The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and p While inhibition of AChE can lead to life threatening intoxication due to cholinergic overstimulation and crisis, inhibition of BChE has no direct adverse effects. For this reason, BChE can be used as the OP bioscavenger and it is considered as a pseudo-catalytic bioscavenger1), trimedoxime (TMB-4) (2), obidoxime (3) and asoxime (HI-6) (4)  (4) 8,9.5), K048 (6) and K203 (7) (in vitro or in vivoThe major drawback of these reactivators is their limited broad-spectrum efficiency against various OPs and their low ability to pass the blood-brain barrier (BBB). That is why the current research is focussing on synthetising derivatives with modified properties, i.e. uncharged reactivators or modified nucleophilesK203 (7)  have bee5), K048 (6) and K203 (7). The pyridinium scaffold with oxime moiety was retained as the α-nucleophile that is able to attack OP-inhibited cholinesterase. Similarly, the second aromatic moiety and the connecting linker remained to be same as in the parent molecules. Differently, the carboxamide moiety was replaced by thiocarboxamide to investigate the sulphur involvement in the binding of reactivator’s molecule within the active site of AChE or BChE. The carboxamide fragment was formerly found to play an important role in hydrogen bonding of charged oximes in the peripheral site of AChEFor this reason, the molecular design of charged pyridinium oximes with thiocarboxamide moiety was proposed from K027 . Melting points were recorded on a Melting Point Apparatus - Büchi B-545  without correction. The 1H and 13C NMR spectra were measured in DMSO-6d solution at room temperature on FT NMR spectrometer Avance NEO 500 MHz (499.87 MHz for 1H and 125.71 MHz for 13C) . Chemical shifts, δ, are given in parts per million (ppm) and spin multiplicities are given as br s (broad singlet), s (singlet), d (doublet) or m (multiplet). Coupling constants, J, are expressed in hertz (Hz). For 1H δ is relative to DMSO-6d (δ = 2.50) and for 13C is relative to DMSO-6d (δ = 39.43). High Resolution Mass Spectrometry (HRMS) was determined by Q-Exactive Plus hybrid quadrupole-orbitrap spectrometer.All chemicals used for the synthesis were purchased from Sigma-Aldrich  in the highest available purity. All used solvents were supplied by Penta Chemicals Unlimited . Organophosphorus compounds 4-nitrophenyl isopropyl methylphosphonate , 4-nitrophenyl ethyl methylphosphonate , 4-nitrophenyl ethyl dimethylphosphoramidate  and paraoxon (POX) were purchased from Chemforase . Thin layer chromatography was performed on Merck silica gel 60 F8–9 were prepared by reaction of 4-hydroxymethylpyridine (32.8 mM) and dibromoalkane (163.8 mM) in acetone (30 ml). The mixture was stirred at reflux for 8 h, then cooled to room temperature and filtered and washed with acetone (3 × 20 ml). The crude product was crystalised from acetonitrile (100 ml per 1 g of product) at reflux and filtered under reduced pressure. The filtrate was evaporated under the reduced pressure to produce monoquaternary salts 8–9,The monoquaternary salts 10 was prepared by reaction of 4-hydroxymethylpyridine (8.20 mM) and (E)-1,4-dibrombut-2-ene (40.90 mM) in acetone (30 ml). The mixture was stirred at reflux for 1.5 h, then cooled to room temperature and crystalline crude product was collected by filtration, washed with acetone (2 × 30 ml). The product was recrystalised from acetonitrileThe monoquaternary salt 8–10 (1.48 mM) in dimethylformamide  was added 4-pyridinethioamide (2.22 mM). The resulting mixture was stirred at 60 °C for 48 h. The solvent was concentrated under the reduced pressure and the crude product was purified by crystallisation from acetonitrile at reflux. Then the solid was filtered, washed with acetonitrile and dried under the vacuum.Secondly, the synthesis of the bisquaternary salt was completed. To a solution of monoquaternary salt 11 was isolated as orange solid, yield 245 mg (36%), m.p. 127.1–129.1 °C. 1H NMR : δ 2.65–2.71 , 4.75–4.80 , 8.28 , 8.34 , 8.47 , 9.13 , 9.24 , 10.29 , 10.74 , 12.85 . 13C NMR : δ 31.4, 56.9, 57.2, 124.1, 124.9, 145.0, 145.1 145.4, 148.5, 152.5, 194.1. HRMS (HESI+): [M + H]2+: calculated for C15H18N4OS2+ (m/2z): 151.0595; found: 151.0594.Compound 12 was isolated as orange solid, yield 364 mg (52%), m.p. 180.3–182.3. 1H NMR : δ 1.96–2.02 , 4.67–4.76 , 8.25 , 8.31 , 8.45 , 9.14 , 9.25 , 10.29 , 10.72 , 12.81 . 13C NMR : δ 26.9, 27.0, 59.2, 59.5, 124.0, 124.9, 145.0, 145.2, 148.3, 152.4, 194.2. HRMS (HESI+): [M + H]2+: calculated for C16H20N4OS2+ (m/2z): 158.0673; found: 158.0673.Compound 13 was isolated as orange solid, yield 431 mg (61%), m.p. 199.1–201.1 °C. 1H NMR : δ 5.35–5.39 , 6.17–6.30 , 8.27 , 8.32 , 8.46 , 9.06 , 9.17 , 10.34 , 10.75 , 12.87 . 