Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain  C. unicolor strain CGMCC 5.1011 produced 122 U/mL laccase.The strain contained 12 expressed laccase isozymes.A novel, thermostable laccase, Lac2, was purified and characterized.Lac2 decolorized malachite green at 50 and 70 ºC whereas another laccase failed.Laccases (EC 1.10.3.2) are copper-containing oxidases catalyzing oxidation of phenolic/non-phenolic lignin-related compounds and recalcitrant environmental pollutants Baldrian . BecauseCerrena genus is not as intensively studied as Trametes, Cerrena species have gained attention as laccase producers  despite incomplete decolorization by Lac7 from Cerrena sp. HYB07 due to heat inactivation. The research presented herein provided a novel laccase with thermotolerance and thermostability that would be desirable for industrial applications.Laccases are widespread in nature; they are found in microorganisms, plants and animals, and white-rot fungi are considered the most efficient laccase producers  at 4 ºC. The fermentation medium for strain 5.1011 contained (g/L): KH2PO4 6 g, MgSO4·7H2O 4.14 g, CaCl2 0.3 g, NaCl 0.18 g, CuSO4·5H2O 0.0625 g, ZnSO4·7H2O 0.018 g, VB1 0.15 g and respective carbon and nitrogen sources. The following carbon sources were tested at 2.0% (w/v): Mannitol, glycerol, glucose, lactose, sucrose, cellulose, maltodextrin, corn dextrin, β-dextrin, soluble starch, corn starch, in combination with 1.5% (w/v) peptone. Next, the following nitrogen source were tested at the level of 1.5% (w/v): ammonium nitrate, ammonium tartrate, peptone, yeast extract, beef extract, ammonium sulphate, and soybean cake powder. The concentrations of the nutrient sources were also optimized. Fermentation of Cerrena sp. strain HYB07 was carried out as described . Transcriptomics analysis was performed by Novogene . Briefly, after RNA quality check, 3 µg RNA was used as input material. Sequencing libraries were generated using NEBNext Ultr RNA Library Prep Kit for Illumina . After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated; then data analysis, gene function was annotated based on the following databases: Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO.Mycelia were collected from 6-d-old E. coli TOP10 competent cells . Four clones of each PCR product were randomly selected and submitted to sequencing analysis.DNA was extracted with E.Z.N.A. HP Fungal DNA Kit . TransScript One-Step Removal and cDNA Synthesis SuperMix  was used to synthesize the first strands of cDNA. PCR was carried out with 2 × EasyTaq PCR SuperMix . Thermal asymmetric interlaced PCR (TAIL-PCR) was used to clone the flanking sequences of incomplete laccase genes. Primers used are listed in Supplementary Table S1. PCR products were inserted into pMD18-T vector , and the recombinant vectors were transformed into N-glycosylation sites (Asn-X-Ser/Thr) were identified with ScanProsite , guaiacol  or catechol (ε = 1260 M−1 cm−1) as the substrate by following absorbance change at 420, 470 and 400 nm, respectively. One unit of enzyme activity was defined as the amount of enzyme needed to oxidize 1 μmol substrate in 1 min. All measurements were carried out in triplicate.Laccase activity was assayed with ABTS 2SO4 was collected by centrifugation , resuspended in buffer A  and dialyzed in buffer A. The dialyzed crude enzyme solution was applied at 5 mL/min to a HiTrap DEAE column pre-equilibrated with buffer A. Adsorbed proteins were eluted with 0.15 NaCl in buffer A. The purified protein was analyzed by SDS-PAGE for homogeneity and stained with Coomassie Brilliant Blue R-250. Deglycosylation was carried out with Peptide N-glycosidase F  according to manufacturer’s instructions. Protein identification with MALDI-TOF MS/MS was performed by APT SHANGHAI Applied Protein Technology .The fermentation broth was harvested by centrifugation at 12,000The effect of pH on laccase activity was determined between pH 2.5 to 6.5 at 40 ºC. pH stability was studied by incubating the enzyme at pH 2.5–9.0 at 30 ºC for 48 h. Residual laccase activity was quantified with ABTS as the substrate. Buffers used included citrate–phosphate buffer (pH 2.5–8.0), and glycine–NaOH buffer (pH 9.0).For optimum temperature, laccase activity was measured at the optimum pH and temperatures from 20 to 70 ºC. Thermostability was analyzed by incubating the enzyme at different temperatures (40–70 ºC), and residual activity was assayed with ABTS at optimum pH and temperature. All experiments were performed in triplicate.3+, Ca2+, Ce3+, Cu2+, Fe2+, K+, Li+, Mg2+, Mn2+ and Zn2+ were in form of sulfate, Cd2+, Hg2+, Ni2+ and Co2+ in form of nitrate, and Pb2+ in form of subacetate. Individual inhibitor or metal ion was incorporated in the enzyme assay, and activity was determined with ABTS at optimal temperature and pH. Enzyme activity in absence of metal ions was regarded as 100%.Effect of metal ions on activity of the purified enzyme was investigated. Metal ions AlOrganic solvents, namely methanol, ethanol, acetone, isopropanol, acetonitrile and dimethyl sulfoxide (DMSO), was added individually to the enzyme activity assay to the final concentration of 10% or 25%, and the laccase activity assay was carried out at the optimal temperature and pH with ABTS as the substrate. Enzyme activity in absence of organic solvents was regarded as 100%.C. unicolor strain CGMCC 5.1011 and Cerrena sp. HYB07, respectively. The decolorization mixture contained 50 mM citrate–phosphate buffer (pH 6.0), 100 mg/L MG and 20 U/mL laccase. The mixture with heat-inactivated laccase was used as the negative control. After decolorization, the reaction mixtures were subjected to UV–visible analysis with a Hitachi U-2910 UV–Vis spectrophotometer . Decolorization efficiency was monitored at 618 nm and calculated with the following formula:0 and A1 are the absorption of MG before and after laccase treatment, respectively.Decolorization of MG was carried out at 30, 50 and 70 °C with laccase from Homologous modeling was conducted with Phyre2  to 2406 bp (for Lac9). The deduced protein sequences of the 12 laccases were of length typical of fungal laccases (from 371 aa for Lac12 to 627 aa for Lac9), and all were predicted to contain a signal peptide. The intron numbers of the laccase isozymes varied between 6 and 12. A phylogenetic tree was constructed to show the evolutionary relationship of CGMCC 5.1011 laccases with reported laccases 2SO4 precipitation and anion exchange chromatography. The protein appeared as a smear on SDS-PAGE between 58.4 and 74.0 kDa. After deglycosylation with peptide N-glycosidase F, a single band was shown around 50 kDa, indicating the laccase was heterogeneously glycosylated   and Lac7 (from Cerrena sp. HYB07) in MG decolorization, both in the absence of a redox mediator. We have previously shown MG is sequentially demethylated by laccase to colored intermediates desmethyl MG, didesmethyl MG, tridesmethyl MG and tetradesmethyl MG  than HYB07 laccases. CGMCC 5.1011 laccases were predicted to have 3–13 glycosylation sites per protein, with Lac2 having 13, while HYB07 laccases were predicted to contain 0–6 glycosylation sites per protein.Fungal laccases exist in gene families, which may be derived from duplication-divergence events of a small set of ancestral enzymes. Laccase gene families have been analyzed in genes Li . LaccaseCerrena sp. HYB07  and six substrate-binding loops  is provided in Additional file Botrytis cinerea laccase from high-temperature denaturation  is also attributed to higher glycosylation, and the glycan moieties played a crucial role in the laccase activity (Garg et al. Cerrena sp. HYB07, which has a 7.2% carbohydrate content (Yang et al. There are several putative molecular mechanisms underlying protein thermotolerance or thermostability (Hilden et al. Hydrophobic interactions play a governing role in stabilizing the protein 3D structure (Christensen and Kepp th residue near the substrate-binding loop B1–B2, 267th residue in B7–B8 and 334th residue in C1–C2, corroborating the lower flexibility and higher stability of Lac2. Residues 98–102, upstream to the fungal laccase signature domain L2, were associated with lower RMSF values in Lac2, meaning they might help maintain the rigidity of the L2. Regions 262–273 and 331–342 corresponded to substrate-binding loops B7–B8 and C1–C2, respectively, they showed higher RMSF values in Lac7 than in Lac2. A unique salt bridge in Lac2, His326-Asp340, flanking the substrate-binding loop C1–C2, might contribute to lower the flexibility around loop C1–C2 in Lac2, whereas the majority of other salt bridges in the two proteins were conserved (Additional file The MD simulation of the two laccases also shined light on the molecular determinants of Lac2 thermostability. The overall RMSD value of Lac2 were lower than that of Lac7, indicating the whole flexibility of Lac2 was smaller than Lac7 (Fig. C. unicolor CGMCC 5.1011 achieved a maximum activity of 121.7 U/mL after fermentation for 15 d. Strain CGMCC 5.1011 contained 12 laccase isozymes, and a major laccase, Lac2, was purified from the fermentation broth. Lac2 was reactive over a wide range of temperatures, was pH- and temperature-stable and tolerant of organic solvents. Lac2 decolorized MG whereas a less thermostable laccase failed, corroborating the thermostability and operational stability of this novel laccase. High-level glycosylation and structural rigidity might account for the high stability of Lac2.In summary, a white-rot fungal strain Additional file 1. Additional figures and tables.


Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat  Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world’s population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis. Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis. This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis. This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets. Mycobacterium tuberculosis is the leading cause of death due to a single infectious agent worldwide  until a single active fraction was identified by bioactivity-guided purification. The structures of the four compounds were determined by nuclear magnetic resonance (NMR) analysis. Three compounds were determined to be novel, since they did not have matches in AntiBase or SciFinder. The fourth was determined to be the previously identified compound marfomycin D, purified from a deep-sea Streptomyces species. However, its activity against mycobacteria had not been reported , total correlation spectroscopy (TOCSY), 1H-13C/15N heteronuclear single quantum coherence (HSQC), 1H-13C heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY)/rotating-frame nuclear Overhauser effect spectroscopy (ROESY) experiments. For elucidation of the structure of streptomycobactin, additional triple-resonance experiments on a universally 13C- and 15N-labeled sample were used. 13C chemical shifts of the peptidyl backbone were mapped using HNCACB and HNCOCACB experiments, while the side-chain 13C and 1H chemical shifts were assigned using (H)CCCONH and H(CCCO)NH experiments, respectively  13C DEPT135; (C) 1H-1H COSY; (D) 1H-13C HSQC; (E) 1H-13C HMBC; (F) NOESY. Download FIG S2, TIF file, 0.3 MB.Key NMR spectra of amycobactin. (A) Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.3FIG S31H; (B) 13C; (C) 1H-1H COSY; (D) 1H-13C HSQC; (E) 1H-13C HMBC; (F) 1H-15N HSQC; (G) HNCACB; (H) HN(CO)CACB; (I) CCCONH; (J) 15N TOCSY-HSQC. Download FIG S3, TIF file, 0.5 MB.Key NMR spectra of streptomycobactin. (A) Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.4FIG S41H; (B) 13C; (C) 1H-1H COSY; (D) 1H-13C HSQC; (E) 1H-13C HMBC; (F) 1H-1H TOCSY; (G) 1H-15N HSQC; (H) 1H-1H ROESY. Download FIG S4, TIF file, 0.5 MB.Key NMR spectra of kitamycobactin. (A) Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.5TABLE S11H and 13C NMR data of amycobactin . Download Table S1, DOCX file, 0.02 MB.Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.6TABLE S21H, 13C, and 15N NMR data of streptomycobactin . Download Table S2, DOCX file, 0.02 MB.Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.7TABLE S31H, 13C, and 15N NMR data of kitamycobactin . Download Table S3, DOCX file, 0.01 MB.Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.8TEXT S1Text S1, DOCX file, 0.01 MB.Detailed data interpretation of structural determination of amycobactin. Download Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.9TEXT S2Text S2, DOCX file, 0.01 MB.Detailed data interpretation of structural determination of streptomycobactin. Download Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01516-20.10TEXT S3Text S3, DOCX file, 0.01 MB.Detailed data interpretation of structural determination of kitamycobactin. Download Copyright © 2020 Quigley et al.2020Quigley et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Amycolatopsis sp., has an exact mass of 762 Da, and is the smallest of the compounds identified. It has an unusual structure, featuring a ketal moiety within a macrolactone backbone. Kitamycobactin and streptomycobactin are semicyclic peptides with exact masses of 2,259 and 1,735 Da, respectively. Streptomycobactin is a 20-amino-acid peptide (VIITVLLVRAVLATVRIERV) produced by a Streptomyces sp. Kitamycobactin, produced by a Kitasatospora sp., is a lasso peptide (GFGRIKADELVGRLIP) and belongs to the same class as lassomycin. Antimicrobials have been identified from these genera previously. However, our in situ cultivation method utilizing the iChip increases recovery 50-fold  and mc26020 . We included susceptibility data for marfomycin D as well, since this compound had not been tested against mycobacteria . The NTM family of organisms encompasses >150 different species of opportunistic pathogens that cause various diseases ranging from pulmonary infections similar to tuberculosis to soft-tissue and skin infections  of 16 to 32 μg/ml against HepG2 and NIH/3T3 cells. Given the streptomycobactin MIC of 0.03 μg/ml against M. tuberculosis, this creates a significant therapeutic window. However, the therapeutic window for amycobactin is considerably smaller, given its MIC of 4 to 8 μg/ml against M. tuberculosis. Overall, the compounds displayed good selectivity against M. tuberculosis and mycobacteria, with limited cytotoxicity.The detrimental effects of antibiotic treatment on the gut microbiome are a well-recognized phenomenon . With th reuteri . The limM. tuberculosis strain mc26020 in both the exponential- and stationary-growth phases. Amycobactin was bacteriostatic in exponential phase  in the related species Mycobacterium smegmatis. The amycobactin MIC against wild-type (WT) M. smegmatis was 1.5 μg/ml, while the MICs against N28R1 and N28R2 were 12.5 μg/ml and >100 μg/ml, respectively. Sequencing revealed that both mutants contained in-frame deletions in the general protein secretion translocase SecY (MSMEG1483), which, together with SecE and SecA, forms the core of the Sec protein translocation machinery . N28R2 contained a 9-bp deletion in secY, resulting in the deletion of amino acids 407 to 409 (FGG)  . Given tM. smegmatis using site-directed recombineering  was used to re-create these deletions in M. tuberculosis. The deletion of amino acids 407 to 409 was successful; it was confirmed by sequencing, and the mutant was named MtbΔsecY 3AA. We were unsuccessful in deleting amino acids 222 to 227 in M. tuberculosis despite several attempts. MtbΔsecY 3AA grew similarly to WT M. tuberculosis (M. tuberculosis SecY with 100% confidence to the highest-scoring template (PDB ID 2ZQP). The 6- and 3-amino-acid deletions were mapped to the predicted structure and are shown in red in Escherichia coli is restricted by a hydrophobic core ring and a channel plug requiring ATP hydrolysis via SecA for secretion  is conserved in both E. coli and S. aureus. However, while M. tuberculosis SecY shares 88% identity with M. smegmatis SecY, it shares only 41% and 40% identity with the E. coli and S. aureus SecY proteins, respectively. It is likely this overall dissimilarity that accounts for the selectivity against mycobacteria.Deletions corresponding to those in N28R1 and N28R2 were made in a clean background of WT ineering , 20. Theeletions . We nextrculosis  and had rculosis  was usedecretion , is showecretion  and e. TM. smegmatis engineered to secrete the E. coli ′BlaTEM-1 β-lactamase via the Sec translocation machinery  was significantly greater in the untreated sample than in the amycobactin-treated sample CF  and 5% glycerol. Lysine (80 μg/ml) and pantothenate (24 μg/ml) were added to mc26020 medium. Tyloxapol was added to liquid cultures at a final concentration of 0.05%. Mycobactin J (Allied Monitor) was added to M. paratuberculosis cultures at a final concentration of 10 mg/liter. Bacteroides fragilis ATCC 25282D-5 and Lactobacillus reuteri ATCC 23272 were grown in brain heart infusion broth supplemented (per liter) with 5 g yeast extract, 10 ml 10% (wt/vol) l-cysteine HCl, 15 mg/liter hemin, and 66 ml 1.5 M 3-(N-morpholino)propanesulfonic acid  in an anaerobic chamber. Kanamycin was used at a final concentration of 50 mg/liter where appropriate.The Soil samples (1 g) were vigorously agitated in 10 ml of deionized water for 10 min and were then allowed to sit for 10 min to allow large soil particles to settle. The supernatant was diluted into molten SMS medium . Aliquots were then dispensed into the wells of 96-well microtiter plates or iChips (2O per liter of deionized H2O [pH 7.0]) and incubated with vigorous agitation at 28°C for 5 to 12 days until the culture was turbid . The time to turbidity depends on the isolate. The seed culture was then diluted 1:20 into three different fermentation media. After 11 days of growth at 28°C with agitation, the cultures were dried down. Dimethyl sulfoxide (DMSO) was added to the dried biomass and mixed, and the crude extracts were tested for activity against S. aureus. A 5-μl aliquot of each extract was applied to a lawn of S. aureus growing on Mueller-Hinton agar. After overnight incubation at 37°C, the presence of a clearing zone indicated hit activity.Isolates from the NovoBiotic collection were transferred to seed broth  of 0.4 to 0.5. The culture was diluted to an OD600 of 0.003 and 148.5 μl of culture added to the wells (1:100 dilution). After 7 days of incubation with agitation at 37°C, growth was monitored by measuring the OD600 and by measuring fluorescence with excitation at 580 nm and emission at 610 nm. Extracts that demonstrated ≤25% growth relative to that of the DMSO positive-growth controls were cherry picked and retested under the same assay conditions. These extracts were also tested at a 1:100 dilution in a broth microdilution assay against S. aureus. Extracts without any effect on the growth of S. aureus that repeated the inhibition of the growth of M. tuberculosis were considered confirmed hits.The extracts were tested against 4)2SO4, 10 g CaCO3, and 20 g glycerol in 1 liter] and required a total of 40 liters of production for the isolation of sufficient material to characterize amycobactin. IS019923 and IS008612 were fermented in R4 fermentation medium , 10 g Pharmamedia (Traders protein), 1 g . The cell pellet was extracted with acetone and filtered. The acetone pellet extract was dried onto HP20 resin by rotary evaporation. The HP20 resin containing the pellet extract was washed with deionized water (1 liter) and eluted using a step gradient . Amycobactin was determined to be present in the 80% fraction of the supernatant and in both the 60% and 80% fractions of the pellet extract. These fractions were combined, dried completely, and then reconstituted in methanol. This mixture was further separated on a column of LH20 resin (eluted in methanol), yielding 3 fractions containing amycobactin. These fractions were again combined and dried completely. The residue was dissolved in DMSO and was separated via HPLC  . Amycobactin eluted at 19 min, and a second purification via HPLC was performed  . Purified amycobactin eluted from the column at 10 min. Fractions containing amycobactin were lyophilized in preparation for structural analysis and biological testing.The fermentation broth of IS019924 (10 liters) was centrifuged at 17,700 × g for 45 min. The cell pellet was extracted with 1 liter of acetone. The supernatant was