for its high-efficiency catalytic activity , as well as significantly decreasing the intracellular levels of lactate, pyruvate, and ATP. Cancer cells seriously depend on aerobic glycolysis for a cellular process under a normal oxygen environment . It is eIn summary, this is the first study to identify direct human protein targets of P7C3, which provides many important scientific clues for the research on the functional mechanism of P7C3 in many fields. We demonstrated that P7C3 is an important regulator of energy metabolism in glioma cells, suggesting that P7C3 regulates aerobic glycolysis by directly targeting PGK1. The underlying mechanism of P7C3 involves direct binding to PGK1 at lysine and asparagine residues, and the specific interaction induces accelerated PGK1 protein degradation through the autophagy-lysosome-mediated pathway. Of note, this is the first study to demonstrate that neurogenic compound P7C3 exhibits an inhibitory effect on malignant growth in glioma and has lesser damaging effects on normal glial cells.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by ethics committee of the Southern Medical University and The Seventh Medical Center of General Hospital of PLA.WC and RX conceived and designed the experiments. WC, WJ, CW, LC, KS, JW, BD, and NL performed the experiments. WC, RX, WJ, and CW collected and analyzed the data. WC and RX wrote the original manuscript. RX reviewed and edited the manuscript. All authors agree to be accountable for the content of this work. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the National Natural Science Foundation of China (Grant No. 81573774), and the Military Medical Science Research Project (16CXZ001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The complexes 1 and 2 were found to undergo continuous reversible phase transitions as well as switch dielectric phase transitions. Strikingly, intensive red luminescence and green luminescence were obtained under UV excitation respectively to reveal potential application of the two complexes in multi-functional materials along with dielectric switches and so on.The luminescence of dielectric phase transition materials is one important property for technological applications, such as low-energy electron excitation. The combination of dielectric phase transitions and luminescence within organic–inorganic hybrids would lead to a new type of luminescent dielectric phase transition multifunctional material. Here, we report two novel A The luminescence of dielectric phase transition materials is one important property for technological applications, such as low-energy electron excitation. For example, the Al3+-added SrTiO3 : Pr3+ with a perovskite structure shows red cathodoluminescence, which can be excited at a low anode voltage; even below 10 V. The cathodoluminescence at such a low accelerating anode voltage makes this material attractive for potential applications in flat-panel display, field emission display (FED) and vacuum fluorescent displays.15–17Triggered by external stimuli like temperature, pressure and light, solid-state phase transitions are usually accompanied by dramatic variations in crystal structures and some physical properties, such as the thermal, optical, dielectric, magnetic and even ferroelectric properties.18 These advantages make them suitable for a variety of low-cost optoelectronic devices, such as light emitting diodes19–22 and solar cells.23–25 Meanwhile, much progress has been achieved in ferroelectricity in organic–inorganic hybrid complexes.26–40 Our group reported a series of AMnX3-type luminescent ferroelectrics with excellent performances, including (pyrrolidinium)MnCl3, (pyrrolidinium)MnBr3, (3-pyrrolinium)MnCl3, and trimethylchloromethylammonium trichloromanganese(ii) (TMCM–MnCl3).41–44 Among them, TMCM–MnCl3 has shown a large piezoelectric coefficient d33 of up to 185 pC/N, which is comparable with that of classical piezoceramics, such as barium titanate .44 Their photoluminescence (PL) can be attributed to the 4T1–6A1 electronic transition of the octahedrally coordinated Mn2+ ions, which typically produces a red emission.45 Additionally, several luminescent molecular ferroelectrics based on lanthanide ions have been reported recently.46–48Along with the development and applications of organic–inorganic hybrid complexes in various fields in the past decade, a large number of luminescent materials based on hybrid organic–inorganic hybrid complexes have been developed because they combine facile synthesis with intriguing and tunable optical properties.49–51 The construction of molecular compounds with quasi-spherical organic cations has become a universal method for designing phase transition materials.52 Hybrid metal halide perovskite-type complex with general formula of A2BX4 was a good case . Such hexagonal stacking perovskites involve a large class of complexes .53 Among them, the Mn-complexes have been discovered to fluoresce brightly due to the existence of the luminous activator Mn2+ ion.54 Therefore, we tried to design A2BX4-type complexes, and found two new phase transition materials: [Me3NVinyl]2[MnX4] , which exhibit intense luminescence respectively under an UV excitation. Here, their synthesis, structural phase transitions, and photoluminescence properties are described as follows.In the search for new phase transition materials with excellent dielectric properties, quasi-spherical organic cations are usually selected as the building block for organic–inorganic hybrid complexes because they could trigger ordered-disordered phase transition and thus exhibit desired properties.2.2.155 Trimethylvinyl ammonium bromine was purchased from Sigma Aldrich with a purity of 99%. Complex 1 was obtained as block-shaped green crystals by slow evaporation of the mixture solution of manganese chloride and trimethylvinyl ammonium chloride in a molar ratio of 1 : 1 in methanol solution at room temperature, mp ∼ 230 °C. Complex 2 was synthesized as block-shaped orange single crystal by slow evaporation of the mixture solution of manganese bromide and trimethylvinyl ammonium bromide in a 40% hydrobromic acid solution, mp ∼ 250 °C. Single crystal suitable for X-ray diffraction analysis was selected and studied. Polycrystalline samples were prepared by grinding the air-dried crystals into fine powder. The exposure to air for complexes 1 and 2 is not good for testing due to hygroscopicity.All reagents and solvents were commercially available without further purification. Trimethylvinyl ammonium chloride was synthesized according to the literature.2.2α radiation (λ = 0.71073 Å) by using an φ–ω scan mode at 200 K, 303 K and at 348 K. X-ray data for complex 2 was collected at 173 K and at 223 K. Data processing including empirical absorption corrections was performed using the Crystal Clear software package . Absorption corrections were applied using the Sadabs program.56 The structures were solved by direct methods57 with the Shelxtl program and refined by full matrix least-squares techniques on F2 with the Shelxtl program.57–59 Non-H atoms were refined anisotropically using all reflections with I > 2σ(I). All H atoms were generated geometrically and refined using a “riding” model with Uiso = 1.2 Ueq (C and N). The