13C NMR : δ 60.3, 60.6, 124.0, 124.9, 129.6, 130.6, 145.0, 145.1, 145.3, 148.7, 152.9, 194.2. HRMS (HESI+): [M + H]2+: calculated for C16H18N4OS2+ (m/2z): 157.0595; found: 157.0594.Compound The stability of tested compounds was evaluated in two different media – demineralised water and phosphate buffered saline  as simulation of physiological environment. Compounds were dissolved in particular solvent  and incubated at 37 °C. The amounts of analysed compounds were determined at time intervals of 0, 1, 2, 3, 4 and 5 h by UHPLC Infinity II 1290 system  coupled with DAD detector. The stability was evaluated as the amount of remaining compound (%) in sample after specific time interval. GraphPad Prism version 8.2  was used for the statistical data evaluation and visualisation.aK) of prepared compounds were determined spectrophotometrically using buffers of given pH (range from 4.5 to 10.0 with 0.5 unit increment). Ten microliters of tested oxime (1 mg/mL) were dissolved in 490 µL of particular buffer and absorbance spectra of various dissociation states were scanned in range 200–400 nm using Carry-60 UV-VIS spectrophotometer  at 20 °C. The paK values were calculated from the sigmoidal dependence of the absorbance of the dissociated form of the substance on the pH value using GraphPad Prism 8.2 software .The negative decimal logarithms of the dissociation constants (phrAChE) and butyrylcholinesterase (hrBChE) were prepared at the Department of Chemistry, Faculty of Science, University of Hradec KralovehrAChE/hrBChE was determined by standard Ellman method adapted for 96-well plateshrAChE  or hrBChE (220 ng/mL of protein), water solution of tested compound at appropriate concentration (the range of 1 µM to 500 µM for hrAChE and of 50 µM to 1500 µM for hrBChE) and solution of 5,5′-dithiobis-2-nitrobenzoic acid  in 20 mM Na-phosphate buffer. The mixture was pre-incubated for 15 min at 37 °C. Afterwards the substrate acetylthiocholine iodide (ATCI) or butyrylthiocholine iodide (BTCI) was added to the final concentration of 1000 µM. The total volume of reaction was 100 µL. Creation of the product 5-thio-2-nitrobenzoic acid (TNB), formed during the reaction was determined by observing its absorbance at specific wavelength 436 nm. The catalytic activity of enzyme was evaluated as amount of product (%) formed in the reaction after 10 min of incubation at 37 °C. IC50 values of compounds were calculated using non-linear regression by GraphPad Prism 8.2 . Data were calculated from three individual experiments which were made in triplicate.Recombinant forms of human acetylcholinesterase (hrAChE/hrBChE) were inhibited by 25 µM POX, NEMP, NIMPor NEDPA for 30 min to obtain >99% inhibition. The excess of organophosphate was removed by dialysis against 25 mM Na-phosphate buffer (pH 7.4) for 16 h with three buffer exchanges. The tested oxime (10 µM or 100 µM) was incubated with inhibited enzyme for 15 or 30 min at 37 °C. The reaction mixture (100 µL) contained 10 µL of inhibited enzyme , 20 µL of DTNB (2.5 mM), 10 µL of relevant oxime solution and 50 µL of Na-phosphate buffer . In parallel, reaction mixture containing no enzyme was followed as blank reaction to reflect oximolysis. The reaction was started by addition of 10 µL of substrate ATCI or BTCI (10 mM). The catalytic activity of enzyme reactivated by oxime was determined spectrophotometrically at specific wavelength 436 nm using Spark multimode microplate reader Tecan . Data were calculated from three individual experiments which were made in triplicate.Recombinant enzymes (hrAChE/hrBChE) was incubated for eight different times (0.5 to 15 min) with seven different concentrations of tested oxime (varying from 1 to 1200 µM) at 37 °C. As the blank the mixture containing no enzyme (to control oximolysis) was used. Acquired data were analysed by non-linear regression analysis according to Worek et al.The selected compounds with promising reactivation ability were further tested to investigate reactivation kinetics parameters. Inhibited enzyme (8–10 were formerly prepared by our research groupN2) of these monoquaternary salts with 4-pyridinethioamide in aprotic polar solvent DMF allowed creation of bisquaternary salts 11–13 (11 (K487) which was probably caused by steric hindrance of two close pyridinium rings. The highest yield was obtained for compound 13 (K489), which contains but-2-en-1,4-diyl chain. In this case, the substrate 10 react