extracted with 2 liters of n-butanol, and the aqueous layer was discarded. The acetone pellet extract and the n-butanol extract of the supernatant were added to separate round-bottom flasks, and HP20 was added to each flask. The organic solvents were removed via rotary evaporation. The HP20 resins were washed with deionized water and eluted using a step gradient . Streptomycobactin was found in the 50% and 100% elutions. These elutions were then combined and dried, leaving a brown residue. Hexane was added to the residue, which was then sonicated and centrifuged. The hexane extract was then discarded, and the residue was dissolved in DMSO and separated via HPLC  . The fractions containing streptomycobactin were combined and lyophilized to leave a white powder.The fermentation broth (8.5 liters) was centrifuged at 17,700 × 2·6H2O, 4 g CaCl2·2H2O, 0.2 g K2SO4, 5.6 g of 2-ethanesulfonic acid [TES free acid], 10 g of 13C-labeled glucose , 10 ml of 0.25-g/ml 13C-, 15N-labeled Celtone base powder ) with the pH adjusted to 7.0. The culture was placed on a rotatory shaker at 200 rpm and 28°C for 7 days. The culture was monitored visually daily for robust growth that attached itself tightly to the sides of the flask at the liquid-air interface. A 2% inoculum of this culture was transferred into 500 ml of modified CM-R4 medium in a 2-liter, 6-baffle flask at 28°C, shaken at 200 rpm, and harvested after 9 days.Grown biomass of IS019923 was inoculated into 20 ml modified CM-R4 . Labeled streptomycobactin was found in the 50% and 100% elutions, which were dried separately, leaving brown residues. These residues were dissolved in DMSO, separated via HPLC  . Fractions containing labeled streptomycobactin were combined and lyophilized to leave a white powder. The powder was dissolved in DMSO, and the sample was then repurified via HPLC  . The fractions containing 13C- and 15N-labeled streptomycobactin were combined and lyophilized to leave 10.4 mg of a white powder.The fermentation broth (500 ml) was centrifuged at 17,700 × g for 45 min. The supernatant was decanted into a separatory funnel, and the remaining cell pellet was extracted with acetone before centrifugation. The acetone extract was transferred to a round-bottom flask, and the acetone was removed via rotary evaporation. The remaining water layer was added to the supernatant, and then the mixture was extracted with n-butanol. The water layer was discarded, and the n-butanol was transferred to a round-bottom flask. The n-butanol was removed via rotary evaporation. The remaining residue was dissolved in DMSO and then purified via HPLC  . Kitamycobactin eluted at 24 min. The fraction was then lyophilized to leave a white powder.The fermentation broth was centrifuged at 17,700 × d6  and heated to 40°C for data acquisition. Structural assignments of amycobactin  in a sample tube, and the sample was heated to 35°C for data collection. The structure of streptomycobactin was assigned based on a series of 1D, 2D, and 3D NMR experiments using the streptomycobactin sample  and lysine (80 μg/ml) were added to H37Rv mc26020 cultures. Plates were incubated at 37°C for either 7 days , 3 days , or 20 h . B. fragilis and L. reuteri were grown under anaerobic conditions. The MIC was defined as the lowest concentration of antibiotic with no visible growth.MICs were determined by broth microdilution. Cell concentrations were adjusted to an ODExponentially growing NIH/3T3 mouse embryonic fibroblasts (ATCC CRL-1658) in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine calf serum and HepG2 cells (ATCC HB-8065) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum were seeded into 96-well flat-bottom plates. After 24 h of incubation at 37°C, the medium was replaced with fresh medium containing 2-fold serial dilutions of test compounds. After 72 h of incubation at 37°C, viability was determined with the CellTiter 96 AQueous One Solution cell proliferation assay kit  according to the manufacturer’s recommendations.M. tuberculosis to mid-exponential phase  and then back diluting to an OD600 of 0.003. For stationary-phase cultures, M. tuberculosis was grown for 2 weeks to an OD600 of >1.5. Cultures were challenged with either 4× MIC (amycobactin) or 10× MIC  of the compound at 37°C. At intervals, 100-μl aliquots were removed, washed once in phosphate-buffered saline (PBS), serially diluted, and plated onto 7H10 medium to determine the CFU count per milliliter.Exponential-phase cultures were prepared by growing M. smegmatis were selected by plating onto 7H10 medium containing amycobactin at 10× wild-type MIC. Briefly, 10 ml of wild-type M. smegmatis was grown to an OD600 of ∼1.0. The culture was washed once and was concentrated to 1/10 the original volume (1 ml). The culture was then plated onto five 7H10 plates containing 10× MIC amycobactin. Mutants were streaked onto nonselective medium, and MICs were determined. Mutants were sent for whole-genome sequencing, and variant analysis was conducted by MR DNA. Targeted mutations were made in M. smegmatis and M. tuberculosis via single-stranded recombineering as in reference ΔsecY 3AA, 5′-CCCTGCTCGACGAACACCACGCCGATGATGATCACGGCGGTGAACACGACGCCGCCGCGGCTCTCCAGGA-3′ for MsmΔsecY 6AA, and 5′-CCAAACCGACACCGATCATGATCAGCACCGCGGTAGGCAGGTTCTGCACGGTTCCACCGGCGCCGATCTG-3′ for MtbΔsecY 3AA. Targeted mutations were confirmed via PCR and Sanger sequencing.Mutants resistant to amycobactin in M. smegmatis ′BlaTEM-1 reporter strain were grown in minimal medium with 0.02% glucose and 0.05% Tween 80 to late-exponential phase. The cultures were washed twice in minimal medium , was used for protein depiction.Analysis was performed with GraphPad Prism, version 7.


Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State . Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene ; δ-elemene ; elemol  and α-muurolol, bicyclogermacrene, and cubebol . The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g Piper is the largest from the Piperaceae, with about 3000 species identified, and is widely distributed in tropical and subtropical regions of the planet, and in Brazil, there are around 44 varieties reported, distributed among 292 species [Piper nigrum L. (black pepper), which is considered the “King of Spices” and is commonly used as a condiment around the world, but is also applied in traditional medicine in several countries, mainly in Asia, Africa, and South America [The genus  species ,2. The m America ,6,7,8.Piper species are distinguished by the presence of nodes in the shoots, inflorescence spikes, and their aromatic smell [ic smell . Black pic smell . Cultivaic smell ,12,13. Tic smell ,15,16,17Fusarium solani f. sp. piperis) and yellow wilt (Fusarium oxysporum), have resulted in price instability in the international market of this condiment [India, Brazil, Indonesia, Malaysia, Vietnam, China, and Sri Lanka are the largest producers of black pepper ,19,20. Iondiment ,23,24.Secondary metabolites are defined as products of metabolism that are non-essential for the survival of a plant, but they play an important role in the interaction with the environment in which the plant is inserted, including protection against phytopathogenic microorganisms ,26. TheyEscherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis [P. nigrum exhibited potential usability as a natural antioxidant and food supplement, and presents a pharmacological potential [Some secondary metabolites of black pepper are used to fight infections caused by microorganisms, such as viruses and fungi, that are called phytoalexins ,30. Studsubtilis . Additiootential .The metabolomic analysis of plants against these stimuli can help in the identification of secondary metabolites involved in different plant physiological processes, and can be used as biomarkers for the discrimination and characterization of the species ,34,35,36Fusarium solani f.sp. piperis and F. oxysporum are the most notably responsible for the fungal diseases in black pepper that negatively interfere in its fruit production and the plant quality [The soil-borne pathogens  quality ,40. More quality . StrategP. nigrum cultivars cultivated in the Pará State (Brazil), and to evaluate the potential volatile phytoalexins by molecular docking analysis on Fusarium protein targets. The discrimination and characterization of the cultivars in chemical groups can give more insight into plant–pathogen interactions, so as to develop new strategies in plant disease control.Therefore, this study aims to identify the secondary metabolic profile in the The chemical analysis by gas chromatography–mass spectrometry (GC-MS) resulted in the identification of 114 components see , represePiper species, such as P. amalago (5.0–9.3%) [P. caldense (9.0%) [P. cernuum Vell. (5.8%) [P. manausense (7.6%) [P. sp. aff. aereum (5.8%) [The “Bragantina” cultivar showed a predominance of oxygenated sesquiterpenoids (50.55 ± 10.35%), followed by sesquiterpenoid hydrocarbons (34.62 ± 12.97%). The major components were α-muurolol (20.63 ± 2.81%) A, bicycl.0–9.3%) , P. calde (9.0%) , P. cern. (5.8%) , P. manae (7.6%) , and P. m (5.8%) .E)-β-caryophyllene (5.72 ± 0.93%), and muurola-4,10(14)-dien-1-β-ol (4.83 ± 0.64%). “Iaçará” and “Kottanadan” cultivars displayed similar chemical profiles with quantitative differences. The main compounds were δ-elemene (50.89 ± 1.64% and 32.1% ± 7.44%), β-elemene (9.92 ± 0.58% and 6.10 ± 1.32%), viridiflorene (8.87 ± 0.84% and 12.42 ± 2.28%), and β-selinene (8.17 ± 0.57% and 7.54 ± 2.59%) for the “Iaçará” and “Kottanadan” cultivars, respectively. In black pepper, δ-elemene (53.3%) was previously reported as the main component in the leaves of “Cingapura” cultivar. The other main constituents identified in the cultivar were (E)-β-caryophyllene (4.7%), and muurola-4,10(14)-dien-1β-ol (4.9%) [Piper species that present δ-elemene as one of the main constituents are P. aequale (19.0%) [P. amalago (6.82%) [P. aleyreanum (8.2%) [P. artanthe (11.69%) [P. dilatatum (7.6%) [P. fimbriulatum (9.4%) [P. cernuum (5.89–12.0%) [P. hispidum (7.6%) [P. marginatum (5.9–9.7%) [P. madeiranum (7.1%) [P. molicomum (9.9%) [In the “Cingapura” cultivar, sesquiterpene hydrocarbons 58.48 ± 0.94%) were predominant, followed by oxygenated sesquiterpenoids (20.36 ± 0.80%). The major components identified were δ-elemene (37.17 ± 1.90%) B, linalol (4.9%) . Other P (6.82%) , P. aleym (8.2%) , P. arta-elemene .3% was p ± 0.94% m (9.4%) . Moreove9–12.0%) ,57,58, P.9–9.7%) ,61. For m (7.1%)  and P. mm (9.9%)  showed hPiper species present γ-elemene as one of the main components in their EOs, such as P. gaudichaudianum Kunth (5.4%) [P. marginatum (3.75%) [P. nemorense (6.8%) [P. vicosanum (14.16%) [“Equador” and “Guajarina” cultivars presented