asymmetric units and the packing views were drawn with DIAMOND . The angles and distances between some atoms were calculated using DIAMOND, and other calculations were carried out using SHELXLTL. For complex 1, a summary of the crystallographic data and detailed structure refinements at 200 K, 303 K and at 348 K are given in Table S1.X-ray data for the title complex 1 was collected on a Bruker Smart APEX II diffractometer with Mo K2.3−1 over the temperature ranges of 150–355 K and 160–240 K, respectively.Differential scanning calorimetry (DSC) experiments were performed on a Perkin-Elmer Diamond DSC instrument. The polycrystalline samples of complexes 1 and 2 were heated and cooled in aluminium crucibles with a rate of 20 K min2.4ε′) of the complex dielectric constants  were measured on a Tonghui TH2828A impedance at various frequencies and within the temperature range of 180–280 K, 305–350 K, respectively. As for complex 2, the real parts (ε′) of the complex dielectric constants were measured at the respective frequencies of 1 kHz, 100 kHz and 1 MHz in the temperature range from 170 to 230 K.The polycrystalline samples were pressed into pellets. The pressed-powder pellets deposited with silver conducting glue were used as electrodes in the dielectric measurements under an applied AC electric field of 1 V. For complex 1, the real parts  and [Me3NVinyl]2[MnBr4] (2), they displayed distinct phase transition temperatures (Tc). For [Me3NVinyl]2[MnCl4] (1), complex 1 shows a small endothermic peak in the heating run at 334 K and a small exothermic peak in the cooling run at 316 K, indicating a reversible structural phase transition. Meanwhile, a couple of continuous reversible phase transitions below room temperature 254/224 K and 237/212 K were also observed in the DSC curve. For convenience, we take the transition temperature in the heating run as different phases, the temperature above 334 K as α phase, the temperature ranging from 254 to 334 K as β phase, the temperature ranging from 237 to 254 K as γ phase and the temperature below 237 K as δ phase.Generally, DSC measurement is an effective and useful way to detect whether a crystal displays a reversible phase transition triggered by the external temperature. For the purpose of examining the phase transition characteristics of complexes 1 and 2, DSC measurements were carried out on them. As presented in 3NVinyl]2[MnBr4] (2) was found to only undergo two continuous reversible phase transitions below room temperature. A couple of continuous reversible phase transitions around the temperature 213/194 K and 199/179 K were observed in the DSC curve , with cell parameters of a = 18.23(10) Å, b = 12.60 (4) Å, c = 18.13 (10) Å, β = 119.22(7)°, and V = 3637.2(4) Å3  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bond distances are essentially identical, which are 1.281(9), 1.294(8), 1.299(9) and 1.304(7) respectively as shown in Table S2.ab, each Mn atom situates at 21 axis, and the four symmetric operations  in the δ phase could be observed, as shown in To gain further insight into the details of the structural phase transitions of two complexes, variable-temperature X-ray structures of complex 1 were performed at 200, 303, and 348 K, and crystal structures of complex 2 were measured at 173 and 223 K, respectively. At 200 K (corresponding to δ phase), complex 1 crystallizes monoclinic crystal system  Table S1. Each as Fig. S4a. Each MnPnma (no. 62). It is noteworthy that the molecular component in the β-phase is the same as that at 200 K in the δ-phase, while the basic unit of the crystal structure at 303 K has halved the content compared to that of the δ-phase at 200 K, with cell parameters of a = 13.13(9) Å, b = 8.89(10) Å, c = 16.20(14) Å, β = 90°, Z = 8 and the volume reducing twice from 3637.2(4) Å3 to 1889.5(3) Å3 ° to 90°, accompanied by the transition of crystal system from monoclinic to orthorhombic. In the β phase, the thermal ellipsoids of most atoms are accordingly larger than those in the δ phase, corresponding to a definitely disordered phase. With the temperature increasing from 200 K to 303 K, the swaying motions of the three chloride atoms are activated, while the ordered chloride atoms become disordered and occupy two positions, respectively  have doubled to eight symmetric operations  , the structure parameters are remarkably dissimilar to those found in the δ-phase. At 303 K, complex 1 crystallizes in the orthorhombic system space group  Table S1. Each as Fig. S4b. The ris Fig. S4b. The Mn– Fig. S4b.Pnma with cell parameters of a = 13.10(14) Å, b = 9.04(12) Å, c = 16.18(19) Å, β = 90° and the volume expanding from 1889.5(3) Å3 to 1916.4(4) Å3. The asymmetric unit remains the same as that at 303 K in the β phase, consisting of one [MnCl4]2− anion and two organic ammonium cations, with the same formula of A2BX4, as shown in Fig. S4c.C bond distances are larger than that in the β phase and δ phase, which are 1.342(9) and 1.337(9) Å. Compared with the β phase, the Mn–Cl bond distances and the Cl–Mn–Cl bond angles change slightly, resulting in the structure phase change, also confirmed by the DSC results. Although space group does not change from β phase to α phase, the structural phase transition of complex 1 belongs to isomorphic phase transition.Interestingly, with the rise of temperature, the structure at 348 K (α phase) still adopts the same orthorhombic system and the same space group With the temperature rising from δ phase to α phase, the hydrogen bond networks of complex 1 changed, as shown in Fig. S5–S7.P21/c (no. 14), with cell parameters of a = 9.23(6) Å, b = 15.19(10) Å, c = 13.74 (8) Å, β = 91.49(6)°, and V = 1926.2(2) Å3  to 2.535(3) Å and Br–Mn–Br angles of adjacent Br atoms vary from 105.50(11) to 118.26(12)°  can be observed in the pack structure, as shown in Similarly, the variable-temperature X-ray structures of complex 2 were performed at 173 and 223 K. At 173 K (γ phase), complex 2 crystallizes in the monoclinic crystal system the space group  Table S5. As show Table S6. The fouPnma (no. 62), with cell parameters of a = 13.45(3) Å, b = 9.114(2) Å, c = 16.42(5) Å, β = 90°, and V = 2012.4(9) Å3  and 1.26(3) Å in the γ phase to 1.263(17) and 1.221(18) Å in the α phase. During the transition from γ phase to α phase, the symmetry elements of the crystallographic symmetric operations  have doubled to eight symmetric operations . Rich hydrogen bond networks of complex 2 are also observed in Fig. S9 and S10.†When the temperature rises to 223 K (α phase), complex 2 crystallizes in the orthorhombic system space group  Table S5. Similar Fig. S8b. Each Mndiagram.  