rapidly by the SN2 mechanism because the π-system of the adjacent double bond can stabilise the transition state by conjugation. The final compounds were determined by NMR  and HRMS analysis. Based on NMR and HRMS analysis, the non-calibrated purity of all products was ≥95%.The monoquaternary precursors ts 11–13 . The yie11), K488 (12) and K489 (13) and analogous oximes K027 (5), K048 (6) and K203 (7) were tested for stability in unionised water and PBS at 37 °C for 5 h, when the buffered environment and selected temperature are better mimicking human relevant conditions and the selected time is close to a full elimination of similar oximes from human organism. All compounds showed high stability in both media after 5 h (13) and K203 (7) was found in PBS buffer, but still with residual >90% oxime after 5 h. Stability is very important parameter for the compound use and also for maintaining the biological activity for the necessary time i.e. first 1–2 h after OP intoxication for charged oximesPrepared compounds K487 , K488 (12), K489 (13), pralidoxime (1), asoxime (4) and for parent oximes (5–7). The paK is an important parameter in pharmaceutical development to rationalise the physicochemical and biopharmaceutical properties of the drug molecule. It allows to detect protonated/deprotonated form of the compound under physiological conditions. In case of oximes deprotonated form so called oximate anion is important, because it is the active form of the oxime reactivatoraK values of oxime moiety (The pe moiety . This re11–13 (K487-K489) and oximes K027 (5), K048 (6), K203 (7), pralidoxime (1) and asoxime (4) were tested for in vitro inhibition of hrAChE and hrBChE. While standard and parent oximes resulted as poor AChE and BChE inhibitors, compounds K487 (11), K488 (12) and K489 (13) resulted as relatively stronger inhibitors of both enzymes , while inhibition of hrBChE resulted to be relatively lower in high µM scale (IC50∼189–320 µM). Oxime K488 (12) was found to be the most potent hrAChE (IC50∼7 µM) and hrBChE (IC50∼189 µM) inhibitor. This increase of inhibitory ability is most probably caused by sulphur atom. Although we did not study particular interaction with ChEs active sites, we suppose that bulkier sulphur atom has different binding and interactions at the active site gorge (e.g. hydrogen bonding properties) i.e. the sulphur atom is probably occupying more space within the peripheral site of the active site of AChE, which leads to stronger interactions with present aromatic amino acids and consequently increases the compound’s inhibition potency. The inhibition of BChE was less pronounced since BChE has less aromatic amino acids in its active site.All novel compounds  enzymes . The inh,Supplementary Table 2). For NIMP-AChE, the best reactivation was obtained for 6 (∼73%) followed by asoxime . Generally, the thiocarboxamides (11–13) were found as less potent reactivators when compared to carboxamide analogues (5–7). For NEMP-AChE, the best reactivation was observed with carboxamides  followed by thiocarboxamide K487 . The NEDPA-inhibited AChE was well reactivated by carboxamides  and thiocarboxamide K487 . The tested oximes were found to be poor reactivators of POX-inhibited AChE, and only K203  was able to restore AChE activity to some extent.All compounds were screened for reactivation ability on both ChEs inhibited by nerve agent surrogates NIMP, NEMP, NEDPA and POX1) and asoxime (4) were found to be generally less potent reactivators of NEMP-, NEDPA- and POX-inhibited AChE, when compared to carboxamides (5–7), while thiocarboxamides (11–13) resulted as weaker reactivators if compared to carboxamides. This result is most probably related to the stronger inhibition of AChE provided by thiocarboxamides which decreases overall activity of reactivated enzyme.Taken together, pralidoxime (Supplementary Table 3). In case of NIMP-BChE conjugate, carboxamides  were found as the best reactivators followed by thiocarboxamides  and pralidoxime . The most potent reactivators of NEMP-BChE conjugate were carboxamides 12 (∼37%) which was found to be the most potent reactivator of NEDPA-BChE as well. Surprisingly for NEDPA, thiocarboxamide 12 (∼25%) showed the best reactivation followed by carboxamides . The POX-BChE was reactivated almost equally by carboxamides  and thiocarboxamide 12 (∼31%). Taken together, pralidoxime (1) and asoxime (4) were found to be generally less potent reactivators of NIMP-, NEMP-, NEDPA- and POX-inhibited BChE, when compared to carboxamides (5–7) and equally or slightly less effective thiocarboxamide 12. The better