Pseudomonas sp. has been examined for producing L- methionine gamma-lyase enzyme. The identity of the tested bacteria was verified by VITEK2, and MALDI-TOF analysis in addition to molecular confirmation by 16S rDNA sequence and submitted in Genbank under accession number ON993898.1. Production of the targeted enzyme was done using a commercial medium including L-methionine, as the main substrate. This obtained enzyme was precipitated using acetone (1:1v/v) followed by purification with Sephadex G100 and sepharose columns. The specific activity of the purified enzyme (105.8 µmol/ mg/min) increased by 1.89 folds after the purification steps. The peptide fingerprint of the native MGL was verified from the proteomics analysis, with identical conserved active site domains with database-deposited MGLs. The molecular mass of the pure MGL denatured subunit was (>40 kDa) and that of the native enzyme was (>150 kDa) ensuring their homotetrameric identity. The purified enzyme showed absorption spectra at 280 nm and 420 nm for the apo-MGL and PLP coenzyme, respectively. Amino acids suicide analogues analysis by DTNB, hydroxylamine, iodoacetate, MBTH, mercaptoethanol and guanidine thiocyanate reduced the relative activity of purified MGL. From the kinetic properties, the catalytic effectiveness (cat/kmK) of Pseudomonas sp. MGL was 10.8 mM -1 S-1 for methionine and 5.51 mM -1 S-1 for cysteine, respectively. The purified MGL showed highly significant antiproliferative activity towards the liver carcinoma cell line (HEPG-2) and breast carcinoma cell line (MCF-7) with half inhibitory concentration values (IC50) 7.23 U/ml and 21.14 U/ml, respectively. No obvious signs of toxicity on liver and kidney functions in the examined animal models were observed.A soil inhabiting  The lack of methionine synthase activity in tumors, a distinct metabolic criterion that explains the reliance of cancer cells on extrinsic plasma L-methionine, elaborates the rationality of MGL's focused anticancer effect . Unlike normal cells, methionine dependency is a physiological criterion for various tumor cells including prostate, melanoma, colon, kidney and fibrosarcoma cells . Thus, methionine depletion selectively represses the different biological processes of tumor cells particularly cellular gene methylation, cell cycle transition, and cellular protein synthesis , preventing cells from dividing at the G2 phase and resulting in cell death .The enzyme Methioninase , which is dependent on pyridoxal 5′-phosphate, catalyzes the transformation of L-methionine into ketobutyrate, methanethiol and ammonia . L-Methi. The lacr effect . Unlike ma cells . Thus, mynthesis , preventll death .Aeromonas sp., Pseudomonas putida; Brevibacterium linens; Citrobacter freundii; Lactococcus lactis as well as Clostridium sporogenes . As well as, MGL has been extensively characterized by Aspergillus flavipes . From the crystal analyses, MGL from C. freundii, P. putida  and A. flavipes. Purified MGL from A. flavipes was chemically modified by polyethylene glycol, dextran and co-immobilized with glutaraldehyde dehydrogenase to scavenge the toxicity of released ammonia . Briefly, the medium includes 0.25 % L-methionine, 0.1 % polypeptone, 0.1 % glycerol, 0.1 % KH2PO4, 0.1 % K2HPO4, 0.01 % MgSO4·7H2O, and 0.02 % yeast extract. A loop full of the bacterial isolate was inoculated to 100 ml Erlenmeyer conical flask with 25 ml of medium, then kept at 37 °C for three days at 120 g. Bacterial pellet was collected by centrifugation for 10 min at 5000 g, then washed with 10 mM sterile phosphate buffer (pH 7.8).A Gram-negative bacteria collected from a non-cultivated soil sample in Daqahlia Governorate, Egypt, was isolated and purified on nutrient agar media. The potency of isolated bacteria for producing MGL was evaluated by the modified method of The cellular crude protein was extracted according to 2.22.2.1). Briefly, the components of the reaction include phosphate buffer with 1 ml of 20 mM L-methionine (pH 7), 10 mM PLP, and 0.25 mM DTNB reagent. Separate blanks for the substrate and the enzyme were prepared. The reaction was kept warm for 30 min at 37 °C, and the released yellow coloring was assessed at λ420 nm. The quantity of resulting methanethiol served as a proxy for the activity of MGL, which was calculated using DTNB under identical circumstances using a standard curve with varying cysteine concentrations. Under ideal test circumstances, the quantity of enzyme that introduces 1 M of methanethiol/minute was expressed by one unit (U) of MGL. The enzyme activity (U) per mg protein served as a measure of the enzyme's specific activity.The demethiolating activity of MGL was calculated from the amount of liberated methanethiol by using the 5,5-dithiol-bis-2-nitrobenzoic acid (DTNB) assay . Briefly2.2.2. After 1 h of incubation at 25 °C, the mixture was halted with 5% Tri carboxylic acid (TCA). The mixture was whirled for ten minutes at 5000 g, and then a volume of 50 µl of Nessler's reagent was added. This evolved coloration was determined at about 500 nm after 15 min. Under ideal test circumstances, the quantity of enzyme required to produce one mole of ammonia/minute was used to represent one unit of MGL.The direct Nesslerization test was used to quantify the MGL activity. The assay combination in a total volume of 0.5 ml, l-methionine (10 mM) was added, PLP(0.02 mM), phosphate buffer (50 mM)(pH 7.8), and>0.015 U of the enzyme . After 12.2.3Folin's test  was used2.3). The bacterial isolate was identified using the VITEK automated system  located at 57,357 Children’s Cancer Hospital Foundation, Egypt and the MALDI-TOF MS spectrometer, Autof MS2000 system , located at 57,357 Children’s Cancer Hospital Foundation, Egypt. Identity of tested bacteria was confirmed using 16S rDNA sequence using the universal primer 5′-27F (5′AGAGTTTGATCCTGGCTCAG3′) and 1492R(5′GGTTACCTTGTTACGACTT3′) . Re-suspended bacterial colonies in 50 ml of lysis solution, which contains Tris-HCl (10 mM), Tween 20 (0.1%), and KCl (50 mM) (pH 8.3), were incubated for 10 min at 99 °C. The bacterial lysate  was utilized as a PCR template for amplification. The PCR reaction is composed of 10 µl of the master mixture (2x) , 1 µl of forward and reverse primers (10 pmol), 2 µl of bacterial lysate and Polymerase chain reaction committed to 20 µl total volume with sterile distilled water. The PCR was set up to have 35 cycles, starting with denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, followed by annealing at 55 °C for 10 s, extension at 72 °C for 30 s, with a final extension at 72 °C for 2 min.The bacterial isolate was identified based on its biochemical parameters . The bacd system  located 0 system , locatedGACTT3′) . Re-suspThe PCR amplicons were assessed in comparison to a DNA ladder  using agarose gel (1.5%) in Tris/Borate/EDTA(TBE) buffer (Ambion Cat# AM9864).). Target sequences' of the phylogenetic tree was produced by MEGA 6.0′s neighbor-joining approach with 1000 bootstrap replications  were collected after incubation and washed with sterile distilled water and the intracellular crude protein was extracted according to . Five volumes of pre-chilled absolute acetone (at −20 °C) were added to one volume of the sample and kept for 15–20 min then vortexed for 30 s. The precipitate was separated by centrifugation at 10,000 g for 10 min. Protein pellets were collected, and 10 ml potassium phosphate buffer  was used to dissolve them. By using dialysis bag with a 20 kDa cut-off , the precipitated proteins were selectively concentrated against polyethylene glycol 6000 till reached 2 ml total volume. By using gel filtration and ion exchange chromatography, the enzyme was purified . A sephadex column G200 (240 cm) was loaded by the crude protein after being pre-equilibrated with 50 mM potassium phosphate buffer (pH 7.5) at a rate of flow 0.5 ml/min. The same buffer was used to elute the enzyme fractions (1 ml), and the standard test was used to assess the enzyme activity and concentrations. Dialysis against polyethylene glycol 6000 was used to collect and concentrate the most active fractions. The first step partially purified enzyme was further refined using ion exchange chromatography column of DEAE-sepharose (2 × 30 cm), with potassium phosphate buffer pre-equilibrated . Following gel permeation, the column was loaded with the sample and then equilibrated with a flow rate of 1 ml/min through the same buffer. This buffer containing gradient concentrations of NaCl (100–300 mM) was used to elute the enzyme. The recovered fractions' activity and protein content were assessed as previously mentioned. The MGL fractions with the highest activity were gathered and concentrated using a 10 K ultra-centrifugal membrane. The pure MGL was held at 4 °C pending assay. The purified MGL's subunit structure and molecular homogeneity were checked by running over 12% SDS-PAGE . Peptide elution to mass spectrometry from a column was carried out using a linear cascade of acetonitrile (ACN) buffer (5–60%) flowing at a rate of 20 L/min. The MS/MS data were obtained independently, and the MS/MS data is being obtained from m/z for 50–2000 Dalton and MS1 data being obtained for 250 ms at m/z for 400–1250 Dalton . After extracting the MS/MS data files, Protein Pilot 4.0 was used to analyze and identify the peptides (ABSCIEX)  matching the Pseudomonas sp. proteome. Five peptide segment ions with at least/protein of E-values under 0.05 were required for identification.The peptide fingerprint of the pure enzyme was examined by the Liquid Chromatography-Tandem Nanospray Ionization Mass Spectrometry (LC-MS/MS) at the Proteomics and Metabolomics Facility Core, 57,357 Children’s Cancer Hospital Foundation, Egypt. The MGL was removed from the SDS-PAGE gel, and the gel fragment was ground before being destained by 200 ml of 50 mM ammonium bicarbonate (AB)-acetonitrile (1:1 v/v) and 500 ml of acetonitrile. After the extra acetonitrile was vacuum-extracted, the gel was re-swollen in 100 mM AB with 10 mM dithiothreitol (DTT) for 30 min at 60 °C, then in 100 mM AB with 50 mM iodoacetamide. Using trypsin, the dried gel fragments were broken down  for 12 h at 37 °C, under shaking conditions. The supernatant was combined, added to 100 ml of extraction buffer which was made of 5% formic acid and 1:2 acetonitrile, and then kept standing for 15 min at 37 °C. The peptides were desalted and analyzed by the nanospray ionization using Triple-TOF 5600 mass spectrometer, interfaced by nano-scale RP-HPLC . Peptide0 Dalton . After eABSCIEX)  matching2.62.6.1Kr) and half-life time (T1/2) were calculated according to Lowry et al, 1951). The pH where the protein was precipitated most readily, served as an indicator of the isoelectric focusing (pI).The biochemical characteristics of the purified MGL from the bacteria, including optimum temperature, thermal stability, reaction pH, pH stability and MGL inhibitors were examined. The enzyme reaction mixture was incubated at various degrees of temperatures , and the MGL activity was then detected using the standard test to determine the temperature at which the purified MGL was most active. MGL thermal stability was assessed by keeping at 37, 45 and 50 °C for 30, 60, 120 and 180 min, respectively, and the remaining demethiolating activity of MGL was then assessed by the standard test. Thermal kinetic parameters such as the thermal inactivation rate  against a 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM EDTA, then enzyme preparations were desalted. Several cations such as K of 1 mM . Several2.6.2maxV), Michalis-Menten constant (mK) and turnover number (catK) were calculated  of 10 µM PLP, in one ml total volume, and then the demethiolating and deaminating enzyme activity was assayed, as mentioned previously. The catalytic and kinetic parameters such as maximum velocity (lculated .2.6.3). Triplicates were conducted for each enzyme preparation.The intrinsic UV-spectra of the pure enzyme was assessed . The purEl-Sayed et al., 20122.6.4.The antiproliferative activity of the purified l-methioninase was examined against breast carcinoma (MCF7) and Hepatocellular carcinoma (HPG2) at Al-Azhar University, The Regional Center for Mycology and Biotechnology .2.6.5Pseudomonas sp. MGL were evaluated in vivo using female Swiss Albino mice weighing 25 g. The experimental protocols were approved by the IAEC   and under the recommendations for the proper care and use of laboratory animals. The mice were intravenously injected using a single dosage (50 µl) of the MGL (50 mg/kg) and allowed to adapt for three days. Three mice are employed in each treatment group. Untreated mice were kept as control group. The biochemical characteristics of blood samples taken via tail vein were analyzed. MGL activity in recovered sera from tested mice was determined using previously mentioned standard test.Cytotoxic properties of 2.6.6The collected blood samples from treated and untreated mice were used for the estimation of biochemical parameters, denoting liver and kidney functions, such as AST, APL, ALT, albumin, total protein, globulin, creatinine and urea, as markers to evaluate the toxicity of MGL. These markers were analyzed as follows:2.7340 nm. AST and ALT activities were calculated according to the instructions of the working kit .Transaminases such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed using SPINREACT Clinical Systems Kit. Briefly, the reaction mixture contained 500 µl working solution ) for AST assay. For the ALT assay, the working solution was  of 110 mM, then added to 50 µl of serum for each enzyme treatment, comparing to control. The alteration in absorption spectrum was measured per minute at Aking kit .Activity (μkat/l) = A/min. × 1746 × 0.0167.2.7.1405 nm , 0.6 mM magnesium ions and 20 µl serum. After one min of incubation, the resulting color was evaluated at A405 nm . The rea2.7.2580 nm  and 10 µl serums and leave for 5 min at room temperature. The developed color was assessed at A580 nm . Serum a2.7.3546 nm . The concentration of protein (gm/dl) = A sample/A standard × 6.Total protein was assessed using SPECTRUM Kit. The reaction mixture contained 1 ml of working solution  and 20 µl of serum. The reaction was kept for 10 min and the released color was detected at A546 nm . The con2.7.4578 nm (Urea was assessed using DIAMOND Kit. The reaction included 1 ml of reagent A (phosphate buffer 50 mM (pH 6.7), 2 mM EDTA, 400 mM sodium salicylate and 10 mM sodium nitroprusside), 50 µl of 3000 U Urease and 10 µl of serum and leave at 37 °C for 3 min. Then, 200 µl of reagent B (140 mM sodium hypochlorite and 150 mM sodium hydroxide) was added and kept for 5 min at 37 °C. The resulting coloring was assessed at A578 nm .Urea concentration = Asample/Astandard × 50 (Standard conc.).2.7.5492 nm . The concentration of creatinine (mg/dl) = Asample/Astandard × 2.Creatinine was assessed according to the instruction of the SPECTRUM kit. One ml of 25 mM picric acid with 1 ml of 0.4 mM sodium hydroxide was mixed with 100 µl of mice serum and the change in absorbance was measured per min at A492 nm . The con2.7.6http://WWW. Easy calculation.com/statistics/fiishers-Isd-calculator).All data were represented by three replicates and expressed as mean ± STDV.  using SPSS software (version 20.0) .3Pseudomonas sp.The Gram-negative bacterial isolate confirmed as a potent MGL-producing isolate was identified using the automated VITEK- 2 system and analyzing its peptide sequence using the MALDI TOF system, as  of the obtained sequence revealed that the tested isolate displayed 99 % similarity with Pseudomonas species encoded: OM669957.1, MW477013.1, MW876240.1, LC507991.1, LC507995.1, CP077094.1, OK135828.1, 0 K147819.1, KM187448.1, MN519638.1, MT255196.1, MT255227.1, MT255228.1 with E-value zero and query coverage 98%. The result is coincident with the biochemical and metabolic identifications from VITEK 2 and MALDI-TOF identification systems and the isolate is identified as Pseudomonas sp. and submitted under accession number (ON993898.1) in Genbank.The identification of the tested isolate was molecularly verified depending on the sequence of the 16S rRNA gene, using its genomic DNA as a template for PCR. The amplicons of PCR were visualized with an apparent size of 1500 bp. The specific PCR amplicon amplified using universal primers was purified, sequenced and non-redundantly BLAST searched on the database. The phylogenetic tree built from the alignment analysis  of the o3.1Pseudomonas sp. after being grown in a commercial nutrient medium described by Methionine gamma-lyase (MGL) enzyme produced by tested Obtained results revealed an increase in MGL enzyme activity by 1.725 folds, and a yield of 40.48% after gel-filtration using Sephadex G100. Most active fractions (6–20) were gathered and further eluted by 150 mM NaCl through ion exchange DEAE-sepharose. MGL assessment indicated an increase in enzyme activity by 1.89 folds with a recovery yield of 37.5% with an overall specific activity of 105.8 µmol /mg/min.3.2Pseudomonas sp. was > 40 kDa when detected by SDS-PAGE. Meanwhile, the entire molecular mass was ∼ 140–150 kDa, as revealed from the native PAGE. These results emphasize that MGL produced by Pseudomonas sp. has four identical subunits in molecular mass and charges (i.e. homotetrameric identity).The purified MGL enzyme was further checked for its molecular mass and subunit structure by the denatured and native PAGE. The molecular mass of the subunit structure of MGL of Pseudomonas sp. were determined at wavelengths 200–600 nm. Results in Spectroscopic analyses of purified MGL enzyme produced by Pseudomonas sp. MGL was assessed by LC-MS/MS, after trypsinization followed by nanospray ionization. The extracted raw peptides MS data were examined, identified, and normalized to the Pseudomonas sp. proteome by Protein Pilot 4.0 (ABSCIEX). The identifying criterion required at least five peptide segment ions of E-values < 0.05) per protein. The obtained sequences of peptide were categorized by matching with MGL sequences from various microbial origins (a&b). The numerous aligning analysis revealed that the partial peptide sequence of Pseudomonas sp. MGL had 80% similarity with the MGL from Streptomyces malaysiensis ATL87061.1, C. botulinum WP061310508.1, Arabidopsis thaliana AEE34271.1, Azorhizobium caulinodans BAF89241.1, Porphyromonas asaccharolytica AEE12456.1, B. wiedmannii EEK65504.1, Erwinia pyrifoliae CAY73263.1, Porphyromonas gingivalis ERJ88601.1, B. thuringiensis EEM45798.1, B. cereus EEL43444.1 and EEL04005.1.The peptide fingerprint of the purified  origins a&b. The Pseudomonas sp. peptide sequence in the database submitted MGLs sequences was observed. Phylogenetic analysis indicated the peptide sequence of tested Pseudomonas sp. MGL displays high proximity to Arabidopsis thaliana AEE34271.1 and Azorhizobium caulinodans BAF89241.1 (b).The phylogenetic evaluation of partial F89241.1 b.Pseudomonas sp. MGL  matched with that in MGL from species of P. asaccharolytica AEE12456.1, B. wiedmannii EEK65504.1, E. pyrifoliae CAY73263.1, P. gingivalis ERJ88601.1, B. thuringiensis EEM45798.1, B. cereus EEL43444.1 and EEL04005.1 (c).The partial peptide sequence's conserved active sites domains of L04005.1 c.3.33.3.1Pseudomonas sp. were estimated. The reaction mixtures of MGL were incubated at temperatures 20 to 60 °C, and the activity of the enzyme was calculated by standard test. The reaction temperature profile  of purified MGL from the  profile , indicatPseudomonas sp. has been determined by keeping the enzyme without substrate at 4, 20, 37 and 50 °C for 30, 60, 90 and 120 min, then assessing the residual enzymatic activity. Remarkably, the catalytic efficiency and enzyme structural stability sharply decreased using the higher pre-incubation temperature. The overall thermal kinetic parameters of MGL from Pseudomonas sp. are summarized in Pseudomonas sp. was 349.3 h, ensuring the proximity of storage stability of Pseudomonas sp. MGL. Meanwhile, T1/2 of MGL from Pseudomonas sp. at 20 °C (101.2 h) showed 71% decrease in enzyme activity, while at 37 °C decreased by 88.2% with T1/2 value as (20.66 h). After incubation at 50 °C, 98.5% of MGL activity was lost with T1/2 (5.39 h). In conclusion, the thermal denaturation rate of the MGL from Pseudomonas sp. displayed an obvious high conformational stability towards increased storage temperatures. At 37 °C, the thermal denaturation rate (Kr) of MGL from Pseudomonas sp. was 0.59x10-3 min. The half-life temperature (Tm) of the MGL of Pseudomonas sp. was 101.3 °C after 60 min by about 1.18 folds increment of structural stability. Meanwhile, recorded Tm values were 79.54 °C after 90 min and 60.34 °C after 120 min. Meanwhile, after 120 min, the maximum thermal denaturation rate (Kr) of the tested enzyme (0.59x10-3 min & 5.16 x10-3 min) was observed at 37 °C & 50 °C, respectively.The thermal stability of the purified MGL from 3.3.2(a), the highest enzyme activity was reported at pH 6.0–7.0, with a marked decrease in activity at pH 3.0 and pH 10.0. The MGL activity was reduced by about 80% at pH 3.0, suggesting the denaturation of catalytic subunits of MGL or dissociation of enzyme PLP, consequently forming inactive apo-MGL.The purified MGL activity was assessed in response to various pH values of the reaction mixture. From the results a, the hib) revealed that maximum activity was recorded at pH value 6. Meanwhile, obvious reduction of enzyme activity (45–20%) was detected at pH 3.0 and pH 9.0, assuming the drastic effect of acidic pH on the enzyme conformational structure than the alkaline one (b).The purified MGL pH stability was assessed by keeping the enzyme at various pH (3–10) for 3 h at 4 °C, then detecting the MGL residual activity. The pH stability pattern b revealeline one b.pI = 5).Purified MGL enzyme was dissolved in buffers adjusted at pH values from 3 to 10 for 24 h at 4 °C. Thereafter, the precipitated protein was estimated where maximum protein concentration was determined