And the diagram. . And the3.3Tc, while the magnitude of the variations is related to the characteristics of the phase transition. In consideration of the above-mentioned reversible phase transitions confirmed by the DSC and single crystal X-ray diffraction results, complexes 1 and 2 are expected to display interesting dielectric responses triggered by temperature.Usually, the dielectric properties of crystalline materials exhibit distinctive anomalous changes of temperature-dependent dielectric constants in the vicinity of ε′) has a value of about 5.9 when the temperature is ranging from 305 to 330 K, which is corresponded to β phase. At 334 K, the dielectric constant value (ε′) sharply increases. When the temperature is ranging from 340 to 350 K, the dielectric constant value (ε′) has a value of about 9.8, which is corresponded to α phase. That is, the magnitude of ε′ after phase transition is almost 1.6 times that before phase transition, demonstrating a noticeable step-like anomaly at around 334 K. Furthermore, the changes of ε′ at lower frequencies are more pronounced than those at higher frequencies, revealing that the dielectric constant is very sensitive to the external frequencies  at different frequencies upon a heating–cooling cycle shown in ε′ kept stable with a value of about 9.6 when the temperature was decreasing from 227 to 196 K at 1 MHz, which corresponds to a high dielectric state. When further cooling, the ε′ value rapidly decreases to reach a value of about 6.9 at 186 K, transferring to a medium dielectric state with a step-like anomaly around the 194 K. Correspondingly, the change of dielectric constant means the structural phase transition from β phase to α phase in the heating process. The ε′ value at the high dielectric state is approximately 1.4 times as high as that at the medium dielectric state. Immediately, as temperature further declines, it displayed an abrupt decrease down to about 6.3 at 176 K, which corresponds to a low dielectric state. The change of dielectric constant equally means the structural phase transition from γ phase to β phase when heating. Furthermore, at 1 kHz, 100 kHz and 1 MHz upon cooling, the dielectric constant is sensitive to the external frequencies, consistent with the results recorded in As for complex 2, the real part 2(t2)3–(e)1(t2)4 electronic transition.60In addition to the dielectric properties, the optical absorption properties of complexes 1 and 2 were investigated by obtaining a solid state UV-vis transmission spectrum at room temperature. As shown in Then by quenching the heated components, the luminescence data were collected at 80, 185, 200, 300 and 344 K, respectively. As shown in 60For complex 2, when subjected to a UV lamp at 365 nm, the orange crystals emit intense green light, as shown in Then by quenching the heated components, the luminescence data of complex 2 were collected at 80, 160, 223 and 310 K, respectively. As shown in m (P21/c) to point group mmm (Pnma) belonging to the 94 species of ferroelastic phase transitions with an Aizu notation of mmm F2/m in complexes 1 and 2, corresponding to one ferroelastic–paraelastic phase transitions. The orthorhombic–monoclinic transition lowers the symmetry from the mmm to the 2/m. Eight symmetry elements in mmm decrease to four in 2/m. Hence the possible domain number is 8/4, that is, two. The Curie symmetry principle tells us that the space group at the ferroelastic phase should be the subspace group at the paraelastic phase. The number of spatial symmetry operations decreases from 8 to 4 during the symmetry breaking process, as shown in In addition, we noticed that the point group changes from point group 2/4.2BX4 novel organic–inorganic hybrid complexes: [Me3NVinyl]2[MnX4] , which exhibit multiple structural phase transitions along with photoluminescent properties. Complexes 1 and 2 underwent continuous reversible phase transitions, which were confirmed by the DSC results, variable-temperature structural analyses and dielectric measurements. The results indicate that the dielectric phase transitions are related to the motion of ions under the stimuli of variable temperature. It is worth mentioning that accompanying the phase transitions, complex 1 and 2 also display notable step-like dielectric anomalies, which are characteristics of switchable dielectrics. Additionally, the two complexes reveal striking intensive fluorescence properties with strong emissions observed in the vicinity of 540 nm. Combining the photoluminescent property, structural flexibility and dielectric properties, it is expected that our findings will not only throw light on the search for new dielectric photoluminescent materials but also accelerate their potential practical applications such as dielectric switch, etc.In summary, the present work has successfully demonstrated two AThere are no conflicts to declare.RA-011-D0RA08795E-s001RA-011-D0RA08795E-s002


Gordonia sp. are members of the actinomycete family, their contribution to the environment improvement and environmental protection by their biological degradation ability, but there are few studies on the antimicrobial activity of their secondary metabolites. Our team isolated and purified an actinomycete WA 4-31 from the intestinal tract of Periplaneta americana, firstly identified the strain WA 4-31 by the morphological characteristics and the phylogenetic analyses, and found it was completely homologous to the strain of Gordonia terrae from the Indian desert. Meanwhile, actinomycin D (1), actinomycin X2 (2), mojavensin A (3) and cyclic (leucine-leucne) dipeptide (4) were obtained from the EtOAc extract from the broth of WA 4-31. Compounds 1–4 showed anti-fungus activities against Candida albicans, Aspergillus niger, A. fumigatus and Trichophyton rubrum, also anti-MRSA and inhibited Escherichia coli in different degree. Interestingly, we found when 3 was mixed with 4 with ratio of 1:1, the activity of the mixture on anti-Candida albicans was better than the single. Besides, compounds 1–3 had varying degrees of antiproliferative activities on CNE-2 and HepG-2 cell lines. These indicated that Gordonia rare actinomycete from the intestinal tract of Periplaneta americana possessed a potential as a source of active secondary metabolites.The online version contains supplementary material available at 10.1186/s13568-021-01272-y. Streptomyces species. But over time, the continual rediscovery of known compounds from secondary metabolites of actinobacteria has resulted. Consequently, special extreme environment and growing conditions for bacteria and Fungi raised much concerns in recent years , Actinomycin X2 (2), Mojavensin A (3) showed better anti-fungal and anti-cancer activities. The study reported the antimicrobial compounds isolated from Gordonia from the intestinal tract of Periplaneta americana for the first time. These indicated that Gordonia rare actinomycetes possessed a potential as a resource of active secondary metabolites.Rare actinomycetes have attracted more attention with a hope to discover new antibiotics. At present, some antibiotics that have been used in clinic, such as gentamicin, erythromycin, vancomycin and rifampicin, are all from rare actinomycetes  was isolated from the intestinal tract of Periplaneta americana and deposited in Guangdong Microbial Culture Collection Center (GDMCC NO.1.2521). The tested strain including four kinds of pathogenic fungi: Candida albicans ATCC 10231, Trichophyton rubrum ATCC 60836, Aspergillus niger ATCC 16404, Aspergillus fumigatus ATCC 96918, and four kinds of pathogenic bacteria: methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pneumobacillus ATCC 13883, were obtained from Guangdong Institute of Microbiology. CNE-2 Human nasopharyngeal carcinoma cell line, HepG2 human hepatocellular carcinoma cells were purchased from Experimental Animal Center of Sun Yat-sen