reactivation profile of thiocarboxamide 12 in case of BChE is related to the lower intrinsic inhibition of BChE and thus the overall higher activity of reactivated enzyme.The reactivation of OP-inhibited BChE was screened with 100 µM concentration of oxime after 15 min  and the ability to remove the OP residue from the active site of the enzyme (reflected by rk) were determined. Afterwards, the specific overall second-order reactivation rate constant (r2k) was calculated (r2k=rk/DK). The highest affinity for NEDPA-inhibited AChE, as well as the fastest dephosphorylation of catalytic serine (rk) was exhibited by oxime K027 (5), which therefore resulted with the highest r2k constant. Thiocarboxamide 11 was found to be a better reactivator of NEDPA-AChE than 2-PAM (1) and asoxime (4), but less effective than K027 (5) (5 and 11 towards NEDPA-AChE was directly compared by using 50 µM concentration of the reactivator (11 showed markedly lower reactivation which is caused by its stronger inhibition of AChE (IC50∼10 µM), which indicates more convenient use of carboxamide moiety instead of thiocarboxamide one when using the same (trimethylene) linker.The affinity of relevant oxime towards OP-inhibited recombinant enzyme (reflected by K027 (5) . In addictivator . In this5) and its analogue thiocarboxamide 11. Furthermore, due to the high reactivation rate (rk) these oximes can be singled out as the best reactivators of NIMP-inhibited BChE with an overall reactivation rate of about 6-fold higher than 2-PAM and HI-6 , NEMP (VX-surrogate), NEDPA (tabun-surrogate) and paraoxon. From reactivation screening, compound K487 (11) showed ability to reactivate AChE inhibited by NEMP and NEDPA. For BChE screening, K487 (11) and K488 (12) were found to have ability to reactivate all tested OPs. The reactivation kinetics was further determined for thiocarboxamides K487 (11) and K488 (12). Oxime 11 resulted as slightly weaker reactivator of NEDPA-AChE or NIMP-BChE if compared to its direct carboxamide analogue 5, which is caused by its higher intrinsic inhibitory ability for both enzymes. Taken together, the tested thiocarboxamides resulted as equally or less effective charged oxime reactivators when compared to their carboxamide analogues. Therefore, the introduction of thiocarboxamide moiety doesn’t bring much benefit to the overall cholinesterase reactivation.Three pyridinium mono-oximes containing thiocarboxamide group were designed, synthetised and tested. The novel compounds were evaluated for stability, and all compounds resulted as highly stable in both media (water and PBS). Further, pClick here for additional data file.


To study whether ropivacaine inhibits the proliferation and migration of colon cancer cells through ITGB1 (Integrin beta-1). First, the effect of ropivacaine on cell proliferation and migration was detected by MTT and Transwell. DAPI staining, annexin V staining and Western blot were used to detect the expression of apoptosis-related proteins to investigate the effect of ropivacaine on cell apoptosis. Using bioinformatics software to predict the potential drug targets of ropivacaine. RT-PCR, Western blot and immunofluorescence verify the distribution and expression of the drug target ITGB1, and detect its downstream-related proteins to further prove that ropivacaine affects colon cancer cells by acting on ITGB1 protein. 1. Ropivacaine significantly inhibited the proliferation of colon cancer cells and promoted their apoptosis 2. Ropivacaine could interact with ITGB1 protein, and inhibited the expression of ITGB1 protein in colon cancer cells, thereby affecting its downstream signaling pathway. Ropivacaine regulates the function of colon cancer cells by targeting the expression of ITGB1 protein and affecting the activation of its downstream signaling pathways.Abbreviation: Integrin beta-1 (ITGB1); 3-(45)-dimethylthiahiazo (-z-y1)-35-di- phenytetrazoliumromide (MTT); 4. 