the same chemical profile, only with quantitative differences. The main compounds were γ-elemene (38.33 ± 3.80% and 44.36 ± 4.06%) D, curzerh (5.4%) , P. marg (3.75%) , P. nemoe (6.8%) , and P. (14.16%) .Curzerene was widely found in “Equador” (23.21 ± 4.96%) and “Guajarina” (23.39 ± 4.45%) cultivars, and is considered a derivative of furanodiene, which is produced from a reversible thermal rearrangement known as the Cope rearrangement . It is aPiper species, this oxygenated sesquiterpenoid can be found in P. dilatatum (13.8–28.7%) [P. hispidum (4.9–12.9%) [Eugenia uniflora, commonly known as “pitanga”, an arboreous plant widely distributed in Brazil and other South American countries and possesses antifungal activity against Candida spp., and its EO presents antiproliferative and cytotoxic effects against some cancer strains [In 8–28.7%)  and P. h9–12.9%) ,69. Anot strains ,71.GC-MS analysis identified 104 components, representing 99.45% of the total composition see . MonoterE)-β-caryophyllene (10.7–18.4%) were observed [E)-β-caryophyllene (16.8–39.1%), sabinene (4.3–18.8%), myrcene (2.0 9.6%), α-phellandrene , δ-3-carene , α-cubebene , and even the oxygenated sesquiterpene elemol  [E)-β-caryophyllene (12.8%), sabinene (11.2%), α-pinene (5.6%), and β-pinene (6.7%), while the EO of black pepper from S. Tomé e Príncipe displayed limonene (18.8%), (E)-β-caryophyllene (15.4%), sabinene (16.5%) and β-pinene (10.7%) as majorities [The EO chemical composition of black pepper cultivars can present quantitative and qualitative differences according to their geographic occurrence. For example, in cultivars from India , quantitative variations in amounts of α-pinene (7.3–16.7%), β-pinene (13.2–13.6%) and limonene (15.2–16.2%), δ-3-carene (9.2–32.6%), α-phellandrene (2.9–8.9%), and (observed . Fruits ultivar) . In Camejorities ,77.Multivariate analysis based on the leaf volatiles classified the cultivars into four groups . Group IE)-β-caryophyllene (5.72%) as the majority. This group showed a similarity of 45.9% with group III. The cultivars “Clonada” and “Uthirankota” were grouped in Group III with a similarity level of 98.1% and high content of elemol (40.55–49.78%), and presented a similarity of 43.9% with group IV. Finally, group IV separated only the “Bragantina” cultivar from the others, due to its composition rich in α-muurolol (20.63%), bicyclogermacrene (7.55%), and cubebol (6.49%). Regarding volatiles from the fruits, two clusters were identified with a high similarity between them (~93.5%), displaying no significant variation among the cultivars.In addition, cultivars “Iaçará” and “Kottanadan” presented significant amounts of viridiflorene (8.87–12.42%), β-selinene (7.54–8.17%) and β-elemene (6.10–9.92%). In contrast, the cultivar “Cingapura” exhibited spathulenol (5.85%) and , (E)-β-caryophyllene (4.24–4.69%), and valencene (3.02–3.11%). Group II was composed only of the variety “Bangla” due to the presence of eugenyl acetate (1.50%) and α-humulene (1.35%) in its composition. The varieties “Sanchi” and “Khasia” were separated in Group III, which was characterized by γ-muurolene (1.74–4.49%) and (E)-β-caryophyllene (1.74–4.93%) in their compositions [The use of metabolic profiling as a tool for the discrimination of cultivars of several crops has been reported in the literature ,79. Prinositions .Citrus varieties as tolerant and moderately tolerant from those susceptible to Candidatus Liberibacter asiaticus (CLas). Tolerant and moderately tolerant cultivars showed higher amounts of volatiles than susceptible varieties. These compounds include aldehydes  and some monoterpenes such as linalool (0.1–68.3%), d-limonene (0.3–55.3%), myrcene (0.1–8.5%), α- (0.2–3.1%) and β-phellandrene (0.1–11.4%) [PCA analysis of volatile profiles was able to distinguish fourteen 1–11.4%) .F. oxysporum (yellow wilt) [Previous studies reported the different degrees of the susceptibility of black pepper cultivars to ow wilt) ,82,83. OFusarium inoculation given to each variety are required to confirm this relationship. Moreover, an antimicrobial assay with the main compounds is recommended for understanding the role of specific compounds in each cultivar’s plant defense mechanism.In comparison to the fruits, the volatile profiles of leaves functioned as suitable biomarkers for the discrimination of cultivars, and their resistance or susceptibility to yellow wilt. However, further experiments with P. nigrum extracts showed great variation in the leaves (44.75 ± 1.60 to 140.53 ± 14.01 mg GAE·g−1)  A. HoweveGAE·g−1) B. AccordPiper species  were extracted with n-hexane, chloroform, methanol, and water. Methanol extracts from “IISR Malabar Excel” and “Panchami” varieties, and a chloroform extract of P. colubrinum displayed higher phenolic contents, with values of 50.85, 38.74, and 100.6 mg GAE·g−1 of the extract [The levels of total phenolics were higher in the leaves in comparison to the fruits. These differences can be related to the harvesting and processing steps involved in sun-drying, which alter the interaction between the enzymes related to phenolic production in the black pepper fruits . In addi extract . TherefoF. solani f. sp. piperis, showed a significant variation in total phenolics in the leaves and roots from the “Cingapura” cultivar (tolerant to Fusarium), which can be attributed to the response to the infection in the early stages, since the first symptoms were noted after two weeks of inoculation [Plants that are under biotic or abiotic stresses can exhibit changes in their phenolic contents in the early stages of development . Black pculation . Moreoveculation .−1)  . The litIn plants, the carotenoids are involved in different areas, such as antioxidants, accessory pigments that absorb light, and molecules that act as attractants for pollinators and seed dispersers, in addition to the plant response to environmental stresses . Thus, iP. nigrum cultivars exhibited a considerable variation in PAL enzymatic activity, ranging from 20.19 to 57.22 µU·mL−1  and two strains of endophytic fungi (Trichoderma viride and Trichoderma asperellum), an increase in PAL activity induced the systemic resistance of black pepper against P. capsici [An increase in PAL activity can result from a sensitive response that occurs in plants when they are infected by pathogens or during an injury ,100. Sigr stress . After 4 capsici . TherefoFusarium resistance, we have carried out a molecular docking analysis of the major P. nigrum volatile components with Fusarium protein targets that were available from the Protein Data Bank (PDB): F. oxysporum cutinase (PDB 5AJH), F. solani cutinase , F. oxysporum endoglucanase (PDB 4OVW), F. oxysporum feruloyl esterase (PDB 6FAT), and F. oxysporum xylanase (PDB 5JRM). In addition, F. solani ornithine decarboxylase was prepared by homology modeling using murine ornithine decarboxylase (PDB 7ODC) as a template, F. oxysporum glucosamine-fructose-6-phosphate aminotransferase was prepared from human glutamine fructose-6-phosphate amidotransferase (PDB 6R4E) as a template, F. odoratissimum β-glucosidase was prepared using Neurospora crassa β-glucosidase (PDB 5NBS) as the template, F. oxysporum guanine nucleotide-binding protein subunit β was prepared using human guanine nucleotide-binding protein subunit α1 (PDB 6CMO) as the template, and F. vanettenii thiamine thiazole synthase was prepared using N. crassa thiazole synthase (PDB 3JSK) as the template. Docking energies for the essential oil ligands and 10 Fusarium target proteins are listed in In an attempt to correlate volatile phytoalexins with Fusarium-resistant P. nigrum cultivars  were δ-elemene , viridiflorene, β-selinene, β-elemene (abundant in “Iaçará” and “Kottanadan”), spathulenol and (E)-β-caryophyllene (abundant in “Cingapura”). δ-Elemene, viridiflorene, β-elemene, and (E)-β-caryophyllene, all showed selective docking (most exothermic docking energies) to F. oxysporum endoglucanase. Furthermore, F. oxysporum endoglucanase had the lowest docking average energy for the 40 essential oil components, and was the most targeted protein with 13 ligands showing preferential docking to this protein. Endoglucanase is one of the enzymes responsible for breaking down cellulose by hydrolyzing the β-1,4-glycosidic bonds in the cellulose polymer [Fusarium. Interestingly, the essential oil components all dock into a hydrophobic pocket removed from the site of the co-crystallized ligand (4-(β-D-glucopyranosyloxy)-2,2-dihydroxybutyl propanoate) but still in the active canyon of the enzyme  were provided by EMBRAPA Amazônia Oriental . The plants were acclimatized and maintained in a greenhouse with a daily watering regime. Adult leaves were collected during the fruiting stage and stored at −20 °C for further experimentation. For the fruits, the collection time varied from July to October, according to its ripeness for each cultivar. After harvesting, the fruits were sun-dried for about three days until the humidity was around 13%, and preserved at room temperature for further experimentation.The leaves and fruits of “Bragantina”, “Cingapura”, “Clonada”, “Equador”, “Guajarina”, “Iaçará”, “Kottanadan”, and “Uthirankota” cultivars of n-pentane (3 mL) for two hours. After extraction, an aliquot (1.0 μL) of the organic phase was injected in a gas chromatography coupled with mass spectrometry (GC-MS) apparatus. The analysis was performed in GC-MS equipment  according to the following settings: Rtx-5MS (30 m × 0.25 mm × 0.25 µm film thickness), silica capillary column ; programmed temperature, 60–240 °C (3 °C/min); injector temperature, 250 °C; carrier gas, helium with linear velocity of 32 cm/s (measured at 100 °C); injection type, split (1.0 µL); split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 mL/min; EIMS, electron energy, 70 eV; temperature of the ion source and connection parts, 200 °C. A homologous series of n-alkanes  was applied to calculate the retention index (RI) [Leaves and fruits were extracted by the simultaneous distillation–extraction process using a Likens–Nickerson apparatus to obtain the volatile concentrate. The solvent of the extraction was dex (RI) , which wdex (RI) ,106. TheP. nigrum were extracted by percolation (96 h) with 100 mL of methanol. Every 48 h, the samples were placed on ultrasound for 10 min. At the end of 96 h, the solvent was evaporated. The extracts were used for Folin–Ciocalteu total phenolic and total carotenoids determination [Samples (2.0 g) of leaves and fruits of mination ,108,109.