University and Shanghai Cell Biology Institutes respectively.The Microbiological media were purchased from Guangdong Huankai Microbial Co.. The genomic DNA isolation kit was purchased from TIANGEN Biotech . Silica gel used for extraction and column chromatography were obtained from Qingdao Ocean Chemical Co. . Methanol and Acetonitrile for High-performance liquid chromatography (HPLC) were purchased from ThermoFisher . Other solvents were obtained from Guangdong Guanghua Sci-Tech Co..Gordonia strain WA 4–3 was inoculated on Gauze’s No. 1 medium and incubate at 28 °C for 3 days. Three days later, the single colony was selected and cultivated in the ISP-1 seed medium at 28 °C for 2 days. The morphology and surface characteristics of the bacterial colonies were examined using a scanning electron microscope . Whole genomic DNA of the strain was extracted according to the manufacturer’s instructions of the genomic DNA isolation kit. The 16S rDNA gene was then amplifified by PCR, using universal bacterial primer (27F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5’-TAC GGC TAC CTT GTT ACGACT T-3’). PCR products were visualized by electrophoresis and was sequenced in Invitrogen . The resulting sequence was compared to all sequences available in GenBank using the BLAST software from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The phylogenetic tree was constructed by MEGA.The The ISP 2 medium was used as the production culture. A 9 mL portion of the seed culture was transferred into a 500-mL Erlenmeyer flask containing 300 mL of the ISP 2 medium and incubated on rotary shakers (160 rpm) at 28 °C for 21 days. The total 63L of culture broth was extracted 3 times with an equivalent volume of ethyl acetate. After extraction and evaporation, 13.4 g crude extract was obtained, which was isolated to given 20 fractions (1–20) using silica gel column with mobile phase dichloromethane:methanol . The crude extract and every fraction were tested for antimicrobial activity against Candida albicans ATCC 10231 by Oxford Cup method . Selecting appropriate 600μL of deuterated reagent to dissolve 10.0 mg of the compounds, the proton and carbon nuclear magnetic resonance (NMR) spectra were recorded at 600 MHz using a spectrometer .6 CFU/mL) were added to the 96-well plates. Then 100 µL compounds were added to the 96-well plates respectively. The final concentrations of each sample in the wells were 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 µg/mL. Amphotericin B, Vancomycin , Ampicillin  and Ciprofloxacin  were used as positive drugs for fungi, MRSA, Pneumococcus and Escherichia coli respectively. The MIC value of the compounds was determined as the lowest concentration that completely inhibited bacterial growth after 48 h with incubation at 37℃, while the fungi was incubated at 28℃. All experiments were performed in triplicate.Minimum inhibitory concentration (MIC) measurements of compounds against the tested strain were performed by a microdilution method. The compounds were dissolved in DMSO and then diluted by the twofold dilution method. 100 µL bacterial suspension or fungi suspension  were taken, soaked sufficiently in different samples for 5 min and set aside. Fungi suspension was dipped with sterile cotton, and uniformly applied in the culture media. Fully soaked filter paper was placed in the C. albicans containing plates and cultured for 24 h at 28 °C. Amphotericin B (32 μg/mL) was used as positive control. The experiment was performed in triplicate and the mean of the diameter of the inhibition zones was calculated.Antifungal activity against 3 cells suspended in 100 μl DMEM medium were added into a 96-well plate and treated with different concentrations of compounds for 24 h /48 h /72 h. After then, 10 μL of 0.5 mg/mL MTT reagent was added to each well for an additional 4 h. The purple formazan crystals were dissolved with 100 μL DMSO per well for further quantitative measurement at an absorbance of 570 nm using a microplate spectrophotometer . Experiments were independently repeated three times.3--2,5-diphenyl tetrazolium bromide  assay was carried to evaluate cell viability. 5 × 10Gordonia terrae from a cold desert of the Indian (EU333873) by 100% , compound 2 (24.2 mg), compound 3 (34.1 mg) and compound 4 (35.9 mg).The crude ethyl acetate extract about 13.4 g) was firstly separated by silica gel column chromatography eluting with a gradient system of CH3.4 g was1H NMR and 13C NMR spectral data , Actinomycin X2 (2), Mojavensin A (3) and cyclic (leucine-leucine) dipeptide (4) by comparing the microspectrum, SciFinder and related references  -Leucine) dipeptide has the strongest inhibitory effect on E. coli at 32 μg/mL. In addition, compounds 1 Actinomycin D and compounds 2 Actinomycin X2 have the best effects against MRSA , with an inhibitory effect of 0.25 μg/mL.The MIC experiment found that compounds 1–4 have broad spectrum antimicrobial activity Table , especiaCandida albicans  dipeptide, which were separated from the tenth fraction with the strong activity against ans Fig. , mixed won Table .Fig. 4Ac2 and Mojavensin A for HepG-2 and CNE-2 cells was performed by MTT at different time. Results show that compounds 1–3 could inhibited cell proliferation in a time-dependent manner  of the compounds on HepG-2 and CNE-2 cells were calculated using the probability distribution of SPSS13.0  and was especially effective against S. aureus, with MIC50, 0.002 μg/ml; MIC90, 0.017 μg/ml  dipeptide were isolated and purified from the secondary metabolites of the strain WA 4-31. We determined their antimicrobial activity and inhibitory effect on the growth of CNE-2 cells and HepG-2 cells. In addition, we found that Mojavensin A and cyclic (leucine-leucine) dipeptide had a synergistic effect after being mixed in different proportions, which could enhance the anti-Candida albicans activity. This phenomenon was a good enlightenment for us, maybe we can continue our in-depth study in the follow-up study on antimicrobial activity.Through our work, we identified a rare actinomycete strain WA 4-31 from the intestinal tract of Additional file 1: Fig. S1. Identification of compound 1: Actinomycin D. (a) Mass spectrometry. (b) Proton (1H) nuclear magnetic resonance. (c) Carbon (13C) nuclear magnetic resonance spectrum of the compound 1 in Chloroform-d. Fig. S2. Identification of compound 2: Actinomycin X2. (a) Mass spectrometry. (b) Proton (1H) nuclear magnetic resonance. (c) Carbon (13C) nuclear magnetic resonance spectrum of the compound 2 in Chloroform-d. Fig. S3. Identification of compound 3: Mojavensin A. (a) Mass spectrometry. (b) Proton (1H) nuclear magnetic resonance. (c) Carbon (13C) nuclear magnetic resonance spectrum of the compound 3 in DMOS-6d. Fig. S4. Identification of compound 4: cyclic (leucine-leucine) dipeptide. (a) Mass spectrometry. (b) Proton (1H) nuclear magnetic resonance. (c) Carbon (13C) nuclear magnetic resonance spectrum of the compound 4 in Chloroform-d.Additional file 2: Table S1. NMR data correlations of Actinomycin D (Compound 1). Table S2. NMR data correlations of Actinomycin X2 (Compound 2). Table S3. NMR data correlations of Mojavensin A (Compound 3). Table S4. NMR data correlations of cyclic (leucine-leucine) dipeptide (Compound 4).