6-diamimo-2-phenyl indole (DAPI); Reverse transcrption PCR (RT-PCR); Colorectal cancer (CRC); Local anesthetics (LA); voltage-gated sodium channel (VGSC); dulbecco s modifed eade medium (DMEM); propidium iodide (PI); dodecyl sulf ate, sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE); Polyvinylidene Fluoride (PVDF); BCL2 associated X (Bax); Focal Adhesion Kinase (FAK); extracellular signal-regulated kmase (ERK); alpha serme threcnime-proteim kinase (AKT); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Tris-buffered salme with 0.1% Tween 20 (TBST); Similarty ensemble approach (SEA)  ColorecIn recent years, more evidence has shown that the risk of cancer spread after surgery has increased ,6. TumorIn this study, we investigated the effects of ropivacaine on the proliferation, migration and apoptosis of colon cancer cells, and further studied whether ropivacaine regulates the biological function of colon cancer cells through ITGB1.2.2.1.2.1.1.Ropivacaine ; MTT, Crystal Violet and DAPI were purchased from Beijing Solarbio; Rabbit anti-human ITGB1 antibody ; goat anti-rabbit IgG-HRP antibody ; FITC Annexin V Apoptosis Detection Kit (BD Pharming).2.1.2.2.Human colorectal cancer HCT116 cells, SW620 cells were purchased from ZhejiangRuyao Biotechnology, and the cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37°C in a constant temperature culture containing 5% CO2.1.3.2 constant temperature incubator (ThermoFisher); fluorescence microscope ; gradient PCR instrument (Bio-rad company in the United States); 7500 F real-time fluorescence quantitative PCR instrument (ABI company in the United States); electrophoresis instrument electrophoresis system (Shanghai Tanon Bio).CO2.2.2.2.1.The cells were seeded in 96 well plates containing 100uL medium at the density of 4000 cells per well. After 24 hours, the final concentrations of 0, 200, 400 and 800 μm of ropivacaine were added to DMED, with 100 μL DMEM as negative control, three multiple holes were set and three independent experiments were carried out. After 48 and 72 h, MTT assay was used to detect cell proliferation. The detection wavelength was 562 nm with the reference wavelength at 630 nm.2.2.3.2. Samples were observed and photographed at 24, 48 h.Scratch test was performed to observe the cell migration ability. First, a marker pen was used to draw a horizontal line with a ruler on the back of the 6-hole plate and the line evenly crossed the hole. Each hole crosses at least five lines. The cells were added to the wells and after overnight cultivation, a ruler was used to scratch the horizontal line perpendicular to the back with the pipette tip. The cells were washed 3 times with PBS to remove the underlined cells, and serum-free medium was added. Incubated in 37°C incubator with 5% CO2.2.4.4 cells/well were seeded in 200 μL of fetal bovine serum-free medium precoated with Matrigel in the upper chamber. Fill the lower well with 1 mL of completed DMEM medium to attract cell invasion. After incubation for 24 h, the cells on the surface of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet, and observed under a microscope. A total of five random high-power microscopic fields (×200) were taken for each filter, and the number of cells was directly counted.To test whether ITGB1 inhibited migration of HCT116 and SW620 cells, Matrigel invasion test was used after 48 h transfection. 102.2.5.5 cells/well, and crawling culture on the slide. After adhesion overnight, cells were treated with 0, 200, 400, and 800 μM ropivacaine, respectively. After 6 h, the cells on coverslip were washed with PBS, and stained with 1 μg/mL DAPI for 30 s. After being washed with pure water and fixed with fluorescent anti-quencher, the cells were imaged with a 400× fluorescence microscope.Cells were seeded on the 12 well plate with an initial density of 5 × 102.2.6.5) were seeded in 6-well plates for cell culture and adherent overnight. The cells were cultured with 0, 200, 400, 800 μM ropivacaine. Standard growth medium was used for negative control. After incubating for 24 h, the supernatant was poured out from the cells to preserve the floating cells. Adherent cells were washed with PBS and digested with 0.25% trypsin; 5 μL FITC annexin and 5 μL propidium iodide (PI) were added to 100 μL cell suspension containing 105 cells. The mixture was evenly mixed and incubated at room temperature for 15 minutes. After adding 400 μL binding buffer, the cells were analyzed by flow cytometry. All experiments were repeated three times.Cells . The qPCR reaction was performed by ABI 7500 PCR system. The forward sequence of human ITGB1 primer was 5ʹ-TGATTGGCTGGAGGAATGTTA-3ʹ and the reverse sequence was 5ʹ-GTTTCTGGACAAGGTGAGCAA-3ʹ. The forward sequence of the control GAPDH primer was 5ʹ-AGAAGGCTGGGCTAATTTG-3ʹ and the reverse sequence was 5ʹ-AGGGGCCATCCACAGTCTTC-3ʹ. 22.2.8.Western blot analysis was performed on the lysates of cells treated with 0, 8 μM ropivacaine. After SDS-PAGE electrophoresis, the extracted cell proteins were transferred to PVDF membrane and blocked with 5% skim milk containing 0.05% Tween. The following rabbit polyclonal antibodies were used to Western blot the membrane: anti-ITGB1/Bax/caspase-3/a-caspase-3/caspase-9/a-caspase-9/FAK/ERK/AKT/p-FAK/p-ERK/p-AKT/GAPDH. They