at pH 5  confirmi3.3.32+, Cu2+, Zn2+, Fe3+, Ca2+, Hg2+, Al3+, Na+, K+, and Mg2+ at 1 mM total concentration. After incubation, the enzyme activity was determined by the standard test. There was no significant effect on the activity of MGLs towards monovalent cations , divalent cations  and trivalent cations (Al+3and Fe+3), adjusting for the demetallized MGLs residual activity  with regard to methionine as standard.The affinity of MGL for γ-elimination and deamination reactions of various amino acids namely L-cysteine, DL-homocysteine, L-tyrosine, L-asparagine, L-tryptophan, L-ornithine, L-arginine, L-phenylalanine and D-glycine was evaluated regarding L-methionine as blank. In the substrate specificity profile , the purmaxV), Michalis-Menten constant (mK) and turnover number were summarized in Pseudomonas sp. was 4.9 mM for L-methionine and 6.3 mM for L-cysteine. The turnover number (catK) of MGL of Pseudomonas sp. was 53.1 s−1 for the L-methionine substrate and 34.9 s−1 for L-cysteine. The obtained kinetics parameters revealing high catalytic efficiency of MGL from Pseudomonas sp. for L-methionine as a substrate might be due to the molecular conformational structure and active site orientation of purified MGL enzyme.Further kinetic properties for the purified MGL have been evaluated towards L-cysteine compared to L-methionine as the standard substrate. The kinetic and catalytic parameters; maximum velocity  was 21.14 μmol/mg/min with a standard deviation ± 0.98 .The anti-proliferative activity of the purified MGL from bacterial isolates towards breast (MCF-7) and liver (HepG-2) carcinoma cells was determined while using phosphate-buffered saline as negative control. The results in n ± 0.98 .Fig. 5In3.3.6Pseudomonas sp. was determined in vivo using Swiss Albino mice. The collected Blood samples from treated mice were centrifuged and the titer of various biochemical parameters was examined in blood sera. According to the biochemical analysis . The Pseudomonas sp. MGL's entire molecular mass was ∼ 140–150 kD, as shown by the native-PAGE, and > 40 kD under denaturing-PAGE. So, the SDS- PAGE and native, indicated that the molecular identity of MGL has been emphasized to have four identical subunits, i.e. homotetrameric identity. The purified MGL Pseudomonas sp. molecular identity and subunit structure were coincident with the purified MGL from various bacterial and fungal isolates  matching with that in MGL from species of P. asaccharolytica AEE12456.1, B. wiedmannii EEK65504.1, E. pyrifoliae CAY73263.1, P. gingivalis ERJ88601.1, B. thuringiensis EEM45798.1, B. cereus EEL43444.1 and EEL04005.1.The phylogenetic analysis of peptide sequence of tested 196- Gly197-Cys198, and the location of this sequence varied greatly depending on the microbial source of the enzyme, for instance, Tyr114-Gly115-Cys116was present in P. putida, Tyr111-Gly112-Cys113 in Trichomonas vaginalis and Tyr108-Gly109-Cys110 in Entamoeba histolytica . The MGLs from bacteria, fungi, and plants possess moieties Tyr114-Gly115 and Cys116 that substantially constitute the active site .The conserved MGL active sites are usually Tyrtolytica . Goyer eive site .A. flavipes and from other various bacterial isolates . Thus, the purified MGL from this bacterial isolate could have the same reactivity and conformational catalytic structure for binding with the substrate and further catalytic process. The overall thermal structural stability reported for Pseudomonas sp. MGL at 37 °C is consistent with MGL from B. linens .The purified MGLs from this bacterial isolate exhibited the same maximum activity at 37 °C, emphasizing the potential activity of this enzyme for in vivo therapeutic applications. Consistently, the highest activities of MGL at 37 °C were reported for the enzyme from isolates . Thus, t. linens .pI, 5) of the enzyme. Similar results for the pH activity and stability of MGL were reported for MGL from various bacterial  and fungal sources , on MGL activity might be contributed to proximity to precipitation El-Sayed, 2011et al., 2015; El-Sayed ;El-Sayed et al., 2016) were completely blocked by carbonyl reagents; DL-propargylglycine, L-cycloserine, hydroxylamine, which are potent irreversible inhibitors to the PLP, assuring their reliance on PLP. As well as, the MGL was greatly reduced by the reaction between their sulfur amino acids and thiol reagents such as DTNB and iodoacetamide . Lactococcus lactis MGL activity was fully reduced by the carbonyl reagents; hydrazone 3-methyl-2-benzothiazolinone and hydroxylamine . An extreme reduction of demethiolating activity of Achromobacter starkeyi MGL by p-chloromercuri-benzoic acid, iodoacetate, semicarbazide and sodium arsenate was reported . However, MGL from B. linens , P. ovalis and Clostridium sporogenes , exhibited a higher affinity for DL-homocysteine compared to methionine as detected based on the Km values. MGL from E. histolytica  had a 30–20% activity on DL-homocysteine comparing to L-methionine.The purified MGLs displayed the maximum demethiolating and deaminating activities towards L-methionine, followed by L-cysteine and DL-homocysteine. The relative MGL activity from . linens , P. ovalorogenes , exhibittolytica , A. flavPseudomonas sp. MGL antitumor activity was higher towards the HEPG-2, recording IC50 as 7.23 ± 0.59 μmol/mg/min, than that recorded against MCF-7 cell lines (IC50 21.14 ± 0.98 μmol/mg/min). Similar results for the antitumor activity of the purified MGL produced by A. flavipes , and 726 ± 29.31 µg/ml (1.452 U/ml), respectively, ensuring its good catalytic properties along with significant anticancer activity at low concentrations . Thus, reporting A. fumigatus MGL as a probable candidate to be applied in the enzymotherapy field.The antiproliferative activity of purified bacterial MGL was determined for the breast carcinoma (MCF-7) and liver carcinoma (HEPG-2) cell lines. flavipes  and P. p. putida. Recordetrations . Thus, rPseudomonas sp. MGL had any harmful effects on physiological functions. It was found that the examined enzyme had no significant impact on kidneys or liver functions. Titers of albumin, globulin, A/G ratio, total proteins, creatinine, urea as well as ALT, AST and AlP along the experimented time (72 h), were not significantly altered from the normal level of control animals. The designed in vivo experiment using Swiss Albino mice aimed to examine whether the purified 5Pseudomonas sp. was purified to its molecular homogeneity by gel-filtration and ion-exchange chromatography. The obtaind molecular masses of denatured subunit (>40 kDa) and native enzyme (>150 kDa) confirmd its homotetrameric nature.The proteomics analysis of Pseudomonas sp. MGL displayed the peptide fingerprint showing the conserved active site domains , with a slight difference on the N and C-terminals, that might be attributed to the microbial source, or enzyme post translational modification process. The purified MGL enzyme showed high degree of stability at high temperatures up to 50 °C and wide range of pH values (pH 3–10). The enzyme also displayed the highest affinity for the substrate l-methionine than other tested substrates ensuring its promising activity in in-vivo applications in combatting L-methionine-dependent tumor. Significant in-vitro anticancer activity of Pseudomonas sp. MGL towards the HEPG-2 and MCF-7 cell lines, that did not exhibit any overt toxicity in the examined animals, demonstrating the absence of direct effect on either liver or kidney. As a result, the current study indicates that Pseudomonas sp. MGL has potential as a cancer treatment agent.In conclusion, MGL enzyme extracted from a local soil inhabiting The ZU-IACUC committee of the Faculty of Medicine at Zagazig University examined and approved the animal study, which followed NIH rules, with approval number: ZU-IACUC/1/F/220/2022.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Huang (483 m a. s. l) in East China during the summer of 2019 with a 3 h time resolution. The concentrations of nearly all of the detected species, including organic carbon (OC), elemental carbon (EC), levoglucosan, and SIA , were higher at night (19:00–7:00 of the next day) than in the daytime (7:00–19:00). In addition, air pollutants that accumulated by the dynamic transport of the mountain breeze at night were also a crucial reason for the higher BSOA tracers. Most of the BSOA tracers exhibited higher concentrations at night than in the daytime and peaked at 1:00 to 4:00 or 4:00 to 7:00. Those BSOA tracers presented strong correlations with O3 in the daytime rather than at night, indicating that BSOAs in the daytime were primarily derived from the photo-oxidation of BVOCs with O3. The close correlations of BSOA tracers with SO42− and particle acidity (pHis) suggest that BSOAs were primarily derived from the acid-catalyzed aqueous-phase oxidation. Considering the higher relative humidity and LWC concentration at night, the promoted aqueous oxidation was the essential reason for the higher concentrations of BSOA tracers at night. Moreover, levoglucosan exhibited a robust correlation with BSOA tracers, especially β-caryophyllinic acid, suggesting that biomass burning from long-distance transport exerted a significant impact on BSOA formation. Based on a tracer-based method, the estimated concentrations of secondary organic carbon (SOC) derived from isoprene, monoterpenes, and β caryophyllene at night (0.90 ± 0.57 µgC m−3) were higher than those (0.53 ± 0.34 µgC m−3) in the daytime, accounting for 14.5 ± 8.5% and 12.2 ± 5.0% of OC, respectively. Our results reveal that the BSOA formation at the foot of Mt. Huang was promoted by the mountain-valley breezes and anthropogenic pollutants from long-range transport.The molecular characteristics and formation mechanism of biogenic secondary organic aerosols (BSOAs) in the forested atmosphere are poorly known. Here, we report the temporal variations in and formation processes of BSOA tracers derived from isoprene, monoterpenes, and  Equations used in the calculation of OC contributions are illustrated as follows:To assess the relative contributions of primary and secondary sources of organic aerosols, the SOA tracer method can be used to estimate their relative abundances in t et al.  was applg et al.  and Zhang et al. , the cond et al.  and Yttrd et al.  in detaiC-MTs2, CC5-alkane, CMG2-, caryophyllinicC, and levoglucosanC refer to the concentrations of 2-methyltetrols, C5-alkene triols, 2-methylglyceric acid, β-caryophyllinic acid, and levoglucosan in aerosols, respectively; ∑BSOAM refers to the sum of cis-pinonic acid, cis-pinic acid, 3-hydroxyglutaric acid, and 3-methyl-1,2,3-butanetricarboxylic acid.In the above equations, −3 and 0.93 ± 0.23 µgC·m−3 during the day and night, accounting for 12.7 ± 6.0% and 15.1 ± 8.4% of the sum of OC, respectively (−3) was more than 20 times higher than that (0.03 ± 0.03 µgC·m−3) of primary OC (biomass-burning-derived OC). Isoprene-derived SOC was the most abundant species during the daytime and night-time, followed by monoterpene-, β-caryophyllene-derived SOC, and biomass-burning-derived OC. These four species accounted for 6.8 ± 2.8%, 3.6 ± 1.4%, 1.8 ± 0.9%, and 0.5 ± 0.5% of OC during the daytime and 8.8 ± 5.6%, 3.7 ± 2.1%, 2.0 ± 1.4%, and 0.6 ± 0.4% during the night-time, respectively.The calculated OCs are shown in ectively . The diu−3, which was equal to that (0.03 ± 0.01µgC·m−3) over Mt. Fuji. However, the contribution of biomass-burning-derived OC to total OC concentrations was only 0.6 ± 0.5% during the entire sampling period, which was much less than that (24%) in early June over Mt. Tai where organic aerosols were significantly influenced by biomass burning, and smaller than that (2.3%) over Mt. Fuji of Japan where the effect of biomass burning was minor, indicating the negligible influence of biomass burning on organic aerosols at the foot of Mt. Huang. The relative abundance (13.4 ± 6.9%) of estimated biogenic SOC in aerosol OC at Mt. Huang was almost the same as that in the urban aerosol of Jinan (13.3%) [The average concentration of biomass-burning-derived OC was 0.03 ± 0.03 µgC·m (13.3%)  and high (13.3%)  and a ru13.4 ± 6.