−1), followed by dilution in distilled water. Then, an aliquot of 500 μL of extract was added to 250 μL of Folin–Ciocalteu reagent (1 N) and 1250 μL of sodium carbonate (75 g·L−1). The reaction was kept in the dark and after 30 min, the absorbance of the mixture was read at 760 nm using a UV-visible spectrophotometer . The experimental calibration curve was prepared using gallic acid  at concentrations of 0.5 to 10.0 mg·L−1, and the content of total phenolics was expressed as gallic acid equivalents (GAE) in milligrams per gram of extract (mg GAE·g−1).The extracts were solubilized in methanol (20 mg·mL−1. The absorbance (Abs) of the samples was read at 470, 648, and 664 nm using a UV-visible spectrophotometer . Ethanol was used as a blank. The pigment contents  were calculated using Equations (1)–(3). The carotenoid content was expressed in µg of extract per mL of ethanol (µg·mL−1).An aliquot of each extract was prepared in ethanol at the concentration of 1 mg·mLg (15 min at 4 °C), and then an aliquot of supernatant (0.5 mL) was mixed with 1.0 mL of reaction buffer composed of sodium borate buffer , and 0.03 mM L-phenylalanine . After 15 min at room temperature, the absorbance was measured at 290 nm using a UV-visible spectrophotometer . The specific molar extinction coefficient of (E)-cinnamic acid (9630 mol·L−1·cm−1) was used to determine the PAL activity based on its formation from the substrate, L-phenylalanine [Frozen fresh leaves of each cultivar were pulverized in liquid nitrogen. Each sample (100 mg) was homogenized in sodium borate buffer , 1 mM ethylenediaminetetraacetic acid (EDTA) , 1 mM dithiothreitol (DTT) , and 5% polyvinylpolypyrrolidone (PVP) , totalizing 1 mL of extraction buffer. The samples were centrifuged at 12,000× lalanine ,111.Fusarium targets were generated using three-dimensional crystal structures from the Protein Data Bank  using FASTA sequences obtained from the National Center for Biotechnology Information (NCBI) protein database (https://www.ncbi.nlm.nih.gov/protein) (see Homology models for each of the ein) see . ProteinP. nigrum essential oil major components with 7 Fusarium protein targets from the Protein Data Bank : F. oxysporum cutinase (PDB 5AJH), F. solani cutinase , F. oxysporum endoglucanase (PDB 4OVW), F. oxysporum feruloyl esterase (PDB 6FAT), and F. oxysporum xylanase (PDB 5JRM). In addition, homology models of F. solani ornithine decarboxylase, F. vanettenii thiamine thiazole synthase, F. oxysporum glucosamine-fructose-6-phosphate aminotransferase, F. odoratissimum β-glucosidase, and F. oxysporum guanine nucleotide-binding protein subunit β, were also screened. Structures of the ligands were generated using Spartan ’18 for Windows version 1.4.4 . Conformational analyses and geometry optimizations were carried out using the MMFF force field [Molecular docking was carried out on 39 ce field . The ligce field . Solvent® software, version 18.1 , using as variables the chemical components that are exhibited at least once in the composition of the cultivars and can reach above 1%. The data matrix was standardized by subtracting the mean from the individual value of each compound and then subtracting it by the standard deviation. The Euclidian distance, complete linkage, and absolute correlation coefficient distance were selected to obtain the similarity of the cultivars for the hierarchical cluster analysis (HCA). For total phenolics determination, total carotenoids, and PAL activity, in each sample, three independent tests were performed, and the results were expressed as mean ± standard deviation. The data were submitted to a normality test for validation (ANOVA), and subsequently, the Tukey’s test was applied with a 5% probability level (p < 0.05), using GraphPad Prism 6.0 software .The multivariate analysis was performed using the MinitabFusarium enzyme targets, and Fusarium endoglucanase was identified as a potential enzyme target, there are several other mechanisms of anti-Fusarium activity that may be important. Nevertheless, it was possible to correlate the metabolomic analysis with the susceptibility degree to yellow wilt of black pepper cultivars available in the State of Pará, Brazil, and the results highlight the importance of secondary metabolites in understanding the interaction of plants with different environments for the development of new biotechnologies.The present study detailed the qualitative and quantitative composition of volatile concentrates from the leaves and fruits of black pepper cultivars, which revealed about 218 components (114 in the leaves and 104 in the fruits). HCA analysis allowed the chemical discrimination of the cultivars based on the biomarkers identified in the aroma of the leaves. Differences among the cultivars were also observed in the total phenolics content, PAL activity, and total carotenoids. Although molecular docking was used to evaluate the potential inhibition of


Mellitus (T2DM) is a chronic disease which corresponds to 90% of the worldwide cases of diabetes, mainly due to epigenetic factors such as unhealthy lifestyles. First line therapeutic approaches are based on lifestyle changes, most of the time complemented with medication mostly associated with several side effects and high costs. As a result, the scientific community is constantly working for the discovery and development of natural therapeutic strategies that provide lower financial impact and minimize side effects. This review focus on these nature-based therapeutic strategies for prevention and control of T2DM, with a special emphasis on natural compounds that present pharmacological activity as dipeptidyl peptidase-4 (DPP4), alpha-amylase, alpha-glucosidase, lipase, and protein tyrosine phosphatase 1B (PTP1B) inhibitors.Type 2 Diabetes  These studies were done in the presence or absence of simulated gastric fluid and the content of phenolic compounds was determined [o-coumaric acid, and quercetin for Século XXI [−1·g−1) and Paluma against alpha-glucosidase before (28.82 ± 0.02 inhibited enzyme unit in μmol·min−1·g−1) and after exposure (2.59 ± 0.06 inhibited enzyme unit in μmol·min−1·g−1) [−1·g−1) and after exposure (43.33 ± 1.80 inhibited enzyme unit in μmol·min−1·g−1) [The beneficial effect of polyphenols and their subgroups on lipid metabolism is extensively reported on literature. Flavonoids are the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants, mainly on leaves or shells, with the function of protecting them from harmful external influences . Flavonocooking) . Ethanoln effect . Addition effect . Acacia ICR mice . The res loading . Water eat 1 mM) . Simão atermined . The stuculo XXI . The enzn−1·g−1) . Pedro SPlatycodin grandiflorum (isolated platycodin D from radix), Scabiosa tschiliensis (prosapogenin 1b at 0.12 mg/mL), Accanthopanax sessiloside , Panax japonicus, Dioscorea nipponica (dioscin IC50 of 20 μg/mL), and Cyclocarea paliurus (extract present IC50 of 9.1 mg/mL) [50 value was 0.885 mM for Ro; 0.895 mM oleanolic acid; 0.958 mM for botulin; 242 mM for tea saponin; and 388 mM for Rd [Argyranthemum pinnatifidum leaves and flowers exhibit inhibition activity against yeast alpha-glucosidase  and alpha-amylase  [Artemisia argentea leaves and flowers , Helichrysum devium , and Helichrysum melaleucum  [Phagnalon lowei leaves (IC50 = 3.05 ± 0.07 mg/mL) [50 = 25.4 ± 0.6 μg/mL and IC50 = 5.4 ± 0.6 μg/mL, respectively) and methanol  extracts [O-trans-p-coumaroyl actinidic acid isolated from roots of Actinidia arguta (IC50 = 14.95 μM) [Scutellaria baicalensis  [Saponins are composed by sugars attached to a steroid or triterpene, which are primary constituents of the roots and rhizomes of various plants, being responsible for a diversity of biological effects . These p1 mg/mL) . Tea sap1 mg/mL) . Tea sap1 mg/mL) . The EC5M for Rd . In anotctively) . Alpha-actively) . Lipase 7 mg/mL) . Pits frextracts . Other e4.95 μM) ; and baictively) .50 value of 8.6 ± 1.1 mg/mL [Condensed tannins called proanthocyanidins are found in the insoluble fraction of plant-derived foodstuffs , where type-B are the more abundant and type-A are abundant in vegetables like peanut skin, avocados, and cranberries . When th.1 mg/mL .Kappaphycus striatus with 92% inhibition against lipase activity and 88% for fresh Euchema denticulatum against alpha-amylase [Caulerpa taxifolia competitively inhibited 50% of lipase activity using emulsified triolein and dispersed 4-methylumbelliferyl oleate as substrates at 2 mM and 13 μM, respectively [Eisenia bicyclis revealed an inhibitory effect of pancreatic lipase, with IC50 values ranging from 37.2 ± 2.3 to 12.7 ± 1 μM for fucofuroeckol A and 7-phloroeckol, respectively [Saccharina japonica fermented by red mold Monascus purpureus inhibited alpha-amylase (IC50 = 0.98 ± 0.10 mg/mL), rat intestinal alpha-glucosidase  and lipase (IC50 = 4.98 ± 0.85 μg/mL) [Ascophyllum nodosum inhibited pancreatic lipase activity in an oil-based turbidimetric assay  as well as its purified phlorotannin-enriched fraction  [Seaweed are extensively studied for their health benefits; namely, inhibition of certain crucial enzymes for the control/prevention of DM. These studies include: ethanol extract of dried -amylase ; purifieectively . Additioectively . Brown s5 μg/mL) . Austin r assay) .Laminaria hyperborea seaweed has shown the most potent inhibitory activity than High-M alginates from Lessonia nigrescens, revealing that the alginate structure is related to the inhibition [Alginates are dietary fibers found in the cell walls of brown seaweed and certain bacteria and comprised of mannuronic and guluronic acid. Wilcox and colleagues found that alginate inhibited pancreatic lipase by a maximum of 72.2 ± 4.1% with synthetic substrate (DGGR) and 58 ± 9.7% with olive oil as substrate, where High-G alginates from hibition .Streptomyces toxytricini (IC50 = 0.14 μm); panclicins from Streptomyces sp. ; valilactone from Streptomyces albolongus (IC50 = 0.14 nm); ebelactones A and B from Streptomyces aburaviensis ; esterastin from Streptomyces lavendulae (IC50 = 0.2 ng/mL); caulerpenyne from Caulerpa taxifolia ; vibralactone from Boreostereum virans (IC50 = 0.14 μg/mL); and percyquinnin from Basidiomycete stereum complicatum (IC50 = 2 μm) [According to a review by Birari and Bhutani, metabolic products from microorganisms can also inhibit pancreatic lipase . These p = 2 μm) .The enzyme responsible for the reversal insulin receptor auto phosphorylation is a tyrosine phosphatase known as PTP1B (protein tyrosine phosphatase 1B), where inhibition results in a prolonged insulin signaling cascade, increasing insulin sensitivity . HoweverMorus alba showed the best inhibitory effect against PTP1B in a study by Huang and colleagues, with IC50 values of 0.72 ± 0.09, 0.60 ± 0.07, and 0.57 ± 0.16 μM, respectively [Artemisia princeps is a noncompetitive inhibitor with IC50 value of 11.1 μM using ursolic acid (known PTPB1 inhibitor) as positive control (IC50 = 3.1 μM) [Ficus