were washed in Tris buffer containing Tween-20 for 3 times, incubated with goat anti-rabbit secondary antibody at a dilution of 1:10,000 at room temperature for 1 h, and then washed with TBST. Finally, scanning with an ultraviolet imaging system.2.2.9.SW620 and HCT116 cells were inoculated in 6-well plate medium containing coverslips for cell crawling culture. After 24 hours of culture, 800 μM ropivacaine was added. The standard cell culture medium was used as a negative control. Cells were fixed with 4% paraformaldehyde after 8 h incubation in 5% CO2 37°C incubator. The rabbit anti-ITGB1 antibody was diluted with 1:1000 for 4°C overnight and then incubated with fluorescent secondary antibody for 1 h. Leica fluorescence microscope was used to observe and photograph.2.2.10.http://sea.bkslab.org/) to predict potential target genes. Use the Genecards (https://www.genecards.org/) database to search with ‘colorectal cancer’ as the keyword to obtain disease targets.  Then, the gene cluster from the two databases are intersected, and then the potential binding targets of ropivacaine are screened based on experience and literature.Structure and SMILE of ropivacaine were downloaded from NCBI PubChem, and the Similarity ensemble approach (SEA) database (2.2.11.https://pubchem.ncbi.nlm.nih.gov/), and optimize the compound structure in the MMFF94 force field of OpenBabel 2.4.1. The three-dimensional structure of ITGB1 is downloaded from RCSB Protein Data Bank (http://www.rcsb.org/) to obtain the three-dimensional structure of ITGB1 (PDB ID: 4WK0). Use AutodockTools1.5.6 to search and define the compound’s rotatable bond; Repair the ITGB1 protein structure by deleting residues and terminal residues on the polypeptide chain, removing water molecules, adding hydrogen atoms, adding charges, and save it in PDBQT format. Autodock vina 1.1.2 was used for molecular docking research. We used POCASA 1.1  to make a pocket prediction of the protein, thus determining the coordinates of the active site of ITGB1: center_x = 19.5, center_y = 13.0, Center_z = −61.3; size_x = 36.2, size_y = 27.0, size_z = 23.8, and other parameters use the default values. The conformation with the lowest docking energy is selected for docking mode analysis, and Pymol is used for mapping .Download the structure of compound ropivacaine from the PubChem database . After statistical processing, the significance was set at P < 0.05. Statistical significance was determined using Student’s t-test and F-test (F-test was used for the statistics of apoptosis level). The Graphpad Prism 8.0 software was used for the visualization of the statistical.The data were presented as the mean ± standard deviation. The statistical analysis was performed using Student’s 3.3.1.P < 0.05) (P < 0.05). The invasion experiment results showed (P < 0.05).In this study, HCT116 and SW620 cells were treated with different concentrations of ropivacaine , and the growth of HCT116 and SW620 cells after 48 and 72 h was detected by MTT method. The results showed that ropivacaine had a dose-dependent effect on the growth of colorectal cancer cells, and the inhibitory effect of 800 μM ropivacaine on colon cancer cells was the most significant ( < 0.05) . DAPI st < 0.05)  that aft < 0.05) ) that whs showed ) that af3.2.In order to explore the effect of ropivacaine on apoptosis of colon cancer cells, HCT116 and SW620 cells were treated with 800 μM ropivacaine for 24 h and then detected the apoptosis by flow cytometry. The results showed that compared with normal colon cancer cells, the percentage of apoptosis of colon cancer cells increased significantly after adding ropivacaine  for quan3.3.Online prediction websites were used to predict the target gene of ropivacaine. As shown in 4.The aim of this study was to investigate the effects of ropivacaine on proliferation, cell cycle, and apoptosis of colon cancer cells. Objective to investigate the effect of ropivacaine on the proliferation and migration of colon cancer cells by targeting ITGB1. Colorectal cancer is a disease that affects millions of people worldwide ,19. BundBaptista-Hon et al.  detected5.This study showed that under the action of ropivacaine, the expression of ITGB1 in HCT116 and SW620 cells was significantly reduced, and ropivacaine can inhibit the proliferation, migration, and invasion of colorectal cancer cells. In addition, we confirmed for the first time that ropivacaine regulates the biological function of colon cancer cells through ITGB1.Click here for additional data file.Click here for additional data file.Click here for additional data file.