% of esti (13.3%) , Mt. Gon (13.3%) , and a r (13.3%)  in the sMt. Huang  is situated in the southwest of the Yangtze River Delta (YRD), East China. Climate in this area is heavily regulated by the East Asian monsoon with a marked seasonal variability in temperature, cloudiness, and relative humidity. More than 90% of the mountainous land is covered by vegetation, which comprises broad-leaved forests at low altitudes and coniferous forests at high altitudes. The climate of Mt. Huang varies significantly on a daily basis, owing to the contrasting topography of high mountains and deep valleys.2.5 sampling at a 3 h time resolution was conducted from July 27 to August 3 in 2019 using a high-volume air sampler  coupled with prebaked (450 °C for 6 h) quartz fiber filters at an airflow rate of 1.13 m−3 min−1. Two blank samples were collected at the beginning and the end of the sampling period by installing the filters onto the sampler for about 15 min without pumping any air. A total of 53 PM2.5 samples and 2 field blank samples were collected in this study. After sampling, each filter was sealed in an aluminum bag and stored at −20 °C prior to analysis.The monitoring site is located at the Anhui Academy of Forestry of Mt. Huang  and meteorological parameters including temperature, relative humidity, and wind speed/direction at the foot of Mt. Huang were retrieved from the website of Chinese Air Quality Monitoring System  at a one-hour time resolution. Planetary boundary layer (PBL) height was obtained from the website of Copernicus Meteorological Center .Gaseous pollutants  were added and ultrasonic extraction was carried out three times (each time for 15 min). The extracts were filtered through glass wool in a pasteurized dropper and transferred to a pear-shaped bottle; they were then concentrated by a rotary evaporator in vacuum state and dried with pure nitrogen gas. After reacting with a 60 µL mixture of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine  at 70 °C for 3 h, the derivatives were diluted with 40 µL n-hexane containing the internal standard (C13 n-hexane) before GC/MS analysis. The derivatized fraction was determined by GC/MS  equipped with a fused silica capillary column .Detailed methods for extraction, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis have been reported in our previous studies ,60. HalfI) tracers such as two 3-MeTHF-3,4-diols , three C5-alkene triols , 2-methylglyceric acid, two 2-methyltetrols (2-methylthreitol and 2-methylerythritol), monoterpene-derived SOA (SOAM) tracers , and 3-methyl-1,2,3-butanetricarboxylic acid (MBTCA)), and one β-caryophyllene-derived SOA (SOAC) tracer (β-caryophyllenic acid).A total of thirteen species of BSOA tracers and levoglucosan were determined , includi2 filter membrane was placed in the analyzer sample boat and the PM2.5 samples were heated sequentially to 140 °C (OC1), 280 °C (OC2), 480 °C (OC3), and 580 °C (OC4) in an oxygen-free pure helium (HE) environment. The temperature was gradually increased to 580 °C (EC1), 740 °C (EC2), and 840 °C (EC3) under the condition of He containing 2% oxygen. The limits of detection for OC and EC are 0.08 μg·m−3 and 0.06 μg·m−3, respectively.OC and EC were analyzed using a DRI Model 2015 Carbon Analyzer following the Interagency Monitoring of Protected Visual Environments (IMPROVE) thermal/optical reflectance (TOR) protocol. A 0.5026 cmInorganic ions were analyzed by ion chromatography . An aliquot of each sample filter was extracted with 30 mL of Milli-Q water using an ultrasonic bath three times and then filtered through PTFE filters to remove particles and filter debris, and finally identified using by ion chromatography.2.5 samples, and no obvious target compounds (less than 3%) were found in the field blanks. The recovery experiment was performed by spiking the standard solution onto blank filters and recoveries of the target compounds ranged from 85% to 120%. Method detection limits for 2-methylthreitols, 3-methyl-2,3,4-trihydroxy1-butene, 2-methylglyceric acid, 3-HGA, and β-caryophyllinic acid are 0.04, 0.05, 0.08, 0.05, and 0.12 ng m−3, respectively.Field blanks were extracted and measured in the same way as the PMis) in the PM2.5 samples were calculated using the ISORROPIA-II model. This model treats Na+-NH4+-K+-Ca2+-Mg2+-SO42−-NO3−-Cl− as a system [In this study, liquid water content (LWC) and particle in situ pH . Meteorological data were retrieved from NOAA air resources laboratory . These trajectories were run eight times every day  with the arrival level at 1000 m. The method of the trajectories used the GIS-based software TrajStat 1.5.4 [Air mass backward trajectories arriving at Mt. Huang were calculated using the Hybrid Single-Particle Lagrangian Integrated Trajectory Model  and SOAC tracers (2.1% and 1.9%). The night-time concentrations of levoglucosan, OC, EC, and SIA were also higher at night than during the day. Nearly all of the BSOA tracers exhibited higher concentrations at night than in the day. In addition, the cumulative effect of the dynamic transport of atmospheric pollutants through the mountain breeze at night was another crucial reason for the higher BSOA tracers. There was a positive correlation between BSOA tracers and O3 during the day rather than at night, indicating that BSOA tracers were primarily generated from the photo-oxidation of BVOCs with O3 only during the day. BSOA tracers were strongly correlated with pHis and SO42−, indicating that BSOAs were produced from the acid-catalyzed aqueous-phase oxidation. The robust correlation of levoglucosan with BSOA tracers, especially β-caryophyllinic acid, suggested that biomass burning from long-distance transport exerted a significant effect on BSOA formation.The diurnal variations in BSOA tracers derived from isoprene, monoterpene, and


Ismarus Haliday are rarely collected parasitoids in the small family Ismaridae. In this study, two new species are described from China’s Yunnan Province: Ismarusrobustus Chen & Yan, sp. nov. and Ismarusunisulcus Chen & Yan, sp. nov. An updated key to the Chinese species of the genus is provided.The genus  Ismarus Haliday belongs to the small parasitoid wasp family Ismaridae, with 59 described species worldwide .This work is based on the specimens collected by Malaise trap set in Gaoligongshan National Nature Reserve, Yunnan Province, China. All the studied specimens are deposited in the insect collection of theA1, A2, ... A12: antennomere 1, 2, … 12; POL: posterior ocellar line (shortest distance between posterior ocelli); OOL: ocular ocellar line (shortest distance between posterior ocellus and compound eye); T1, T2, ... T8: metasomal tergite 1, 2, ... 8. Morphological terminology otherwise follows Ismarus from China have been thoroughly reviewed and keyed , with irregular longitudinal costae dorsally; tergites smooth with scattered fine punctures; base of T2 with several short costae and short median furrow, extending 0.27× length of T2; sutures between tergites complete and deeply impressed.Male. Unknown.Named after the comparatively robust body of this species.China (Yunnan).Taxon classificationAnimaliaHymenopteraIsmaridaeChen & Yansp. nov.529E0C8A-7098-5A41-B50F-7A305447855Bhttps://zoobank.org/25B2BB9E-00E3-44AC-B9BB-CB6B5A7F1D60Holotype. China•1♀; Yunnan Province, Gaoligongshan National Nature Reserve, Dulong River, GLG13; 27°50'55.81"N, 98°28'3.15"E, 2824 m; 2–15 Jul. 2020; Lang Yi leg.; Malaise trap; SCBG 3044337. Paratype. China•1♀; same locality as holotype, but 2–16 Jul. 2020; Lang Yi leg.; Malaise trap; SCBG 3049369.I.halidayi Förster but can be distinguished by the following characters: A4 slightly longer than A3 ; median furrow of T2 very short, distinctly less than half the length of T2 ; radial cell of fore wing 0.75× as long as marginal vein .This species is most similar to Female. Body length 2.50–2.64 mm.Colour. Body black; A1–A9 brown to dark brown, remainder of antenna dark brown; fore and mid legs yellow-brown, with tarsi becoming darker distally, hind leg mostly dark brown with basal coxae somewhat dark and hind femur, trochanter and basal tibia yellow-brown; tegulae yellow-brown; wings hyaline, veins light brown to black-brown.Head. Head 2.0× as wide as long in dorsal view; vertex abruptly sloping behind ocelli in lateral view; POL as long as OOL; most of frons with scattered setae, except densely setose ventro-laterally; transverse facial carina slightly convex ventrally; A4 slightly longer than A3; A4 1.5× length of A5; A6–A14 with each segment less than 1.5× as long as wide; A15 approximately 2.0× longer than wide.Mesosoma. Dorsal pronotal area rugose-punctate and setose; lateral pronotal area rugose-punctate ventrally, smooth dorsally; mesoscutum smooth, shiny and convex, posterior margin with scattered long setae; mesoscutal suprahumeral sulcus present as a single anterior pit; mesoscutal humeral sulcus as long as tegula, deep and crenulate; mesoscutellum smooth and slightly convex, posterior rim rounded; anterior mesoscutellar pit large and deep, shorter than length of the mesoscutellar disc, sparsely punctate posteriorly, median keel weakly defined; mesopleuron smooth, with area below tegula rugulose; metapleuron rugose-punctate and covered with dense, long whitish setae.Wings. Radial cell closed, moderately large, 5.6× as long as wide and 0.94× as long as marginal vein.Legs. Fore and mid legs slender; hind tibia gradually swollen, its maximum width slightly wider than hind femur.Metasoma. Petiole slightly shorter than wide (8:9), with strong costae dorsally; tergites smooth with scattered fine punctures; base of T2 with two short costae and a short median furrow, extending 0.37× length of T2; sutures between tergites complete and deeply impressed.Male. Unknown.The name refers to the single furrow present on the base of T2.China (Yunnan).