racemosa exhibit inhibitory activity against PTP1B with IC50 values of 22.7 ± 1.7, 12.6 ± 1.6, 21.2 ± 3.8, and 2.5 ± 0.2 μM, respectively [Ircinia, bromophenols from red algae Rhodomela confervoides (IC50 from 0.8 to 4.5 μM), two bromophenols from Indonesian marine sponge Lamellodysidea herbacea (IC50 0.9 and 1.7 μM), and others from red algae Symphyocladia latiuscula  [Laurencia similis, polybromodiphenyl ether derivatives from marine sponge Lamellodysidea herbacea, eckol, and its derivatives isolated from brown algae Ecklonia stolonifera and Eisenia bicyclis [Polyphenols mulberrofurans C, J and F from ectively . Caffeoyectively . Ezzat a 2.7 μM) . Other cbicyclis .Incretins are hormones produced by intestinal enteroendocrine cells on ingestion of glucose ,11. Incr12 deficiency and contraindication in patients with chronic kidney disease [Active GLP-1 has a half-life of 1–2 min and its inhibition constitute an efficient pharmacological approach for T2DM treatment . In fact disease ,5. Anoth disease . Besides disease . In fact disease , increas1 position), non-competitive and uncompetitive  and mixed-type modes of action, exerting their effect by binding either at the active site and/or outside the catalytic center of the enzyme [Dietary proteins have been increasingly recognized as precursors of a variety of bioactive peptides, improving various aspects of human health . These be enzyme . The DPPe enzyme . Other ce enzyme .DPP4 is known to act on substrates with proline or other small-uncharged residues such as serine and alanine at their penultimate amino acid position . Because50 ≈ 4 μM) and can be found in the sequence of k-casein. However, not as effective as diprotin A, the peptides WR, IPIQY, and WCKDDQNPHS found in the sequence of lactoferrin, k-casein, and α-lactalbumin, respectively, are among the most potent food protein-derived DPP4 inhibitors reported to date [50 39.75 ± 0.01 mg/mL; EC50 0.07 ± 0.01 mg/mL; IC50 0.020 ± 0.001 mg/mL respectively) and lactoferrin hydrolysate LFH1 . However, the dipeptide Ala-Leu had bigger superoxide scavenging activity (EC50 1.74 ± 0.01 mg/mL) than Trp-Val, which was the dipeptide with greater DPPH scavenging activity [50 0.882 ± 0.057 mg/mL) [Peptides which were not isolated from hydrolysates but are likely to occur in the sequence of dietary proteins, were synthetically produced and studied for their effect on DPP4 activity . Diproti to date . However to date . Nongoni to date . The stuactivity . Besides7 mg/mL) .Origanum vulgare), marjoram (Origanum majorana), rosemary , and Mexican oregano (Lippia graveolens) [50 = 0.43 ± 0.07 μM), hispidulin (IC50 = 0.49 ± 0.06 μM) and naringenin (IC50 = 2.50 ± 0.29 μM), found in rosemary and Mexican oregano extracts [50 = 0.07 ± 0.02 to > 300 μM) and phenolics resveratrol (IC50 = 0.6 ± 0.4 nM), luteolin (IC50 = 0.12 ± 0.01 μM), apigenin (IC50 = 0.14 ± 0.02 μM) and flavone (IC50 = 0.17 ± 0.01 μM) exhibit the most strongly inhibiting activity, where phenolics present IC50 values lower than diprotin A (IC50 = 4.21 ± 2.01 μM) [The potential of phenolic compounds in the treatment and prevention of obesity is due to their thermogenic effects, which corresponds to the ability to oxidize fat and decrease intestinal absorption of fats and carbohydrates, resulting in the inhibition of digestive enzymes with consequent weight loss . Beneficveolens) . Greenhoveolens) . Accordiextracts . Fan andextracts . Malvidiextracts . Anthocy2.01 μM) . Accordi2.01 μM) .Sargassum binderi, Padina sulcata, and Turbinaria conoides had inhibitory activity against DPP4  [S. binderi, P. sulcata, and T. conoides allowed for the stimulation of GIP secretion of 5.46; 4.92 and 5 pM GIP per million cells per hour at 2.5; 10; 2.5 mg/mL, respectively [S. binderi and P. sulcata allows for the stimulation of GIP secretion of 56.38 and 40.67 pM, respectively, GIP per million cells per hour at 5 mg/mL [Sargassum wightii and Sargassum polycystum has an inhibitory effect against DPP4  and acetone extract with moderate antioxidant activity (43% and 22%) at a concentration of 1000 μg/mL according to DPPH free radical scavenging activity method [Turbinaria conoides possesses inhibitory activity against DPP4  at a concentration of 80 μg/mL and significant scavenging ability on DPPH (65%) at a concentration of 1000 μg/mL of acetone extract [Seaweed  are simple unicellular  or multicellular organisms , with rudimentary conductive tissues, presenting a high range of morphological and reproductive level variation that allows their division into different phyla and classes . The prectively) . Additioctively) . The evaectively . Further 5 mg/mL . Anothery method . The sam extract .Mangifera indica (family of Anacardiaceae). It has strong antioxidant, antilipid peroxidation, immunomodulation, antidiabetic cardiotonic, hypotensive, wound healing, antihyperlipidemic, antiatherogenic, and antidegenerative properties [p < 0.001), triglycerides (p < 0.001), LDL (p < 0.01), and atherogenic index (p < 0.01) were significantly reduced and HDL was increased (p < 0.01) in mangiferin and standard drugs treated groups [Xetospongia muta and sea anemones Bunodosoma granulifera and Bartholomea annulata present inhibitory activity against DPP4  [Mangiferin is a glucosyl xanthone and is the major phytochemical in operties . Suman aoperties . The othoperties . The stuoperties . Mangifeoperties . Additiooperties . Total cd groups . Other mctively) .Pancreatic alpha-amylase allows the hydrolysis of carbohydrates through the breakdown of α-1,4-glycosidic bonds, forming linear and branched oligosaccharides, which are subsequently converted to glucose ,13,65. SAcarbose, miglitol and voglibose are examples of inhibitors, where the former shows an excessive inhibitory activity of pancreatic alpha-amylase with consequent abnormal bacterial fermentation of carbohydrates in the colon, resulting in side effects such as flatulence, bloating, and possible diarrhea . As a reAndrias davidianus) was evaluated for its potential inhibitory activity against alpha-amylase and alpha-glucosidase, with further purification and identification of antidiabetic peptides [50 values of 13.76 × 103, 10.82 × 103, 4.46 × 103, 4.23 × 103 and 2.86 × 103, respectively; and with alpha-glucosidase IC50 values of 206.00, 162.00, 66.90, 63.50, and 42.93 μg/mL, respectively [Lagerstroemia speciosa), ethanol extract of chestnut astringent skin, and a purified compound isolated from white beans (Phaseolus vulgaris) [Protein hydrolysate from Chinese giant salamander (peptides . The pepectively . Garza aulgaris) .50 = 8.7 ± 0.8 μg/mL), followed by green tea extract (IC50 = 34.9 ± 0.9 μg/mL), and white tea (IC50 = 378 ± 134 μg/mL) [50 = 0.5 ± 0.1 μg/mL), followed by grape seed extract (IC50 = 1.2 ± 0.2 μg/mL) and white tea extract (IC50 = 2.5 ± 0.4 μg/mL) [Camelia sinensis [50 = 40.02 ± 4.61 μM) and kaempferol diglycoside against alpha-amylase (IC50 = 0.09 ± 0.02 μM) [Morinda lucida present high inhibitory effect against alpha-amylase with IC50 = 2.30 mg/mL and α-glucosidase IC50 = 2.00 mg/mL through a competitively and mixed noncompetitive mode of inhibition, respectively [Senna surattensis ethanolic extract (IC50 = 123.95 μg/mL for α-amylase) [Picralima nitida (IC50 = 6.50 mg/mL for α-amylase and IC50 = 3.00 μg/mL for α-glucosidase) [Olea europea  [Abutilon indicum (IC50 = 191.64 mcg/mL for α-amylase and IC50 = 207.13 mcg/mL for α-glucosidase) [Ononis angustissima flowers, leaves and stems reveled strong inhibitory activity against alpha-glucosidase  [50 = 2.01 mg/mL and IC50 = 2.52 mg/mL, respectively) [Cissus cornifolia, with IC50 value of 22.75 ± 1.23 μg/mL and 2.81 ± 0.97 μg/mL, respectively, where aqueous root extract revealed IC50 value of 33.70 ± 3.75 μg/mL and 37.48 ± 2.35 μg/mL, respectively [Hibiscus sabdariffa) has potent inhibitory effect against alpha-amylase and alpha-glicosidase  with IC50 values of 41.77 μg/mL for the former and 18.09 μg/mL for the latter [v/v) at 5 mg/mL exhibit the highest inhibitory activity against alpha-glicosidase (66%) and alpha-amylase (58%) [50 values of 25, 42 and 32 μg/mL respectively [Extracts from grape seed, green tea, and white tea were evaluated for their potential to inhibit alpha-amylase and alpha-glucosidase . Grape s4 μg/mL) . However4 μg/mL) . Anothersinensis . The inv0.02 μM) . Aqueousectively . Besidesamylase) , leaf acosidase) , hydroxyamylase)  and ethaosidase) . Aqueousctively) . Howeverctively) . Alpha-aectively . Ethanole latter . Differee latter . “Sweet se (58%) . On the ectively . This stectively .Eisenia bicyclis inhibit alpha-amylase [Grateloupia elliptica shown inhition against Saccharomyces cerevisiae alpha-glucosidase  and Baccilus stearothermophilus alpha-glucosidase  [Ishige okamure, evidenced an inhibitory effect against alpha-glucosidase (IC50 = 0.16 mM) and alpha-amylase (IC50 = 0.53 mM) without cytotoxic effect in human umbilical vein endothelial cells in concentrations from 0.49 to 3.91 mM [Cladophora rupestris and alpha-glucosidase in methanol (67%) and ethyl acetate (61%) extracts both inhibitions at 1000 μg/mL [Palmaria sp. and brown algae Alaria sp. and Ascophyllum sp.  show inhibitory effect against alpha-amylase, where the last also inhibits alpha-glucosidase (IC50 ≈ 19 μg/mL) [Ulva reticulata and Garcilaria edulis [Dieckol and eckol from brown algae -amylase , while bctively) . Further 3.91 mM . The inh00 μg/mL . Phenoli9 μg/mL) . Other sa edulis . In thisT2DM is a serious worldwide disease, occuring mainly because of an unhealthy lifestyle. Its prevention and control are achieved with changes in lifestyle, sometimes coupled with medication with several side effects and high costs associated. As a result, the search for new possible anti-hyperglycemic and anti-diabetic agents from natural sources without, or with less, side effects and at a low cost for the patient, has attracted interest from the scientific community, as shown in this review. The natural sources include extracts from plants, fruits, vegetables and specially from marine macro or microorganisms with bioactive compounds. Indeed, marine organisms such as seaweed from edible species have several compounds with modes of action involving specific mechanisms that can be employed in T2DM treatment. The bioactive compounds include polysaccharides and dietary fibers, fatty acids (MUFA and PUFA), and phenolic compounds. Taking into account that the development of T2DM takes time, the glycemic control can be prevented with the intake of healthy foods like seaweed for glycemic control as preventive measures. However, further research in the future is required to fully understand the anti-diabetic mechanisms of this type of food in the prevention and management of this pathology.