We describe a comprehensive characterization of obtained compounds with respect to their anticancer potency and selectivity towards cancer cells. All four novel compounds exert stronger antiproliferative activity than MBA. Moreover, 3b induce apoptosis in colon cancer cell lines. A detailed investigation of the mechanism of action revealed that 3b activity stems from the down-regulation of survivin and the activation of caspase-3. Furthermore, compound 3b attenuates the clonogenic potential of HCT-116 cells. Interestingly, we also found that depending on the type of the silyl group, compound selectivity towards cancer cells could be precisely controlled. Collectively, we demonstrated the utility of silyl groups for adjusting both the potency and selectivity of silicon-containing compounds. These data reveal a link between the types of silyl group and compound potency, which could have bearings for the design of novel silicon-based anticancer drugs.The pharmacological effects of carbon to silicon bioisosteric replacements have been widely explored in drug design and medicinal chemistry. Here, we present a systematic investigation of the impact of different silyl groups on the anticancer activity of mucobromic acid (MBA) bearing furan-2( With over 19 million new cases and 9.9 million deaths in 2020, cancer remains a worldwide cause of premature mortality . Therefo5H)-one scaffold and its variations are widely present in many bioactive natural products + .Brown solid; yield: 76%; m.p. = 91–93 °C; 1H NMR  δ: 1.11 , 1.12 , 1.18–1.26 , 6.10 ; 13C NMR  δ: 12.07, 17.65, 98.80, 117.61, 146.45, 164.33; ESI-MS: m/z 434.9602 [M+Na+]+ .Yellow oil; yield: 42%; 1H NMR  δ: 1.13 3), 5.88 , 7.40–7.43 , 7.46–7.49 , 7.69–7.72 ; 13C NMR  δ: 19.41, 26.74, 96.55, 123.43, 149.95, 163.35; ESI-MS: m/z 516.9445 [M+Na+]+ .Colorless oil; yield: 60%; 1H NMR  δ: 0.74 , 1.02 , 6.00 ; 13C NMR  δ: 4.72 (3 × CH3), 6.48 (3 × Si-CH2-CH3), 98.79, 117.58 (C3), 146.40 (C4), 164.35 (C2); ESI-MS: m/z 392.9134 [M+Na+]+ .Yellow oil; yield: 27%; −/−  were a gift from Dr. Bert Vogelstein. Other cell lines, namely MCF-7, SJSA-1, U2OS, HepG2, and Hep3B were received from the collection stored at Centre of Biotechnology of Silesian University of Technology. All cells were grown in DMEM with a high glucose medium  supplemented with 10% (v/v) inactivated fetal bovine serum (FBS)  and 1% antibiotics   at 37 °C in humidified 5% CO2. The maximum concentration of DMSO in the culture media was 0.4%.Human cells lines: colon cancer , non-small lung carcinoma (A549), and bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection . Isogenic cell line HCT 116 p533 cells per well for the MCF-7 cell line and 2.5 × 103 cells per well for the other cell lines were seeded in 96-well plates to adhere for 24 h. Afterward, the cells were treated with tested compounds. After 72 hours of incubation, 50 μL 0.5 mg/ml solution of MTT -2,5-diphenyl-2H-tetrazolium bromide) were added to each well and the plates were incubated for 3 h at 37 °C. Next, the MTT solution was removed from the plates and formed formazan crystals were dissolved in 2-propanol  containing 0.04 M hydrochloric acid . The absorbance was measured at 570 nm wavelength using a multiwall plate reader . The experiment was performed at a minimum of 3 independent replicates. Cell viability was set as a percentage versus vehicle control. IC50 was defined as a concentration of a drug that decreased cell viability by 50% relative to the untreated control.5 × 104 cells/well. After 24 h the medium was replaced with tested compounds. The cells were incubated for 24 h at 37 °C. Afterward, floating cells were collected and adherent cells were harvested by trypsinization and centrifuged at 600× g. The cells were washed with PBS   and then fixed in 70% ice-cold ethanol and stored at −20 °C overnight. The next day the cells were centrifuged at 600× g, washed with PBS, treated with 100 μg/mL RNase A solution , 100 μg/ml solution of propidium iodide (PI)  and incubated for 30 min at room temperature in the dark. After staining, the cells were analyzed using a Becton Dickinson FACS Aria III sorter . Experiments were repeated at least twice. Data were analyzed using ModFit LTTM Software .The HCT 116 cells were seeded in 6-well plates  at a density of 5 × 104 cells/well and allowed to attach. After 24 h the medium was replaced with tested compounds. The cells were then incubated for 24 h at 37 °C. Next, both floating and adherent cells were harvested and centrifuged at 600× g. The cells were washed with PBS   and we followed instructions from the manufacturer of the Annexin V-FITC Apoptosis Detection Kit . Fluorescence was measured using a Becton Dickinson FACS Aria III sorter . Experiments were repeated at least twice. Data were analyzed using FCE Express 7 Software .The HCT 116 cells were seeded in 6-well plate  at a density of 5 × 105 HCT 116 wt cells were seeded on 100 mm dishes  and allowed to attach for 24 h. Then the medium was replaced with tested compounds at a concentration corresponding 2 × IC50 value and the cells were incubated in 37 °C. After 60 min the cells were lysed and the lysates were clarified by centrifugation for 30 min at 14,000× g. The total protein concentration was measured using the Protein Quantification Kit-Rapid  according to the manufacturer’s instruction. The next steps followed instructions from the manufacturer of the Human Apoptosis Array Kit. The membranes were then scanned in the G:Box trans illuminator  and the protein expression levels were quantified by densitometric analysis using ImageJ software. The Human Apoptosis Array Kit  was used to determine the levels of expression of apoptosis-related proteins. 