However, current evidence suggests that Cd may induce nephron atrophy, resulting in a reduction in the estimated glomerular filtration rate (eGFR) below 60 mL/min/1.73 m2. Herein, these pathologies were investigated in relation to Cd exposure, smoking, diabetes, and hypertension. The data were collected from 448 residents of Cd-polluted and non-polluted regions of Thailand. The body burden of Cd, indicated by the mean Cd excretion (ECd), normalized to creatinine clearance (Ccr) as (ECd/Ccr) × 100 in women and men did not differ (3.21 vs. 3.12 µg/L filtrate). After adjustment of the confounding factors, the prevalence odds ratio (POR) for tubulopathy and a reduced eGFR were increased by 1.9-fold and 3.2-fold for every 10-fold rise in the Cd body burden. In women only, a dose–effect relationship was seen between β2M excretion (Eβ2M/Ccr) and ECd/Ccr . In men, Eβ2M/Ccr was associated with diabetes (β = 0.279). In both genders, the eGFR was inversely associated with Eβ2M/Ccr. The respective covariate-adjusted mean eGFR values were 16.5 and 12.3 mL/min/1.73 m2 lower in women and men who had severe tubulopathy ((Eβ2M/Ccr) × 100 ≥ 1000 µg/L filtrate). These findings indicate that women were particularly susceptible to the nephrotoxicity of Cd, and that the increment of Eβ2M/Ccr could be attributable mostly to Cd-induced impairment in the tubular reabsorption of the protein together with Cd-induced nephron loss, which is evident from an inverse relationship between Eβ2M/Ccr and the eGFR.The excretion of β The stuhttps://www.cdc.gov/diabetes/basics/getting-tested.html (accessed on 25 June 2023)) or a physician’s prescription of anti-diabetic medications. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg u) was based on the latex immunoagglutination method  or the ELISA method .Second morning urine samples were collected after an overnight fast, and whole blood samples were obtained within 3 h after the urine sampling. Aliquots of urine, whole blood, and plasma were stored at −20 or −80 °C prior to analysis. The assay for urine and plasma concentrations of creatinine ([cr]u) were determined using an atomic absorption spectrophotometer. Urine standard reference material No. 2670  or the reference urine metal control levels 1, 2, and 3  were used for quality control, analytical accuracy, and precision assurance. The limit of detection (LOD) of Cd quantitation was defined as 3 times the standard deviation of blank measurements. When [Cd]u was below its detection limit (0.1 µg/L), the Cd concentration assigned was the LOD divided by the square root of 2 u = urine creatinine concentration (mg/dL). The ratio [x]u/[cr]u was expressed in μg/g of creatinine.Ex was normalized to Ccr as Ex/Ccr = [x]u[cr]p/[cr]u, where x = Cd or β2M; [x]u = urine concentration of x (mass/volume); [cr]p = plasma creatinine concentration (mg/dL); and [cr]u = urine creatinine concentration (mg/dL). Ex/Ccr was expressed as the excretion of x per volume of filtrate  was evident in both women and men (Cd/Ccr) × 105] explained a larger proportion of the variation in the blood Cd concentrations (log([Cd]b × 103) in men (η2 = 0.407) than in women (η2 = 0.105) (A strong positive association between log([Cd] and men a. After = 0.105) b.Cd/Ccr) × 105], smoking, diabetes, and hypertension as independent variables. To further address the variables/factors that may influence the blood Cd levels, we conducted multiple regression and univariate analyses of variance that incorporated age, BMI, logb in women, while the interaction between diabetes and hypertension contributed to 1.6% of the [Cd]b variability. In men, higher [Cd]b values were strongly associated with higher ECd/Ccr (β = 0.581), and were moderately associated with smoking (β = 0.184) and not having diabetes (β = −0.246). ECd/Ccr, smoking, and diabetes accounted, respectively, for 42%%, 5.5%, and 9.5% of the variation of [Cd]b in men, while the interaction between smoking, diabetes, and hypertension contributed to 4.2% of the [Cd]b variability.In women, higher [Cd]2M using multiple linear regression and univariate/covariance analyses, where the indicators of Cd exposure ([Cd]b and ECd) were incorporated as the independent variables together with age, BMI, smoking, diabetes, and hypertension (We assessed the effects of Cd exposure on Eβrtension .β2M/Ccr was associated with age (β = 0.137), ECd/Ccr (β = 0.283), and diabetes (β = 0.323). In women, the associations of Eβ2M/Ccr with these three independent variables were evident. In men, Eβ2M/Ccr only showed a significant association with diabetes (β = 0.279).In all subjects, ECd/Ccr and Eβ2M/Ccr with the scatterplots and the covariate-adjusted mean Eβ2M in subjects grouped by ECd/Ccr tertiles . The Eβ2M in the subjects of the high ECd/Ccr tertile was higher compared with those of the middle and low ECd/Ccr tertiles .The relationship between Esubjects a, as welsubjects c. Howevesubjects b, a signtertiles b. In thetertiles d, a doseβ2M that incorporated age, BMI, log[(ECd/Ccr) × 105], gender, smoking, diabetes, and hypertension as independent variables.β2M/Ccr) × 100 ≥ 300–999 and ≥1000 µg/L filtrate were increased with age, log[(ECd/Ccr) × 105], and diabetes. All of the other four independent variables did not show a significant association with abnormal β2M excretion. For every 10-fold rise in ECd/Ccr, the POR for (Eβ2M/Ccr) × 100 of ≥300 and ≥1000 µg/L were increased by 1.94-fold and 3.34-fold, respectively.Among seven independent variables, the prevalence odds ratios (POR) for  via multiple linear regression and logistic regression analyses, where [Cd]rtension .Cd (β = −0.148), and diabetes (β = −0.109). In the subgroup analysis, inverse associations of the eGFR with these three independent variables  were seen only in women. In men, the eGFR was not associated with ECd, but this parameter showed inverse associations with age (β = −0.506) and hypertension (β = −0.212).In all subjects, the eGFR was inversely associated with age (β = −0.517), E2), age, BMI, log[(ECd/Ccr) × 105], gender, smoking, diabetes, and hypertension were incorporated as independent variables (In the logistic regression of a reduced eGFR (eGFR ≤ 60 mL/min/1.73 mariables .Cd/Ccr) × 105], and diabetes. For every 10-fold rise in ECd/Ccr, the POR for a reduced eGFR was increased by 3.2-fold. There was a 4.2-fold increase in the POR for a reduced eGFR among those with diabetes.The POR values for a reduced eGFR were increased with age, logb in men than in women (3.25 vs. 4.36 µg/L). In men only, the [Cd]b variation was associated with diabetes.[Cd]= 0.661) a, and th. 0.105) b. The va2, among Mae Sot residents was 16.1%, while the prevalence of tubulopathy, referred to as tubular proteinuria, was 36.1% , whereas one of five men had this abnormality.In this cohort, tubular proteinuria affected more than half of women (61.1%) and men (52.6%). One of four women had severe tubular impairment [(Eβ2M/Ccr showed a moderate positive association with age (β = 0.131), and an equally strong association with ECd/Ccr (β = 0.306) and diabetes (β = 0.349) . In the covariance analysis, the contribution of ECd/Ccr to the variability of Eβ2M/Ccr in women was demonstrable together with a dose–effect relationship after the adjustment of the covariates and interactions  and diabetes seen in both men and women is in line with the current knowledge that diabetes adversely affects both glomerular (GFR) and tubular function, termed diabetic tubulopathy [In women, E= 0.349) . In men,ractions d. In connificant d. An assulopathy ,19.2, and the overall prevalence of eGFR ≤ 60 mL/min/1.73 m2 in this cohort was 6.9% (p = 0.097), nor did the difference in the mean eGFR in women and men (p = 0.145). The weaker effect of Cd exposure on the eGFR in men, compared to women, remains to be confirmed with a sufficiently large sample group of men. However, the regression analysis also indicated gender differences in susceptibility to the nephrotoxicity of Cd (Cd/Ccr (β = −0.185), and diabetes (β = −0.128). In comparison, the eGFR in men was not associated with ECd/Ccr, while showing an inverse association with age (β = −0.506) and hypertension (β = −0.212). Adverse effects of hypertension and diabetes on the eGFR have been noted in a cross-sectional study of the general U.S. population, where a Cd-induced GFR reduction was more pronounced in those who had diabetes and/or hypertension [The overall mean eGFR was 90 mL/min/1.73 mwas 6.9% . The difty of Cd . In womertension .We speculate that gender differences in the levels of some protective factors, notably body status of nutritionally essential metals such as iron and zinc, may contribute to the increased susceptibility to Cd nephrotoxicity that was seen in women. Similarly, environmental Cd exposure has been linked to a reduction in the eGFR among participants in various cycles of the U.S. National Health and Nutrition Examination Survey (NHANES) undertaken over 18 years 1999 to 2016) [16 20,2121,22. Li2M with the molecular weight of 11,800 Da is filtered freely by the glomeruli and is reabsorbed almost completely by the kidney’s tubular epithelial cells [2M will result in an enhanced excretion rate of β2M [2M for the following reasons [2M per nephron remains constant, its excretion will vary directly with its production. If the production and reabsorption per nephron remain constant as nephrons are lost, the excretion of β2M will rise [The protein βal cells . Thus, te of β2M ,24,25,26 reasons ,26. Whenill rise .2M will increase when the GFR falls for any causes. Indeed, Eβ2M/Ccr was inversely associated with the eGFR in both women and men . In both women and men, the eGFR was the lowest in those who had (Eβ2M/Ccr) × 100 ≥ 1000 µg/L filtrate, indicative of severely impaired tubular function.It can thus be expected that the excretion of β and men c, althou and men d, the η2β2M/Ccr) × 100 of 300–999 and ≥1000 µg/L filtrate, respectively (β2M/Ccr) × 100 < 300 µg/L filtrate. A substantial loss of nephron function was a likely cause of the massive increases in Eβ2M/Ccr that was seen in those who had an eGFR below 60 mL/min/1.73 m2.Notably, the POR for a reduced eGFR was increased by 8.3-fold and 33.7-fold in those with  × 100 of 300–999 and ≥1000 µg/L filtrate compared to those with (Eβ2M/Ccr) × 100 < 300 µg/L filtrate.The Cen of Cd . The daten of Cd  show thacr-normalized data indicated that the mean ECd/Ecr in women was statistically higher than that of men (4.26 vs. 3.30 µg/g creatinine). They also indicated that the difference in % of women across the three tubulopathy categories was minuscule, and that the % distribution of men across the tubulopathy