Trichoderma harzianum strain THIF08, which degrades COS at concentrations higher than 10,000 parts per million by volume from atmospheric concentrations, and its gene cos (492 bp) was cloned. The recombinant protein purified from Escherichia coli expressing the cos gene converted COS to H2S. The deduced amino acid sequence of COSase (163 amino acids) was assigned to clade D in the phylogenetic tree of the β-carbonic anhydrase (β-CA) family, to which prokaryotic COSase and its structurally related enzymes belong. However, the COSase of strain THIF08 differed from the previously known prokaryotic COSase and its related enzymes due to its low reactivity to CO2 and inability to hydrolyze CS2. Sequence comparisons of the active site amino acids of clade D β-CA family enzymes suggested that various Ascomycota, particularly Sordariomycetes and Eurotiomycetes, possess similar enzymes to the COSase of strain THIF08 with >80% identity. These fungal COSase were phylogenetically distant to prokaryotic clade D β-CA family enzymes. These results suggest that various ascomycetes containing COSase contribute to the uptake of COS by soil.Carbonyl sulfide (COS) is the most abundant and long-lived sulfur-containing gas in the atmosphere. Soil is the main sink of COS in the atmosphere and uptake is dominated by soil microorganisms; however, biochemical research has not yet been conducted on fungal COS degradation. COS hydrolase (COSase) was purified from  Measurements of atmospheric COS fluxes in terrestrial environments are considered to be important because they may be used as a proxy for estimating gross primary production (GPP) due to the clear relationship between the uptakes of COS and CO2 is a substrate for CA (CO2+H2O ⇄ HCO3–+H+), and since COS and CO2 are structurally similar, COS is also hydrolyzed by CA to produce H2S (COS+H2O → CO2+H2S). CA and RuBisCO are both essential for CO2 fixation in plants, abundant in the biosphere, and thus, make the greatest contribution to the consumption of COS in the troposphere . There are four phylogenetically distinct clades within the β-CA family (clades A to D)  .2] hydrolase, EC 3.13.1.5)  and archaea (carbon disulfide [CS.13.1.5) . COSase Mycobacterium, Williamsia, and Cupriavidus and fungi belonging to the Trichoderma, Beauveria and Scytalidium were isolated from forest soils exhibiting COS-degrading activity  as previously described (Genomic DNA was extracted from the mycelia of strain THIF08 using an Isoplant DNA extraction kit (Nippon Gene). The ITS1-5.8S-ITS2 ribosomal RNA (ITS) region was amplified by PCR with the ITS5_primer and ITS4_primer  White e Dees an. PCR prog at 4°C for 20 min, rinsed with 50 mM Tris-HCl, pH 9.0, and then stored at –80°C. Cells (5 g as wet weight defrosted) were re-suspended in 15 mL of 50 mM Tris-HCl, pH 9.0, and disrupted using a three-dimensional bead impact homogenizer  at 4°C in a 25-mL volume with zirconia beads (0.6 mm in diameter) . Cell debris was removed by centrifugation at 20,000×g at 4°C for 20 min, and the cell-free extract was then filtered through glass microfiber filter paper with a pore size of 1.2 μm . A 30-μL aliquot of the filtrate was subjected to native polyacrylamide gel electrophoresis (PAGE) on an 8% gel , and the gel was incubated at 30°C for 2 h in the dark. Bismuth in the solution reacted with sulfide produced by COS hydrolysis and formed a black precipitate at locations on the polyacrylamide gel at which the enzyme was present   were pooled and then incubated at 4°C for 2 h after the addition of ammonium sulfate . After centrifugation at 20,000×g at 4°C for 10 min, the supernatant was loaded onto a column (1×5 cm) packed with a strong anion exchange chromatography resin , equilibrated in 50 mM Tris-HCl, pH 9.0, containing 30% ammonium sulfate. Unbound proteins were obtained by washing the column with 50 mM Tris-HCl, pH 9.0, at a flow rate of 1 mL min–1. Fractions exhibiting COSase activity were loaded onto a strong anion exchange chromatography column (1 mL)  equilibrated in 50 mM Tris-HCl, pH 9.0. Proteins were eluted from the column with a NaCl gradient from 0 to 0.6 M in 50 mM Tris-HCl, pH 9.0, at a flow rate of 1 mL min–1. Active fractions were then loaded onto a weak anion exchange chromatography column (1 mL)  equilibrated in 50 mM Tris-HCl, pH 9.0. Proteins were eluted with 0.6 M NaCl in 50 mM Tris-HCl, pH 9.0, at a flow rate of 1 mL min–1. Fractions exhibiting COSase activity were pooled and subjected to SDS-PAGE.The cell-free extract filtered as described above (10 mL) was loaded onto a column (1×5 cm) packed with a weak anion exchange chromatography resin , equilibrated in 50 mM Tris-HCl, pH 9.0. Proteins were eluted from the column with a KCl gradient from 0 to 0.6 M in 50 mM Tris-HCl, pH 9.0, at a flow rate of 1 mL min2S were measured using GC-FPD as previously described  sealed with butyl rubber stopper containing 200 μL of the reaction mixture of enzyme solution and 50 mM Tris-HCl buffer, pH 9.0, and then incubated at 30°C. The reaction was started by adding COS gas after removing an equal volume of headspace gas to make the final COS concentration approximately 1,000–2,000 ppmv. During the incubation, the headspace gas (50 μL) was sampled at appropriate intervals, and COS and H2 hydrolysis activity was essentially the same as that for COSase activity, except that CS2  was used instead of COS. The initial CS2 concentration in the reaction mixture was 460 μM or 80 mM. The amount of H2S in the headspace gas was measured by GC-FPD.The procedure employed to measure CS–1 protein. The definition for WAU is (Tblank–Treq)/Treq where Tblank and Treq are the times required for the pH of the reaction mixture to change from pH 8.3 to 7.0 with or without an enzyme. Two milliliters of CO2-saturated water was added to glass vials containing 3 mL of ice-cold 20 mM Tris-sulfate, pH 8.3. After the pH of the reaction mixture had reached approximately pH 8.4–8.5, the reaction was started by the addition of 50 μL of ice-cold enzyme solution in 50 mM Tris-HCl, pH 8.5 (at 25°C). The enzyme solution contained 5 μg of GST-fused COSase  or 1 μg of bovine CA Isozyme II (Sigma-Aldrich). pH changes during the assay were recorded with a pH meter  and a probe  with gentle stirring. The experiment was performed at a temperature range between 0 and 3°C. Samples were prepared in triplicate.The protocol for the measurement of CA activity was based on the description by Wilbur and Anderson  with som2 gas was added to the reaction vial containing the enzyme solution and buffer (Nissan Tanaka Corporation) after removing an equal volume of headspace gas to reach a final CO2 concentration of 100, 1,000, or 10,000 ppmv. Headspace air was replaced by nitrogen gas (Taiyo Nissan) prior to the experiment.COg at 4°C for 1 min, the resultant supernatant was subjected to ultrafiltration using Vivaspin 500 3,000K MWCO PES (Sartorius) to decrease the buffer concentration to 10–2. Quantification of the protein solution was calculated by absorbance at 280 nm with the molar absorption coefficient  calculated according to the formula by Gill and Hippel  staining and then sent to the Institute for Protein Research  to elucidate the N-terminal amino acid sequence using Edman degradation.Trichoderma sp. strain THIF08 using the Isoplant DNA extraction kit (Nippon Gene) according to the manufacturer’s instructions. PCR for cloning was performed using Phusion High-Fidelity DNA polymerase (Finnzymes).Genomic DNA was isolated from E. coli XL10-Gold (Agilent) was transformed with the pGEX-6P-1 , pGEX-COS, or pGEX-GPI plasmid and grown to the mid-exponential phase in 100 mL of LB medium containing ampicillin (0.1 mg mL–1). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.2 mM to induce the production of glutathione S-transferase (GST), GST-fused COSase, or GST-fused GPI. Cells were harvested by centrifugation at 3,000×g at 4°C for 10 min, washed with 50 mM Tris-HCl buffer, pH 9.0, and suspended in the same buffer. Cells were disrupted using glass beads (0.3 mm in diameter) with the Mini Bead Beater (WakenBtech), and centrifuged at 20,000×g at 4°C for 20 min. The supernatant was mixed with glutathione-conjugated magnetic beads  to capture GST or the GST-fused proteins of interest. The magnetic beads were washed with PreScission buffer . To cleave GST from the fusion proteins, 1 μL (2 units) of PreScission Protease  was added to the bead suspension and incubated on a rotating wheel at 4°C overnight. The resulting solution containing COSase, GPI, or the beads capturing GST was used to measure COSase activity.To identify COSase activity, 2, CS2 hydrolysis activity, and CA activity, E. coli BL21 (DE3) (BioDynamics Laboratory) was transformed with the pGEX-6P-1 or pGEX-COS plasmid and grown to the mid-exponential phase in 100 mL of LB medium containing ampicillin (0.1 mg mL–1). IPTG was added to the culture at a final concentration of 0.1 mM to induce the production of GST and GST-fused COSase. Cells were harvested by centrifugation at 10,000×g at 4°C for 5 min, washed with 500 mM Tris-HCl, pH 8.0, and suspended in 5 mL of the same buffer. Cells were disrupted by sonication and centrifuged at 10,000×g at 4°C for 5 min. The resultant supernatant was mixed with Glutathione SepharoseTM 4B  to capture GST or GST-fused COSase. Beads (1.2 mL) were washed with 50 mM Tris-HCl, pH 8.0 and resuspended in 4.8 mL of 50 mM Tris-HCl, pH 8.5. The resulting solution containing Glutathione SepharoseTM 4B beads capturing GST-fused COSase or GST was used.In experiments on competitive inhibition by COThe phylogeny of COSase was inferred using the neighbor-joining method . EvolutiThe nucleotide sequences of genomic DNA encoding COSase, GPI, the ITS region, and tef1α region of strain THIF08 were submitted to DDBJ under the accession numbers LC499780, LC501463, LC600319, and LC600467, respectively.Trichoderma sp. strain THIF08 was found during the screening of fungi exhibiting COS-degrading activity from soil isolates ; therefore, strain THIF08 was used for further study.Trichoderma  of Hypocrea lixii (Teleomorph of Trichoderma harzianum), and with the tef1α regions of strain TAMA0431  and DUCC003  of T. harzianum, respectively. In the strain catalog of NBRC, NBRC 31294 is listed under the name Hypocrea nigricans, which is the synonym for H. lixii  of H. lixii . Based o2S (2S is a reaction product of COS hydrolysis by the COSase of T. thioparus strain THI115 (COS+H2O → H2S+CO2) , respectively. A search of the National Center for Biotechnology Information  database using the BLASTp program revealed that the N-terminal sequences of the 20-kDa and 60-kDa proteins were 100% identical, except for X, to those of clade D β-CA (GenBank accession number: KKP06884) and GPI (GenBank accession number: KKP03781) of T. harzianum strain THIF08 contains genome regions with high similarities to CA (97%) and GPI (99.8%) of T. harzianum strain T6776. The cloning and expression of pGEX-COS and pGEX-GPI in E. coli revealed that only the recombinant protein from pGEX-COS caused a decrease in COS accompanied by the emission of H2S in the presence of 1,000 ppmv of COS  . To the best of our knowledge, GPI has not been reported to degrade COS. Since GPI was co-purified with COSase through the purification step, GPI may have similar physicochemical properties to the COSase of T. harzianum strain THIF08. Alternatively, GPI may have bound to COSase through the purification step. Further research on GPI may provide another unknown function in the metabolism of this fungus. These results suggest that the COS-degrading ability and resulting H2S-producing ability of T. harzianum strain THIF08 are attributed to the COSase gene (cos).The overall structure of the elucidated sequence regions of COSase and GPI is summarized in mL vial) . The dec–1 of protein for bovine CA and COSase of T. harzianum strain THIF08 were 673.9 and 136.7, respectively. The time course of the pH decrease in the sigmoid curve of COSase was moderate and it took longer to reach pH 7.0 than bovine CA, which showed a steep sigmoid curve , the pH change was consistent with that of the blank (Data not shown).The specific activity of WAU mgid curve . The spe2 is a substrate for CAs, the COS-degrading activity of COSase was examined in the presence of CO2 . Enzyme activity at 1,000 ppmv of COS  was 106 nmol mg–1 min–1 in the first 10 min and was unaffected by the presence of CO2   . Therefocos gene from T. harzianum strain THIF08 encoded a protein of 163 amino acid residues with a calculated molecular mass of ~18.05 kDa, which is consistent with the molecular mass detected by the SDS-PAGE analysis  that were isolated from forest soil showed a strong ability (consumption rate per biomass) to degrade high concentrations of COS in the ppmv range  Fungal Carbonyl Sulfide Hydrolase of https://doi.org/10.1264/jsme2.ME20058Supplementary Material


Crotalus durissus terrificus, has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04–1.2 mg kg−1) or intraplantarly (0.9–7.5 μg 10 μL−1) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg−1) or intraplantar naloxone (5 μg 10 μL−1) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg−1) or intraplantar (2.5 and 7.5 μg 10 μL−1) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.Crotamine, a toxin found in the venom of the South American rattlesnake  Naja atra (Chinese cobra) and other cobras, has strong analgesic effects in animal models , pH 8.0. The cleaved crotamine was eluted with 2 M NaCl after washing with 100 mM NaCl. The gel filtration column  was equilibrated with buffer containing 20 mM Tris-HCl, 500 mM NaCl, 5% glycerol [v/v], pH 8.0, and applied as the final column to remove any remaining contaminants. The purified crotamine was exchanged with phosphate-buffered saline (PBS) using dialysis. Protein concentrations were measured using the bicinchoninic-acid assay . The protein was stored at −80 °C until use.In this study, only recombinant crotamine from s report . In thisad libitum. The animals were allowed to adapt to the laboratory environment for at least 2 h before testing. Experiments were performed during the light phase of the cycle (10 a.m.