Tinnitus was confirmed in the treated rats via Behaviour Testing of Acoustic Startle Response (ASR) and Gap Prepulse Inhibition Test of Acoustic Startle Reflex (GPIAS). A immunostaining study showed that there is significant loss of anti-CtBP2 puncta (a marker of cochlear inner hair cell (HC) ribbon synapses) in treated animals in apical, middle, and basal turns . The ABR wave I amplitudes were significantly reduced at 4, 8, 12, 14, 16, and 20 kHz . No significant losses of outer HCs, inner HCs, or HC cilia were observed . Thus, our study suggests that loss of cochlear inner HC ribbon synapse after SS exposure is a contributor to the development of tinnitus without changing hearing threshold.Tinnitus is a common auditory disease worldwide; it is estimated that more than 10% of all individuals experience this hearing disorder during their lifetime. Tinnitus is sometimes accompanied by hearing loss. However, hearing loss is not acquired in some other tinnitus generations. In this study, we injected adult rats with salicylate sodium (SS) (200 mg/kg/day for 10 days) and found no significant hearing threshold changes at 2, 4, 8, 12, 14, 16, 20, or 24 kHz (all  Tinnitus is becoming a serious health problem worldwide –3. It waSS, an active component of the nonsteroidal anti-inflammatory drug aspirin, has been commonly used to generate tinnitus in animals . SS is aHere, we present the evidence that an appropriate dose of SS exposure can cause tinnitus with normal audiogram and cochlear HCs, but loss of cochlear ribbon synapse suggesting loss of cochlear inner HC ribbon synapse may largely contribute to SS-induced tinnitus.All studies were approved according to the Institutional Animal Care and Use Committee at the Capital Medical University of China. Wistar rats  were obtained from the animal experimental ministry of Capital Medical University. Animals were divided into two groups according to intraperitoneal injection contents for ten days: (i) control group with injection of saline and (ii) SS-treated group with an injection of 5% (200 mg/kg) SS.ASR and GPIAS were used to measure tinnitus generation. Animals were placed in a permeable sound box resting on a sensitive piezoelectric capable of generating a voltage proportional to the magnitude of the startle responses, evoked by sound stimuli generated by a digital signal processor. The test apparatus was located in a soundproof chamber equipped with a tweeter on the chamber's ceiling about 10 cm above the rat's head. Animals were placed in the box for ten minutes prior to testing for adaptation. Test sessions include gap and no gap trial pair-arranged. The background sound is a pure tone of 12 k-16 k Hz, 70 dB, and the startle stimulus is 120 dB broad-banded noise (20 ms) in each trial. The gap lasting 75 ms was embedded in the background tone 100 ms prior to the startle stimulus. The maximum startle reflex within 250 ms after startle stimulus was recorded. Intertrial time was settled 12 sec to 21 sec randomly. ASR amplitude was recorded as the voltage generated by startle reflex of the tested animals . GPIAS was calculated as a ratio using the formula: 1 − (gap/no gap). Animals with tinnitus have been speculated to demonstrate poorer gap detection ability, which can be measured as a lack of suppression of the startle response in gap trial. Tinnitus rats present a lower GPIAS ratio. We compared the ASR amplitude and GPIAS inhibition rates of the SS-treated and control groups.The evoked response signal-processing System 3 hardware  and SigGen/BioSig software (Tucker Davis Technologies) were used for ABR measurement. Animals were anesthetized with Xylazine (10 mg/kg) and Ketamine (90 mg/kg) and placed in a soundproof chamber. Three needle electrodes were inserted at vertex (active) and beneath of each pinna (reference and ground), subcutaneously. The ABR threshold is the lowest stimulus intensity that produced reliable and reproducible ABR waves. We evaluated the rats hearing status before and at the end of this study and recorded ABR threshold at pure tone frequency of 2, 4, 8, 12, 16, 20, and 24 k Hz. Wave I amplitude was measured peak to baseline, and latency was measured within a specified time window at 90 dB level at each frequency.Animals were decapitated after anaesthesia, and the temporal bones were removed and fixed for 2 h in 10% formaldehyde in phosphate-buffered saline (PBS). After the samples were rinsed in PBS, the basilar membranes of the cochlea were dissected out for immunostaining. Permeabilized with 0.3% TritonX-100 (Sigma-Aldrich) for 30 min and blocked with 10% normal goat serum (Jackson) for 1 h; the tissues were incubated overnight at 4°C with the following primary antibodies: rabbit anti-myosin 7a ; phalloidin 594 ; mouse anti-CtBP2 ; and chicken anti-NF200 . After rinsing with PBS, the samples were incubated in fluorescently labelled secondary antibodies  for 1 h at room temperature. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (AppliChem).μm. Cross- sectioned cochleae were imaged covering the entire inner HC nucleus and areas beyond it in an image stack along the z-axis (z-stack).Laser scanning confocal microscopy was conducted with a 63 X oil immersion objective lens (LEICA TCS SP8). Excitation wave lengths were 488, 568, and 594 nm, and local images were digitally magnified by twofold. Sequence scanning was performed from the apex and obtained at an interval of 0.5 Quantification of ribbon synapses, outer HCs, and inner HCs was performed in the cochlear apex turn, middle turn, and basal turn. Quantification was performed from the top of the cochlea to the bottom. In each basilar turn, we choose three visual fields containing approximately 9-11 inner HCs and 27-33 outer HCs. We selected five samples in each group to calculate the average number of ribbon synapses and HCs. The images were identified inner HC ribbon synapses by red fluorescence spot (indicating a pre synaptic ribbon) that appeared in each image. The total number of red fluorescence marks was obtained using Photoshop software. The number of marks in single inner HC was calculated. In the same way, the numbers of outer HCs and inner HCs in single image were calculated (green fluorescence staining).p values < 0.05 were considered statistically significant.All data are presented as the mean ± SE. Statistical analysis was performed using GraphPad Prism 7 software . Statistical differences between groups in ABR threshold shifts, HC counts, and synaptic counts were analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test. p < 0.05). Next, GPIAS was measured in both groups. The mean ratios in the treated and control groups were 0.5145 ± 0.045 and 0.68 ± 0.0466 (ratio) (p < 0.05), consistent with the previous studies. These results indicate that SS-induced tinnitus.Rats were intraperitoneally injected with SS (200 mg/kg/day) for 10 days. Tinnitus was evaluated via behaviour detections . First,  (ratio) . The meap > 0.05), indicating that SS did not cause hearing loss.To examine whether SS exposure in this study induced hearing loss, ABR thresholds of both groups were measured. Pure tone ABR testing was performed at 2, 4, 8, 12, 16, 20, and 24 kHz 2 h after the last SS injection. For the SS-treated group, the respective results were 33.13 ± 2.10, 29.38 ± 0.62, 29.38 ± 0.62, 28.13 ± 0.91, 28.75 ± 1.56, 32.50 ± 1.33, and 45.00 ± 1.33 dB SPL; for the control group, the respective results were 34.17 ± 1.53, 29.17 ± 0.83, 28.33 ± 1.05, 26.67 ± 1.05, 28.33 ± 1.05, 31.67 ± 1.05, and 43.33 ± 1.66 dB SPL . These findings were not significantly different . This staining was dramatically reduced in all three turns in the SS group Figures . The redp < 0.05), indicating that ABR wave I amplitude decreased significantly in the rats after SS treatment. But no significant difference was found in ABR wave I latency between the two groups.ABR wave I amplitudes were measured at 2, 4, 8, 12, 16, and 20 kHz; respective amplitudes were 462.6 ± 108.0, 433.0 ± 81.2, 320.8 ± 54.2, 362.7 ± 81.4, 217.3 ± 38.1, and 133.2 ± 17.2 nV in SS-treated animals, whereas they were 663.6 ± 90.5, 645.3 ± 90.18, 511.2 ± 71.3, 381.4 ± 48.3, 410.6 ± 54.0, and 254.2 ± 33.0 nV in control animals. These differences were statistically significant  identified changes in auditory innervations. We found swelling fibers of ANs and overlapped puncta  after SS exposure . SS induHCs in the cochlea play a critical role in converting mechanical sound waves into neural signals for hearing –21. MostKujawa and Liberman found that, after noise exposure, mice exhibited significant reductions in ABR wave I amplitudes, TTS, and loss of cochlear ribbon synapses . HoweverPrevious studies reported that different dosages of SS exposure can trigger tinnitus , 36. SS-Loss or damage of cochlear ribbon synapses will result in auditory disorder, regardless of whether hearing thresholds are affected. Cochlear ribbon synapses encode auditory information via fusion of massive neurotransmitter vesicles to achieve fast and tonic releasing , 42. SynNoise exposure causes excessive neurotransmitter release and sodium influx into the postsynaptic terminal of the SGNs, leading SGN nerve (AN nerve) endings to swell. The resulting loss of synaptic connections is irreversible . We founOur study showed that moderate SS doses did not significantly change the numbers of outer or inner HCs, explaining the normal audiogram. It has been shown that moderate doses of SS do not affect auditory sensitivity, but steadily induce tinnitus , 50. Her