categories was statistically significant. These data suggest an association of Cd exposure and the severity of tubulopathy in men only. The logistic regression data  over a five-year period [β2M/Ecr above 300 μg/g creatinine does not reflect an early warning sign of the nephrotoxicity of Cd. The utility of this Eβ2M/Ecr value as a toxicity criterion to derive a toxicity threshold level for Cd is inappropriate.Previously, Eced eGFR ,29. Simir period . Thus, aCd and Eβ2M to Ecr produces an erroneous interpretation of the effect of Cd exposure on the eGFR, while underestimating the severity of Cd-induced tubulopathy, especially among women. These data call into question the utility of Eβ2M/Ecr of 300 µg/g creatinine to represent the critical effect of exposure to Cd in the human diet. New health guidance values need to be established for this toxic metal, and new public measures are needed to minimize the Cd contamination of food chains.In summary, adjusting EIn this cohort, the levels of environmental exposure among the participants were assessed by measuring the blood Cd and urinary Cd excretion rates. Strong correlations between these two parameters were seen in both men and women . Both thn = 84) and low-exposure (n = 30) locations is a limitation. This precludes an analysis of both genders separately from both locations that may help to rule out any other environmental effects on adverse kidney outcomes in women. In addition, the heterogeneity in the hormonal status, notably estrogen, in female participants who are menopausal and post-menopausal is a limitation [The small number of males from high- (mitation .2M above 300 µg/g creatinine (≈2–3 µg/L filtrate) and a reduction in the glomerular function, indicated by an eGFR below 60 mL/min/1.73 m2, are the manifestations of severe kidney toxicities due to chronic exposure to Cd that are more prevalent and more severe in women than men of the same body burden.The excretion of β


Scedosporium and Lomentospora are a group of filamentous fungi with some clinically relevant species causing either localized, invasive, or disseminated infections. Understanding how the host immune response is activated and how fungi interact with the host is crucial for a better management of the infection. In this context, an α-glucan has already been described in S. boydii, which plays a role in the inflammatory response. In the present study, an α-glucan has been characterized in L. prolificans and was shown to be exposed on the fungal surface. The α-glucan is recognized by peritoneal macrophages and induces oxidative burst in activated phagocytes. Its recognition by macrophages is mediated by receptors that include Dectin-1 and Mincle, but not TLR2 and TLR4. These results contribute to the understanding of how Scedosporium’s and Lomentospora’s physiopathologies are developed in patients suffering with scedosporiosis and lomentosporiosis. Scedosporium and Lomentospora are a group of filamentous fungi comprising some clinically relevant species, such as Scedosporium boydii, Scedosporium apiospermum, Scedosporium aurantiacum, and Lomentospora prolificans [Scedosporium prolificans but was recently reclassified [L. prolificans is one of the most virulent species of the Scedosporium and Lomentospora group and has therefore been extensively studied [lificans . The latassified . L. prol studied ,4.Scedosporium and Lomentospora cause a wide spectrum of diseases in humans. In immunocompetent people, they usually cause a localized infection called mycetoma, resulting from a traumatic inoculation of fungal cells. On the other hand, in immunocompromised patients, including those with HIV/AIDS, organ transplant recipients, and people who suffer of hematological diseases, an invasive and disseminated infection might occur, which is hard to treat and presents high mortality rates [ty rates ,3. Scedosporium and Lomentospora infections and to avoid their dissemination [Scedosporium and Lomentospora species [S. boydii, which plays a role in inducing the release of pro-inflammatory cytokines by macrophages and dendritic cells, such as TNF-α and IL-12 [S. boydii involves Toll-like receptor 2, CD14, and MyD88 [Activation of the innate immune response is a key step for the clearance of mination . Underst species . A glycond IL-12 . The recnd MyD88 . Scedosporium and Lomentospora species, little is known about α-glucan structures and the biological roles in species other than S. boydii. In the present work, we describe the structural characterization of an α-glucan from L. prolificans and evaluate its role in the induction of innate immune response.However, although several glycoconjugates have already been described in Lomentospora prolificans FMR3569 was kindly provided by Dr. J. Guarro from Unitat de Microbiologia, Facultat de Medicina and Institut d’Estudis Avançats, Réus, Spain. Fungal stocks were maintained in solid modified Sabouraud (m-SAB) . Cells were inoculated in 200 mL liquid m-SAB and kept for seven days at 25 °C under agitation, after which the mycelium was filtered and washed with distilled water. Conidia were grown on Petri plates containing m-SAB at 25 °C. After 7 days, conidia were obtained after washing the plate surface with sterile phosphate-buffered saline (PBS) by scraping. After filtration, the solution was centrifuged to obtain a pellet containing conidia. Cells were counted using a Neubauer chamber.L. prolificans mycelia as described previously [Polysaccharides were isolated from eviously . Brieflyeviously . The pureviously . 2O . Nuclear magnetic resonance  was performed for polysaccharide fractions (20 mg/0.5 mL D2O) using triple resonance probe . 1H NMR 1D spectra were recorded using 250 scans, with a 1.5 second inter-scan delay at 40 °C. 1H-13C HSQC (heteronuclear single quantum coherence) spectra were collected using time proportion phase incrementation (TPPI) for quadrature detection in indirect dimension. TOCSY was run with 4046 × 400 points with a spinlock field of 10 kHz and a mixing time of 80 milliseconds. Chemical shifts were displayed relative to external trimethyl-silylpropionic acid at 0 ppm for 1H and relative to methanol at 48 ppm for 13C [α-Glucan spectra were obtained using a 400 MHz spectrometer DRX AVANCE (Bruker). The 13C-NMR 100.6 MHz) spectra were obtained using a solution of α-glucan in 99.7% D0.6 MHz sImmune sera were obtained according to Haido and colleagues . BrieflyIn all experiments female Balb/C mice, 6–12 weeks old, were used. All experiments were performed according to Institutional Committee for Animal Care and Experimentation of the Federal University of Rio de Janeiro (UFRJ), Protocol 027/22/CEUA-UFRJ.2. After addition of a solution of 0.01%, neutral red cells were incubated at 37 °C for 3 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde for 10 min. Then cells were lysed in a solution of 1% acetic acid and 50% methanol. The number of neutral red cells was determined by spectrophotometer at 492 nm [Mice peritoneal macrophages were added to 96-well plates and incubated in the presence of 3.1, 6.2, 12.5, 25, 50, 100, and 200 µg/m of α-glucan for 24 h at 37 °C with 0.5% COt 492 nm .The treatment with α-amyloglucosidase will remove α-glucan by cleaving off the →4)- αGlc (1→ units.8) were incubated with 6 UI of α-amyloglucosidase in 120 mM acetate buffer, pH 4.2, for 4 h at 56 °C. Enzymatic digestion was stopped by heating the material at 100 °C for 5 min, after which the conidia were thoroughly washed with pyrogenic saline.Conidia treatment: Conidia (1 × 10Glucan treatment: Glucan (1 mg/mL) was incubated with 6 UI of α-amyloglucosidase in 120 mM acetate buffer, pH 4.2, for 2 h at 56 °C. Then, the mixture was heated at 100 °C for 5 min, to inactivate the enzyme, and centrifuged . The supernatant was concentrated and applied to a HPTLC plate under the same conditions described above .Lomentospora prolificans α-glucan (25 ng/100 µL/well) or 5 × 105 conidia were added into wells of flat-bottomed polyvinyl microtiter plates , which were maintained for 1 h at 37 °C and overnight at 4 °C. After washing the plates with 0.05% PBS–Tween 20, the nonspecific sites were blocked by addition of 5% skim milk in 0.1% PBS–Tween 20. Serial dilution of rabbit immune sera against whole L. prolificans cells in blocking buffer (100 μL) were added to the wells (dilutions 1/100–1/3200) and incubated for 1 h at 37 °C. Then, antibody binding was measured using goat anti-rabbit IgG antibodies conjugate to horseradish peroxidase  at 1/2000 in blocking buffer with incubation for 1 h at 37 °C. Furthermore, O-phenylenediamine was added together with H2O2 as chromogen. The enzyme reaction was followed by measuring the absorbance at 490 nm using an automated reader . For inhibition ELISA test, α-glucan at 25, 50 and 100 µg/mL were separately mixed with rabbit immune sera against whole L. prolificans cells (dilution 1/400) in blocking buffer and pre-incubated for 1 h at 37 °C before the incubation with L. prolificans conidia coated onto wells. The inhibition ELISA test was performed as described above [ed above ,16.6) were treated or not (control) with 6 UI of α-amyloglucosidase for 4 h at 56 °C. Cells were then fixed with 4% paraformaldehyde, washed with PBS, and incubated with 3% bovine albumin for 1 h. Furthermore, samples were incubated with immune sera (1:400) for 1 h. For untreated conidia immune sera were incubated for 30 min with 0, 25, and 50 µg/mL of α-glucan prior to its reaction with conidia. Samples were analyzed using a flow cytometer FACS Calibur . Data were evaluated using “Windows Multiple Document Interface Flow Cytometry Application (WinMDI) 2.8 version software” [Conidia (5 × 10oftware” .5/well) were plated suspended in RPMI 1640 supplemented with fetal bovine sera (FBS) in 24-well microplates and incubated at 37 °C under a 0.5% CO2 atmosphere for 24 h. For the interaction of L. prolificans cells and macrophages, conidia (5:1 ratio) were added to macrophage monolayers and kept for 2 h at 37 °C under 0.5% CO2. The wells were then washed with PBS and the cells were fixed with Bouin solution and 70% ethanol. After staining with Giemsa, the phagocytic index was determined using an optical microscope [A solution of 3% thioglycolate (1 mL) was injected into the peritoneum of Balb/C mice to recruit macrophages. Three days after injection, macrophages were collected from the peritoneal cavity with cold RPMI 1640, centrifuged, and counted using a Neubauer chamber. To obtain monolayers, macrophages  was determined by counting a total of 200 macrophages and using the following equation: PI = percentage of macrophages that phagocyted at least one