–5 p.m.). Six-week old male ICR mice  were used in all experiments. Animals were housed 4–5 per cage in a room maintained at 22 ± 0.5 °C with an alternating 12 h light–dark cycle. Food and water were available n = 7) of mice was placed on a hot-plate  and confined inside a transparent acrylic cylinder on the plate. Response latency (baseline), the time until the first response to the heat stimulus (licking of either front or back paws), was then measured. After 30 s, 100 μL of recombinant crotamine was intraperitoneally injected. Recombinant crotamine doses were 0.04, 0.13, 0.4 and 1.2 mg kg−1 (Each group ( mg kg−1 . Control−1) was subcutaneously administered 10 min [To evaluate the antagonistic effect of naloxone, 100 μL of naloxone (4 mg kgd 10 min  before td 10 min .n = 7) of mice received the same doses of recombinant crotamine  intraperitoneally (v/v) acetic-acid solution was intraperitoneally administered, and mice were immediately returned to the transparent acrylic observation cage (−1) was subcutaneously administered 10 min [Each group  of mice received intraperitoneal doses of recombinant crotamine  (−1). After 20 min, the formalin test was performed, in which, 10 μL of a 2.5% formalin solution was injected subcutaneously into the plantar surface of the left hind paw with a 30 G needle. The mice were immediately returned to the transparent acrylic observation cage and placed over a mirror angled at 40–50° to allow for an unobstructed view of the paws.Each group (mg kg−1) . NegativA formalin-induced pain test is usually divided into two phases; phase 1 lasts about 10 min and phase 2 lasts about 50 min ,18. In p−1) was subcutaneously administered 10 min [−1) or saline.To evaluate the antagonistic effect of naloxone, 100 μL of naloxone (4 mg kgd 10 min  before tn = 7) of mice received intraplantar administration of saline (10 μL) to the left hind paw with a 30 G needle. After 15 min, recombinant crotamine  (−1) instead of recombinant crotamine. After 15 min, the formalin test was performed in the same manner as described above.Each group (10 μL−1)  was inje−1) was subcutaneously administered into the plantar surface of the left hind paw 15 min before the intraplantar injection of recombinant crotamine (2.5 and 7.5 μg 10 μL−1) or saline. There was no reference for how long naloxone should be treated in a mouse paw. Therefore, we waited 5 min longer than subcutaneous administration (10 min) to make sure sufficient diffusion.To evaluate antagonistic effects of naloxone, naloxone (5 μg 10 μLThe swelling of the left hind paw was measured as described previously . The lef−1). The assay kit was used according to the manufacturer’s recommendations. The Bradford protein assay was used to measure the total protein concentration in the serum. ELISA microplates were analyzed using a Tecan Sunrise Microplate Reader  at 450 nm; data were standardized as picograms of TNF-α per 200 μg of total serum protein. The TNF-α concentration was determined based on an appropriate set of internal standard curves created using recombinant mouse cytokines.After 3 h following the formalin injection, blood was drawn from the heart under sevoflurane anesthesia for serum analysis. For TNF-α assays, serum was separated from the blood samples by centrifugation at 4000 rpm for 30 min at 4 °C. These samples were preserved at −80 °C until use. The serum TNF-α levels were measured by a commercially available enzyme-linked-immunosorbent assay (ELISA) kit , mice were placed on the rod while it rotated at 4 rpm. The rotation speed was increased from 4 to 40 rpm over 5 min. The latency required for the mouse to fall from the rod expressed in s, was recorded for each animal. Mice were acclimatized to the acceleration by an initial series of three training runs. The motor-coordination performance of each mouse was determined by measuring the average time for the fourth and fifth training runs. A 15 min resting period was allowed between two successive training runs.Motor ability and coordination were evaluated using a rotarod apparatus (Ugo Basile) for mice  . After 3p < 0.05 was considered statistically significant.All behavioral data are expressed as means ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) and the Kruskal–Wallis one-way ANOVA on ranks compared with the control group at the same time point between groups, followed by a Tukey’s post hoc test for multiple comparisons. The significance of the differences in the responses of treatment groups compared with the preoperative baseline value of each group was determined using one-way repeated-measures ANOVA, followed by a Tukey’s post hoc test for multiple comparisons. Statistical evaluation was performed with SigmaPlot Version 11 . −1) group, response latency gradually increased and peaked at 40 min  and then progressively decreased to the baseline level at 100 min. Recombinant-crotamine pre-treatment  showed significant and dose-dependent antinociceptive effects compared with the saline-control group. In the 0.13 mg kg−1 recombinant-crotamine group, response latency was significantly increased at 40 min. In the 0.4 mg kg−1 recombinant-crotamine group, response latency was significantly increased at 20, 40, and 60 min. Moreover, in the 1.2 mg kg−1 recombinant-crotamine group, response latency was significantly increased at 20, 40, 60, and 80 min points.As shown in −1) were not affected by naloxone pre-treatment  20 min before the acetic-acid injection showed significant and dose-dependent antinociceptive effects on the writhing response  compared with the saline-control group. In the lowest recombinant-crotamine group (0.04 mg kg−1), the number of writhes decreased to 58.4 ± 2.3. However, this was not significantly different from the saline-control group.Intraperitoneally administered acetic acid induced writhing behaviors for 20 min . In the −1) were not affected by naloxone pre-treatment  group, the nociceptive response was significantly suppressed in phase 2, but not phase 1. The AUC was 4.8 ± 0.9 for phase 1 and 28.9 ± 2.5 for phase 2 (p < 0.05). In the low-dose recombinant-crotamine (0.13 mg kg−1) group, animals showed similar nociceptive response and AUC (6.0 ± 0.7 for phase 1 and 54.4 ± 3.2 for phase 2) compared with the saline-control group. In contrast, in the high-dose recombinant-crotamine (0.4 and 1.2 mg kg−1) groups, intraperitoneal recombinant crotamine caused a dose-dependent suppression of the nociceptive response during phase 2, but not phase 1, of the formalin test. The AUC values were 4.8 ± 0.8 and 3.8 ± 0.7 for phase 1 and 25.6 ± 2.8 and 17.0 ± 2.6 for phase 2  was similar to the effect of intraperitoneal indomethacin  group, the mean nociceptive score was 2.0 ± 0.2 and it was similar to that of the saline-control group (2.3 ± 0.1). In contrast, in the high-dose recombinant-crotamine (0.4 and 1.2 mg kg−1) groups, the mean nociceptive scores were 1.4 ± 0.2 and 1.1 ± 0.1, respectively, and the scores were significantly suppressed compared to the saline-control group (2.3 ± 0.1). The mean nociceptive scores of the intraperitoneal recombinant-crotamine (0.4 mg kg−1) group (1.4 ± 0.2) was similar to the score of intraperitoneal indomethacin (10 mg kg−1) group (1.4 ± 0.2).In phase 1, there were no significant differences in AUC between each group. However, the mean nociceptive scores at the first 3 min time point produced significant differences between groups. In the low-dose recombinant-crotamine (0.13 mg kg−1) were not affected by the pre-treatment of naloxone  group, the nociceptive response was significantly suppressed in phase 2 but not phase 1. The AUC was 4.7 ± 1.1 for phase 1 and 28.8 ± 1.8 for phase 2 (p < 0.05). In the low-dose recombinant-crotamine (0.9 μg 10 μL−1) group, animals showed similar nociceptive response and AUC (5.6 ± 0.6 for phase 1 and 49.3 ± 1.9 for phase 2) compared with the saline-control group. Contrastingly, in the high-dose recombinant-crotamine (2.5 and 7.5 μg 10 μL−1) groups, intraplantar recombinant crotamine caused a dose-dependent suppression of the nociceptive response during phase 2, but not phase 1, of the formalin test. The AUC was 4.3 ± 0.6 and 3.3 ± 0.7 for phase 1, and 21.4 ± 2.6 and 13.6 ± 2.0 for phase 2  was similar to the effect of intraplantar indomethacin (50 μg 10 μL−1)  A,B.−1) group (1.3 ± 0.2).In phase 1, there were no significant differences in AUC between each group. However, the mean nociceptive scores at the first 3 min time point produced significant differences between groups. In the low-dose recombinant-crotamine (0.9 μg) group, the mean nociceptive score was 1.9 ± 0.2 and it was similar to that of the saline-control group (2.2 ± 0.1). In contrast, in the high-dose recombinant-crotamine (2.5 and 7.5 μg) groups, the mean nociceptive scores were 1.3 ± 0.2 and 1.1 ± 0.1, respectively, and the scores were significantly suppressed compared to the saline-control group (2.2 ± 0.1). The mean nociceptive score of the intraperitoneal recombinant-crotamine (2.5 μg) group (1.3 ± 0.2) was similar to the score of intraperitoneal indomethacin (50 μg 10 μL−1) were not affected by naloxone pre-treatment  group  group, animals showed a similar degree of paw swelling (28.6 ± 2.9%) compared with the saline-control group (−1) groups, intraperitoneal recombinant crotamine caused a dose-dependent suppression of paw swelling  was similar to the effect of intraperitoneal indomethacin  were not affected by the intraperitoneal naloxone pre-treatment  in the left hind paw was 33.6 ± 3.2% A. The paol group A. Contrareatment A.−1), paw swelling (%) was significantly suppressed  group, paw swelling (%) was similar (54.6 ± 5.1%) to that in the saline-control group (−1) groups, paw swelling (%) was significantly suppressed  compared with that in the saline-control group (−1) was similar to the effect of intraplantar indomethacin (50 μg 10 μL−1) (−1) were not affected by intraplantar naloxone pre-treatment , recombinant crotamine, and formalin were administered to the plantar surface of the left hind paw. Therefore, paw swelling (%) was much higher than that in the case of intraperitoneal administration of saline , and recombinant crotamine B. In theol group B. In theol group B. The an10 μL−1) B. Naloxoreatment B.−1 3 h after formalin injections  compared with that in the saline-control group , the serum TNF-α levels were similar (134.3 ± 11.9 pg mL−1) to those in the saline-control group (−1) groups, serum TNF-α levels were significantly decreased  compared with those in the saline-control group  pg mLjections A. In theol group A. In theol group A. Naloxool group A.−1 3 h after formalin injections  compared with those in the saline-control group (−1) group, serum TNF-α levels were similar (120.5 ± 10.3 pg mL−1) to those in the saline-control group (−1) groups, serum TNF-α levels were significantly decreased  compared with those in the saline-control group -injected groups and the saline group (Higher doses of recombinant crotamine could induce paralysis in mice. To distinguish between paralysis and antinociception in the above assays, the rotarod test was used. In the test, no significant differences were found for the latency time between recombinant-crotamine (0.04, 0.13, 0.4, and 1.2 mg kgne group . TherefoThe present study revealed that intraperitoneal and intraplantar application of recombinant crotamine had antinociceptive effects, which were not influenced by intraperitoneal or intraplantar naloxone pre-treatment. Moreover, naloxone pre-treatment did not affect the anti-inflammatory response of intraperitoneal and intraplantar recombinant crotamine.−1 dose of the native crotamine had an analgesic effect that was 50% of that in the control group in the acetic-acid-induced writhing method [−1 dose of recombinant crotamine had an antinociceptive effect that was 50% of that in the control group. Therefore, the potency of our recombinant crotamine was approximately 10% of the previously reported native crotamine. We do not have an explanation for this difference.The hot-plate test is a specific central antinociceptive test . In the g method . HoweverThe formalin test is the most predictive measure of acute pain and was used as a model for tonic and localized inflammatory pain . IntraplAs shown in In the present study, we investigated the anti-inflammatory effect of recombinant crotamine by measuring changes in paw volume. Compared with the saline-control group, the dose of intraperitoneal recombinant crotamine was significantly proportional to the suppression of paw swelling in a dose-dependent manner.In the high-dose groups of intraplantar recombinant crotamine, paw swelling was significantly suppressed in a dose-dependent manner compared to the saline-control group. The anti-inflammatory effect of intraperitoneal recombinant crotamine was similar to the effect of intraperitoneal indomethacin. The anti-inflammatory effect of intraplantar recombinant crotamine was similar to the effect of intraplantar indomethacin. Taken together, these suggest that intraperitoneal and intraplantar recombinant crotamine have an anti-inflammatory effect.In the present study, we measured serum TNF-α levels as a biochemical surrogate marker of inflammation induced by the formalin test in mice. Inflammatory stimuli release reactive oxygen species such as NO and ytokines ,31. TNF-ytokines ,33,34. CMancin et al. demonstrated that naloxone counteracted the analgesic effects of intraperitoneally administered crotamine purified from venom . NaloxonThe homemade recombinant crotamine demonstrated significant antinociceptive effects consistent with the results by native crotamine purified from snake venom . HoweverThe present study showed that the purified recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear related to opioid receptors. Antinociceptive and anti-inflammatory effects of intraperitoneal and intraplantar recombinant crotamine are related to TNF-α. Recombinant crotamine could be developed as an antinociceptive and anti-inflammatory agent in the central and peripheral sites. Clinically, this study provides a background for the clinical use of recombinant crotamine for treating pain using an opioid-independent pathway.