Inhalational anaesthetics were previously reported to promote ovarian cancer malignancy, but underlying mechanisms remain unclear. The present study aims to investigate the role of sevoflurane- or desflurane-induced microRNA (miRNA) changes on ovarian cancer cell behaviour. The cultured SKOV3 cells were exposed to 3.6% sevoflurane or 10.3% desflurane for 2 h. Expression of miR-138, -210 and -335 was determined with qRT-PCR. Cell proliferation and migration were assessed with wound healing assay, Ki67 staining and Cell Counting Kit-8 (CCK8) assay with or without mimic miR-138/-210 transfections. The miRNA downstream effector, hypoxia inducible factor-1α (HIF-1α), was also analysed with immunofluorescent staining. Sevoflurane or desflurane exposure to cancer cells enhanced their proliferation and migration. miR-138 expression was suppressed by both sevoflurane and desflurane, while miR-210 expression was suppressed only by sevoflurane. miR-335 expression was not changed by either sevoflurane or desflurane exposure. The administration of mimic miR-138 or -210 reduced the promoting effects of sevoflurane and desflurane on cancer cell proliferation and migration, in line with the HIF-1α expression changes. These data indicated that inhalational agents sevoflurane and desflurane enhanced ovarian cancer cell malignancy via miRNA deactivation and HIF-1α. The translational value of this work needs further study. Ovarian cancer is the eighth most common cancer among women patients with poor prognosis ,2. The 1MicroRNAs (miRNAs) are noncoding short nucleotides that affect cancer cell biology via protein post-transcription. miRNAs control the normal cell and cancer cell biology . Interesp < 0.001; desflurane 14.68 ± 1.61, p = 0.036 vs. control 12.34 ± 1.68) and at 8 h  after gas exposure compared to the control group. There were no differences between sevoflurane and desflurane (n = 6)  assay and Ki67 immunofluorescent staining. Sevoflurane or desflurane exposure exhibited procancer effects in SKOV3 cells. In the wound healing assay, sevoflurane and desflurane significantly increased the “wound” gap closure ratio at 4 h  a,b.p = 0.012; desflurane 1.11 ± 0.04, p < 0.001 vs. control 1.00 ± 0.04) after gas exposure compared to the control group. There were no differences between sevoflurane and desflurane (n = 6)  c.p < 0.001; desflurane 42.69 ± 1.35, p < 0.001 vs. control 28.08 ± 1.52). There was a significant difference between sevoflurane and desflurane   d,e.p = 0.020; desflurane 0.61 ± 0.16, p = 0.006 vs. control 1.00 ± 0.18, n = 6, p = 0.009; desflurane 0.89 ± 0.22, p = 0.686 vs. control 1.00 ± 0.22). There was a significant difference between sevoflurane and desflurane , and miR-138 mimic treatment decreased at 8 h after the exposure .To ascertain the involvement of miR-138 and -210 to cell biology changes, the mimics of these miRNAs were transfected to SKOV3 cells before anaesthetic exposure. miR-210 mimic treatment decreased “wound” gap closure ratio at 4 h and 8 h after air exposure as shown in p = 0.010 vs. sevoflurane 15.75 ± 1.31; 8 h after the exposure: sevoflurane + miR-210 mimic 30.66 ± 3.21, p < 0.001 vs. sevoflurane 46.28 ± 1.74, n = 6), and miR-138 mimic treatment decreased at 8 h after the exposure . Under sevoflurane exposure, the same tendency was observed as summarized in p < 0.001 vs. desflurane 46.03 ± 2.63, n = 6, With desflurane exposure, only miR-138 mimic treatment suppressed cell migration at 8 h after the exposure , which significantly lessened with miR-138 and -210 mimic treatment at any anaesthetic condition . miR-138 and -210 are reported to control HIF-1α expression, a key factor of cancer malignancy. As shown in p = 0.905, control + miR-138 mimic 0.78 ± 0.26, p = 0.231 vs. control 1.00 ± 0.15; sevoflurane + miR-210 mimic 1.07 ± 0.13, p = 0.961, sevoflurane + miR-138 mimic 0.87 ± 0.33, p = 0.103 vs. sevoflurane 1.11 ± 0.12; desflurane + miR-210 mimic 1.07 ± 0.09, p = 0.936, desflurane + miR-138 mimic 0.81 ± 0.28, p = 0.224 vs. desflurane 1.01 ± 0.18, n = 6–7, The Western blotting analysis showed similar trend changes but did not reach any statistical significance  demonstrated that inhalational anaesthetic agents exert procancer effects via miR-138 and/or miR-210/HIF-1α modulations. Furthermore, miR-210 and -138 mimic administration attenuated the protumour effects of inhalational anaesthetics with HIF-1α suppression. Taken together, both sevoflurane and desflurane may promote cancer cell migration and proliferation by direct HIF-1α upregulation via miR-138 and -210 suppression.Our data demonstrated that sevoflurane and desflurane, commonly used for surgical anaesthesia, may contribute to the risk of cancer recurrence after surgery that may be involved with the HIF-1α signalling pathway via miRNA mediation . The impSeveral studies showed that HIF-1α was directly regulated by miRNAs including miR-138  and miR-Inhalational anaesthetic isoflurane was reported to increase ovarian cancer cell proliferation and migration , and allThe present study was not free from limitations. Firstly, this is an in vitro study with one cell line of ovarian cancer types. Furthermore, SKOV3 unlikely represents the most common ovarian cancer cell type of high-grade serous carcinoma (HGSC) ,53 and eIn summary, our data indicated that sevoflurane or desflurane promoted SKOV3 cell migration and proliferation via miR-138 and -210 suppression. Although the clinical significance of our work requires further investigation, this study undoubtedly enhances our understanding of the impact of inhalational anaesthesia on cancer cells during the perioperative period and provides supporting evidence for the selection of appropriate anaesthetics for the benefit of patients’ prognosis after surgery. 2 balanced with air in McCoy’s 5A medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine (Sigma-Aldrich) and 1% penicillin (Sigma-Aldrich) for the experiments described below with or without inhalational agent exposure for further analyses. SKOV3 human ovarian epithelial carcinoma cell line  was cultured at 37 °C in a humidified atmosphere containing 5% CO2, 5% CO2 and 3.6% sevoflurane or 10.3% desflurane balanced with N2  in a purpose-built 1.5 L airtight gas chamber, equipped with inlet and outlet valves. The chamber was then placed in an incubator  at 37 °C for 2 h. Other cohort cells were exposed to the same concentration gases without any inhalational anaesthetics and served as controls. After exposure, cells were returned to the normal culture incubator for further study. Both sevoflurane and desflurane were set at the equipotent concentrations approximately equal to 1.7 minimum alveolar concentrations in human. When cultures reached 60%, they were exposed to 21% O4) were seeded into each well , incubated for 24 h before wound was made and then exposed to the experimental gas mixtures as above for 2 h. The gap closure was monitored under the microscope and a digital camera  at 0, 4 and 8 h after gas exposure. Images were analysed using Image J version 1.52a software . Cells (2.5 × 103) were seeded in 96-well plates and exposed to the experimental gas mixture. Cell proliferation was assessed by the CCK8 reagent (Sigma-Aldrich) which was read from culture media at 450 nm  at 24 h after gas exposure. Cell proliferation was expressed relative to the corresponding control.Cells .Immediately after the anaesthetic exposure, total RNA was extracted from cells using QIAzol Lysis Reagent  and an miRNeasy Mini Kit (Qiagen) following the manufacturer’s protocols. RNA quantity and quality were assessed using a BioPhotometer . Samples with an A260/A280 ratio > 1.8 were considered to be sufficient quality for further analysis. A total of 1 ng RNA was converted to cDNA using miScript II RT Kit (Qiagen) with the thermal cycles of 37 °C for 60 min and 95°C for 5 min (Mastercycler-ΔΔCt method. A 2-fold change or over was considered to be of significance.qRT-PCR was performed with the miScript SYBR Green PCR Kit (Qiagen) and Rotor gene Q system (Qiagen). SNORD44 small nuclear RNA was used as an endogenous control. The specific primers, miR-138-5p, miR-210-3p and miR-335-5p, were purchased from Qiagen. Thermal cycles were as follows: 95 °C for 15 min and 40 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Melting curve analysis was used to confirm the specificity of amplification. The relative expressions of miRNAs to SNORD44 were determined using the comparative 2The mimics of miR-138-5p (5′AGCUGGUGUUGUGAAUCAGGCCG) and -210 (5′CUGUGCGUGUGACAGCGGCUGA) were purchased from Qiagen in comparison with All Stars Negative Control siRNA (Qiagen) as the negative control. Cells were transfected with either 50 nM of the mimic or the negative control using the HiPerFect transfection reagent (Qiagen), following the manufacturer’s protocols. After 24 h of transfection, the transfection solution was replaced with fresh medium before the experimental gas exposure.Cells at 24 h after gas exposure were fixed in 4% paraformaldehyde for 10 min and blocked with 10% normal donkey serum (Sigma-Aldrich) for 1 h, followed by overnight incubation at 4 °C in primary antibodies: rabbit polyclonal anti-Ki-67 antibody  and rabbit polyclonal anti-HIF-1α antibody . Cells were incubated in Alexa flour 568-conjugated secondary antibody (ThermoFisher scientific) on the following day. Then, cells were costained for the cell nuclei with Vectashield mounting medium containing nuclear dye 4′,6-diamidino-2-phenylindole–mounting medium (Millipore). Slides were then examined using a BX60 wide-field fluorescence microscope  and AxioCam camera  with Zeiss software under 20× magnification. Images were captured using a camera. Fluorescence intensity was quantified as the mean pixel intensity of relevant antibody staining using Image J . Ten representative regions per slide were randomly selected. Intensity values were calculated and expressed as relative to the control.® 4–12% Bis-Tris Precast Gels (Thermo Scientific) for electrophoresis. After electrophoresis, proteins were transferred onto a polyvinylidenedifluoride membrane using the iBlot®2 Dry Blotting System (Thermo Scientific). Membranes were blocked with 5% nonfat powdered milk in Tris-buffered saline with Tween for 1 h at room temperature, then incubated overnight at 4 °C with rabbit anti-HIF-1α primary antibody  followed by horseradish peroxidase-linked antirabbit secondary antibody  for 1 h. Protein bands were visualised using the Enhanced Chemiluminescence system  and Syngene GeneSnap software . The intensity of grey-scale protein bands was assessed using ImageJ software.The protein was extracted from cell samples using a cell lysis buffer  at 24 h after gas exposure and quantified with a Bradford protein assay . Then, 60 µg of protein of each sample was loaded into NuPAGEp value less than 0.05 was considered to be a statistical significance.All numerical data are presented as dot plots and expressed as mean ± SD. One-way analysis of variance followed by post-hoc Tukey’s test were used for data analyses with Prism version 8.0 . A In conclusion, our data demonstrated that inhalational anaesthetics enhanced some ovarian cancer cell biology via miRNA downregulation. The regulatory mechanisms of HIF-1α expression via miRNAs may contribute to the detrimental effects of inhalational anaesthetics and hence worsen cancer outcomes. This research is a proof-of-concept study with one cell line. In order to investigate “common and general onco-effects of inhalational agents”, more cell lines need to be considered in future studies. These data may warrant more research to refine optimal anaesthetic regimens for cancer patients including ovarian cancer sufferers.