conidium × media of internalized conidia ,13.L. prolificans conidia also treated or not with α-amyloglucosidase at 37 °C with 0.5% CO2. Zymosan was used as a positive control. After 2 h and 18 h of incubation, the supernatant was collected from the wells for the measurement of superoxide and nitric oxide, respectively. Nitric oxide was evaluated by the Griess method reading the absorbance at 540 nm [Monolayers of macrophages were obtained as described above. Macrophages were incubated with 200 µg/mL of α-glucan treated or not with α-amyloglucosidase and t 540 nm , and sup2 for 18 h with α-glucan from L. prolificans  or S. boydii (200 µg/mL) and L. prolificans conidia (5:1 ratio), treated or not with α-amyloglucosidase. An LPS  was used as positive control. After incubation, TNF-α concentration was determined by using cytokines kit  according to the manufacturer’s instructions.Monolayers of macrophages were obtained as described above. Polymyxin B (10 μg/mL) was added 5 min prior to the stimulation to avoid LPS contamination. Macrophages were then incubated at 37 °C under 0.5% CO5 or 2 × 105 B3Z or BWZ cells/well were incubated for 18 h with 240 ng of L. prolificans or S. boydii α-glucan. β-galactosidase (lacZ) activity was measured in total cell lysates using CPRG  as substrate. OD at 560 nm was measured using 620 nm as reference. Curdlan (5 ug/well), Bl-Eng2 glycoprotein (30 ng/mL), and trehalose-6,6-dibehenate (TDB) (25 ng/mL or 5 ug/well) were used as positive controls for Dectin-1, Dectin-2, and Dectin-3 or Mincle, respectively.BWZ and B3Z reporter cells expressing Dectin-1, Dectin-2, Dectin-3, or Mincle have been described previously . In a 96TM hTLR2 and HEK-BlueTM hTLR4 cells  were maintained in growth medium  with 1× HEK-BlueTM Selection (InvivoGen). The parental cell lines HEK-BlueTM Null1 and Null2 (InvivoGen) were used as controls for HEK-BlueTM hTRL2 and HEK-BlueTM hTLR4, respectively. Only cells with 20 passages or less were used. About 5 × 104 cells/well were incubated with 0.5 µg/mL L. prolificans or S. boydii α-glucan for 18 h at 37 °C; 25 ng/mL Pam3CSK4 (TLR2 agonist) and 10 ng/mL LPS (TLR4 agonist) were used as control. After incubation, 20 µL of cell culture supernatant were incubated with 180 µL of QUANTI-BlueTM solution (InvivoGen) at 37 °C for 4 h. Alkaline phosphatase activity (the reporter gene for HEK-BlueTM hTLR4 and hTLR2) was measured by optical density (OD) measurement at 620–655 nm using a microplate reader. Polymyxin B, a cyclic polypeptide antibiotic, was used to eliminate the effects of endotoxin contamination in some experiments with HEK-BlueTM hTLR4 cells.HEK-Bluet-test was used for two-group comparisons. The 90% or 95% confidence interval was determined in all experiments.All experiments were performed in triplicate, in three independent experimental sets. Statistical analyses were performed using GraphPad Prism version 5.00 for Windows . One-way ANOVA was used for multiple comparisons using Turkey’s test (Turkey post-test), and a L. prolificans were extracted with a 2% KOH solution at 100 °C followed by neutralization with acetic acid and precipitation with ethanol. The crude precipitate was applied to a Superdex 200 column which was eluted with a discontinuous gradient of aqueous NaCl. Two carbohydrate-containing fractions, FA and FB, were obtained . The signals between 4.10 and 3.20 ppm were assigned to ring protons (H2 to H5) and carbon six (H6). In structural studies of complex polysaccharides, 1D 1H NMR show several signal overlaps. Using 2D 1H-13C Heteronuclear Single Quantum Coherence Spectroscopy - αGlc (1→ (Unit A)  C. S. boydii [L. prolificans  were used. Consistent with this, flow cytometry showed that serum reactivity with intact conidia was reduced when serum was pre-incubated with soluble α-glucan  led to a dose-dependent inhibition of 20–40% of conidia phagocytosis  and measured the superoxide and nitric oxide (NO) in the supernatant. Intact conidia were able to induce superoxide and NO production as much as zymosan, whereas conidia treated with amyloglucosidase showed a significant decrease in superoxide and NO production  or purified α-glucan, and the supernatant was tested for TNF-α production. L. prolificans conidia were able to stimulate TNF-α production, but there was no decrease in TNF-α release when cells were stimulated with conidia treated with amyloglucosidase -linked glucopyranoside, and that the presence of α-(1→6)-linked glucopyranoside side chains in S. boydii α-glucan hinder the recognition by Dectin-1.In addition to TLR recognition, C-type lectin receptors (CLRs) are crucial to guarantee a proper immune response against fungal infection . To furtDectin-3 . HoweverScedosporium and Lomentospora species, α-glucan and rhamnomannan had their structures characterized and their functions as activators of innate immunity identified [Several studies have demonstrated the importance of polysaccharides in the fungal cell, not only contributing to the architecture and integrity of the cell wall, but also as immunologically active components with great potential as regulators of pathogenesis and the host immune response. Among the fungal polysaccharides in entified ,22.Histoplasma capsulatum masks the recognition of β-(1→3)-glucan by innate immune receptor Dectin-1, which can induce the production of pro-inflammatory cytokines by macrophages [Aspergillus fumigatus α-(1→3)-glucan is exposed during conidia germination and induces the aggregation of their germinating conidia [L. prolificans to be a linear polysaccharide of (1→4)-linked glucopyranose units, based on a combination of several techniques, including HPTLC chromatography, 1D RMN 1H, and (B) 2D 1H-13C HSQC. These results confirmed that, differently from glycogen-like glucans of S. boydii [A. fumigatus [Mycobacterium bovis [L. prolificans α-glucan is a linear polysaccharide.α-Glucans have been isolated from a variety of fungal pathogens, and their chemical structures have been characterized. The α-(1→3)-glucan of rophages . Aspergi conidia . In this. boydii , A. fumiumigatus , Mycobacum bovis , and rabum bovis , purifieL. prolificans conidia with immune serum against whole fungal cells showed that α-glucan is accessible on the conidia surface to bind with antibodies, thus suggesting that conidia interactions with host cells may be mediated by α-glucan.The reduction in reactivity after α-amyloglucosidase treatment of L. prolificans conidia by macrophages relies partially on the recognition of α-glucan. In agreement with these findings, α-glucan from S. boydii inhibited conidia phagocytosis by macrophages in a dose-dependent manner. However, the phagocytic receptor involved in the recognition of S. boydii α-glucan was not investigated [The first line of defense against invading microorganisms is the innate immune system, based on the recognition of PAMPs present in pathogens by phagocytic cells, which are important in control the growth and spread of fungi ,29. Herestigated .2-) necessary for the death of microorganisms by producing protein modifications, nucleic acid breaks, and lipid peroxidation [L. prolificans conidia induced an oxidative response, with a high secretion of H2O2, but more importantly, soluble α-glucan from L. prolificans also induced the release of a large amount of H2O2. However, conidia treated with amyloglucosidase—which removes all the α-glucan from the cell surface—are still capable of inducing a robust H2O2 production, indicating that L. prolificans conidia have other molecules on their surface that activate this microbicidal pathway. Previous results showed that other molecules on the surface of L. prolificans, such as PRM or GlcCer [The recognition of PAMPs by macrophages and neutrophils leads not only to the internalization of the pathogen, but also to the production of microbicidal compounds that damage the invading microorganism. Macrophages and neutrophils produce reactive oxygen species (ROS) through the activation of the NADPH-oxidase complex (NOX), which generates the superoxide anion -glucan is not able to trigger TLR2 intracellular signaling. Members of the C-type lectin receptor (CLR) family are predominant fungal-sensing PRRs that elicit several innate immune responses as well as shape adaptive immunity. Mincle is a C-type lectin receptor (CLR) capable of recognizing mycobacterial cord factor  and its analog trehalose dibehenate, but not mycolate or trehalose alone. A bipolar glycolipid with disaccharide composed of glucose or mannose attached to fatty acids was supposed to represent a potential minimal ligand structure for Mincle [Pneumocystis carinii are recognized by Mincle through its short mannosylated chains [Malassezia furfur [O-acyl-α-D-mannosides in C. albicans [L. prolificans and S. boydii were recognized by Mincle. Deficiency in Mincle correlates with a delayed engulfment of fungal cells and impacts the phagocytosis dynamics [α-Glucans consisting of highly α-(1→6)-branched α-(1→4)-glucan, glycogen-like structures have been reported to possess immunomodulatory activity related to recognition and signaling by TLR2 ,38. Herer Mincle ,40. The d chains , as wella furfur  and steralbicans . In contdynamics , so by mdynamics ,46.L. prolificans α-glucan, but the same was not observed for S. boydii glycogen-like α-glucan. Another glycogen-like highly branched α-glucan obtained from maitake mushroom (Grifola frondosa), however, has been shown to induce DC maturation by a Dectin-1-dependent pathway and not by TLR2, and Mincle, showing different binding properties from S. boydii α-glucan [L. prolificans and S. boydii. Little is known about whether α-glucans can also modulate host defense by mechanisms mediated by dectin-1, the fungal β-glucan receptor. Interestingly, in our study, Dectin-1 recognized α-glucan . It has α-glucan ,48 whichL. prolificans and S. boydii α-glucans, respectively, may influence the distinct immunostimulatory effects of these polysaccharides. The importance of co-stimulation of Mincle and TLR2 in TNF-α production and clearance of Fonsecaea pedrosoi was reported in [L. prolificans and S. boydii α-glucans contributes to their different TNF-α stimulatory capacity.It is noteworthy that cooperative interaction between Mincle and Dectin-1 or Mincle and TLR2 for the recognition of orted in , but nevorted in . PresumaL. prolificans phagocytosis and recognized by Dectin-1 and Mincle. We attempted to show that, probably due to the absence of branching, α-glucan from L. prolificans and that from the related species, S. boydii, are differently sensed by PRRs. In conclusion, we described an α-(1→4)-glucan involved in


Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism. We used IPEC-J2 cells as a model to assess the inhibitory effect of LP-1S on PEDV and to further investigate the relationship between LP-1S, Ca2+, and PEDV. The results showed that a divalent cation chelating agent (EGTA) and calcium channel inhibitors (Bepridil hydrochloride and BAPTA-acetoxymethylate) could inhibit PEDV proliferation while effectively reducing the intracellular Ca2+ concentration. Furthermore, LP-1S could reduce PEDV-induced loss of calcium channel proteins (TRPV6 and PMCA1b), alleviate intracellular Ca2+ accumulation caused by PEDV infection, and promote the balance of intra- and extracellular Ca2+ concentrations, thereby inhibiting PEDV proliferation. In summary, we found that LP-1S has potential therapeutic value against PEDV, which is realized by modulating Ca2+. This provides a potential new drug to treat PEDV infection.Porcine epidemic diarrhea virus (PEDV) mainly invades the small intestine and promotes an inflammatory response, eventually leading to severe diarrhea, vomiting, dehydration, and even death of piglets, which seriously threatens the economic development of pig farming. In recent years, researchers have found that probiotics can improve the intestinal microenvironment and reduce diarrhea. At the same time, certain probiotics have been shown to have antiviral effects; however, their mechanisms are different. Herein, we aimed to investigate the inhibitory effect of  The vir2+ acts as an important second messenger in intracellular signal transduction processes. Ca2+ is essential for the transmembrane transmission of biological signals, the regulation of glandular secretion, and the maintenance of acid-base homeostasis in the body ; (ii) binding to proteins with high Ca2+ affinity to form calcium-binding protein-D9k (CaBPD9k), which in turn moves from the BBM to the basolateral membrane (BLM); and (iii) extracellular excretion via the plasma membrane Ca2+-ATPase (PMCA1b/Ca2+ pump) and Na+/Ca2+ exchanger (NCX1). Among them, TRPV6 and PMCA1b, as calcium channel proteins, have received much research attention because they are the major Ca2+ transport proteins in the intestinal epithelium and have important roles in maintaining intra- and extracellular homeostasis and controlling ions entering and leaving the cell  Crothe cell .Lactiplantibacillus plantarum (Lp) significantly reduced the cytopathic effects of respiratory syncytial virus (RSV), influenza B virus, and HCoV-229E coronavirus . Its main metabolites were found to include acids (33.96%), amino acids (and their derivatives) (32.08%), and polysaccharides (15.1%) through the pristine analysis using gas chromatography–mass spectrometry (GC–MS) (Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism.In recent years, research has shown that a variety of probiotics can maintain microbial homeostasis in the intestinal environment and prevent diarrhea . Antivironavirus . Lp is a (GC–MS)  LP, as a2.2.1.2 cell incubator. PEDV-LJX strain and PEDV N mouse-derived monoclonal antibodies were kindly donated by GuangLiang Liu, Researcher, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Lactiplantibacillus plantarum LP-1 was obtained by pre-laboratory isolation. TRPV6 polyclonal rabbit antibodies , horseradish peroxidase (HRP)-goat anti-mouse IgG (H + L)  and β-actin goat anti-rabbit antibody  were purchased from Proteintech. PMCA1b Polyclonal Rabbit Antibodies  were purchased from Abcam . Fura-2-acetoxymethyl ester (Fura-2 AM) and CaCl2 were purchased from Beyotime Biotechnology Co. . EGTA, Fluo3-AM, and BAPTA-AM were purchased from Solarbio . Bepridil hydrochloride (BP) was purchased from MCE . Ca2+ standard solution  was purchased from National Research Center for Reference Materials, Beijing, China.African green monkey kidney cells  and porcine small intestinal epithelial cells  were purchased from Suer Biotech  and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% fetal bovine serum, at 37°C, in a 5% CO2.2.Lactiplantibacillus plantarum LP-1 was inoculated onto MRS solid medium and incubated at 37°C. After 24 h, single colonies were picked, inoculated in MRS liquid medium, incubated at 37°C with shaking 180 rpm for 12 h until they reached a measured OD595 value between 0.8 ~ 0.9, and centrifuged at 3,000 × g for 10 min. The supernatant was retained and filtered using a 0.44 μm–0.22 μm microporous filter membrane gradient. The filtered supernatant (LP-S1) was stored at −20°C for a short time.2.3.6 cells per well. A positive control group (PEDV-infected group) and a negative control were set, and the adsorption culture was replaced with DMEM maintenance solution after 90 min. When the cytopathic effect (CPE) of the PEDV-infected group reached 70%, the maintenance solution was discarded, the wells were washed three times with 0.01 M phosphate-buffered saline (PBS), 5 mg/mL MTT reagent was added to each well, the plate was incubated at 37°C for 4 h to allow the formation of formazan crystals, and the OD575 values were then measured. Cell viability was calculated, and the IC50 of LP-S1 was calculated: lgIC50 = Xm-I (P- (3-Pm-Pn)/4) .Firstly, LP-1S was diluted with DMEM basal medium to 0, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 times the original concentration. Different dilutions of LP-1S were added to each well of a microtiter plate, containing 3.6 × 102.4.50 of each PEDV group was measured according to the Reed-Muench method , which were blocked in 5% skim milk for 1.5 h, followed by incubation with the corresponding primary antibodies overnight at 4°C. Next day, the membranes were incubated with the corresponding secondary antibodies for 90 min at 37°C. The immunoreactive protein bands were visualized using the FX5 imaging system , and the grayscale values were analyzed.2.6.2.6.1.Total RNA was extracted from cells and reverse transcribed to cDNA using RNAiso plus  and 5 × PrimeScript RT Master Mix .2.6.2.TRPV6, PMCA1b, and ACTB (β-actin genes) were downloaded from GenBank. Primers were designed against the sequences using Primer Premier 5.0 software . The primer sequences are shown in −ΔΔCt method was used to analyze the expression of the corresponding mRNAs (The full sequences of ng mRNAs .2.6.3.PEDV M gene.The PEDV-M primer sequences were downloaded from GenBank . The PED2.6.4.The shRNA sequence was designed and synthesized by Wuhan Jinkairui Biotechnology Co.  and then2.7.6 cells per well) and incubated for 60 min at 37°C with 5% CO2. The wells were washed using 0.1 M HEPES  buffered saline three times, added with HBSS containing 1% fetal bovine serum, and incubated for 30 min. Changes in intracellular calcium ion content were observed using fluorescence microscopy.Fluo3-AM was diluted to a 2 mM Fluo3-AM stock solution using dimethyl sulfoxide and stored at −20°C for later use. The preserved Fluo3-AM stock solution was diluted with 0.1 M Hanks’ Balanced Salt Solution , added to cells in a microtiter plate (3.6 × 102.8.3 + 2 mg/mL K+). Then, the standard concentration was measured and the standard curve was plotted. Finally, the collected cell supernatant and intracellular fluid were diluted 5,000 times with medium solution and then assayed on the flame atomic absorption spectrophotometer. The absorbance and concentration values of each sample were recorded. Changes in extracellular calcium ion content were then analyzed with reference to the standard curve.0.25 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL of a Ca standard solution (10 μg/mL) were added to a 50 mL volumetric flask and added with 50 mL with medium solution  using the statistical software GraphPad Prism 8.0  and IBM SPSS Statistics 23 to determine statistical differences between multiple groups. A p-value > 0.05 (ns) indicates a non-significant difference, a p-value < 0.05 indicates a significant difference (*), a p-value < 0.01 expresses a highly significant difference (**), and a p-value < 0.001 expresses a very significant difference (***).All results were subjected toa 3.3.1.2+ concentrations is unknown. We used the calcium ion fluorescence probe Fluo-3 AM to detect the intracellular Ca2+ concentration under PEDV infection, which showed that the intracellular Ca2+ fluorescence intensity after 2 h, 8 h, 24 h, and 48 h of PEDV infection was extremely significantly higher than that in the negative control group, under fluorescence microscopy  compared with that in the negative control group, in which the calcium ion concentration in the extracellular fluid was significantly lower in the 2 h, 8 h, and 24 h infection groups compared with that in the negative control group  and the CaTT assay . IPEC-J2TT assay ; the PEDTT assay . Thus, BTT assay ; the PEDTT assay .3.5.2+ transmembrane transport mainly involves membrane transporter proteins, carrier proteins, and channel proteins. To further investigate the role played by calcium channel proteins during PEDV infection, we selected two typical Ca2+ transport channels, TRPV6 and PMCA1b, as the subjects of our study. Firstly, we constructed vectors for RNA interference of TRPV6 and PMCA1b (shTRPV6/shPMCA1b) separately to determine the association between TRPV6/PMCA1b and PEDV. The results showed that both interfering vectors were stably expressed in IPEC-J2 cells and significantly reduced the protein levels of TRPV6 and PMCA1b  at 12 h , calcium channel inhibitors (BP and BAPTA-AM), and calcium channel proteins (TRPV6 and PMCA1b). Pretreatment with 2 mM EGTA significantly inhibited PEDV M expression, and PEDV expression M was significantly increased when the cells were supplemented with Ca2+, suggesting that EGTA effectively reduced the inward flow of extracellular free Ca2+ by chelating it, thereby inhibiting PEDV proliferation. In contrast, CaCl2 supplementation induced an increase in intracellular Ca2+ flow, leading to an increase in PEDV expression , among which PMCA1b was more sensitive to PEDV infection than TRPV6. LP-1S might precisely enhance the regulation of Ca2+ by PMCA1b and TRPV6 to reduce the intracellular Ca2+ concentration and maintain an intracellular low calcium state, thus exerting an anti-PEDV effect  can be found in the article/supplementary material.Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.ZK, SZ, and GL: conceptualization. ZK: methodology. ZN: software. ZK, SZ, and ZN: validation. ZSu and XL: formal analysis. XH: investigation. ZSo: resources, supervision, project administration, and funding acquisition. YZ: data curation. SZ: writing—original draft preparation. ZN: writing—review and editing. SX, JZ, and HZ: visualization. All authors contributed to the article and approved the submitted version.