2.5 × 104 cells/well) were plated in 6-well plates  and treated with the tested compounds for 24 and 72 h. Cells were harvested by trypsinization and then re-seeded on the new 6-well plate  with a density of 2 × 103 cells/well. After 10 days the cells were washed with cold PBS , fixed in ice-cold ethanol (−20 °C) for 3 minutes, air-dried, washed again with cold PBS and stained with 0.01% crystal violet  in dH2O (deionized water) for 15 min. Next the plates were washed in dH2O and allowed to dry. Colonies were counted with a microscope . Aggregates of 30 cells or more were considered as colonies. Photos were made using the G:Box transilluminator .The HCT116 wt cells (5 × 10t-test. All statistics calculations were carried out in GraphPad Prism software.The results were expressed as means ± S.D. and performed by unpaired 1. Interestingly, compounds showed to be most active against colorectal cancer cell lines, which makes them potential candidates for the development of novel treatments for CRC. The strategy reported here provides the ability to control both hydrophobicity and the selectivity of novel derivatives of MBA. Silylation of 5-hydroxyl group in the MBA ring leads to set of novel compounds with increased cytotoxic potency against cancer cells in comparison to lead molecule In conclusion, this study not only presents a set of potent and highly-cell-active compounds, but it also sheds light on the importance of silyl groups as crucial factors in addressing mechanisms of action and selectivity towards cancer cells. Taking into account the availability of various silylating agents, many further optimizations could be imagined in order to obtain compounds with an appropriate balance between potency and cancer cell selectivity.


Short-chain enoyl-CoA hydratase (ECHS1) is involved in amino acid and fatty acid catabolism in mitochondria and its deficiency causes Leigh syndrome or exercise-induced dystonia. More than 60 patients with this condition have been reported till date. The accumulation of intermediate metabolites of valine is assumed to be responsible for the cytotoxicity. Since protein restriction, including valine reportedly improves neurological symptoms, it is essential to consider the possible incidence of and diagnose ECHS1 syndrome in the earlier stages. This study reported the liquid chromatography with tandem mass spectrometry (LC-MS/MS) urine and plasma metabolite analysis in six cases, including four new cases with ECHS1 deficiency. The values of urine cysteine/cysteamine conjugates from valine metabolites, S-(2-carboxypropyl) cysteine/cysteamine from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine/cysteamine from acryloyl-CoA were separated between six patients and six normal controls. The LC-MS/MS analysis revealed that these metabolites can be used for the early diagnosis and evaluation of diet therapy. ECHS1 catalyzes the conversion of methacrylyl-CoA, acryloyl-CoA, tiglyl-CoA, crotonyl-CoA, and short-chain 2-rnoyl-CoA to 3-Hydroxyisobutyryl-CoA, 3-hydroxypropionyl CoA, 2-Methyl-3-hydroxybutyryl-CoA, 3-Hydroxbutyryl-CoA, and short-chain 3-hydroxycarbonyl-CoA, respectively. The defect of ECHS1 results in the accumulation of biologically active intermediate metabolites, including four cysteine/cysteamine conjugates from valine metabolites as follows: S-(2-carboxypropyl) cysteine (SCPC) and S-(2-carboxypropyl) cysteamine (SCPCM) from methacrylyl-CoA and S-(2-carboxyethyl) cysteine (SCEC) and S-(2-carboxyethyl) cysteamine (SCECM) from acryloyl-CoA.ECHS1 has a wide specificity for substrates such as methacrylyl-CoA , acryloyl-CoA , tiglyl-CoA (isoleucine), and crotonyl-CoA  . The def22.1Six patients with ECHS1 deficiency were included in the valine metabolite analysis, including four newly diagnosed patients . The pat2.22.2.12.2.1.1Acetonitrile and formic acid were obtained from Fujifilm Wako Pure Chemical Co. . S-(2-carboxypropyl)-cysteine and S-(2-carboxypropyl)-cysteamine were purchased from Angene International Limited . S-(2-carboxyethyl)-cysteine, S-(2-carboxypropyl) D2-cysteine, and S-(D3–2-carboxyethyl)-cysteine were chemically produced by Tokyo Chemical Industry . S-(2-carboxyethyl)-cysteamine was obtained from Sigma-Aldrich .2.2.2Our analytical method was modified from a previous report 2.2.3w/v) Triton X-100, and 1:1000-diluted protease inhibitor cocktail . The supernatant was collected after centrifugation at 14,000 rpm for 20 min at 4 °C. The assay conditions for ECHS1 activity were as follows: 100 mM potassium phosphate (pH 8), 0.1 mg/mL bovine serum albumin, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and 30 μM crotonyl-CoA −1·cm−1 for ECHS1 activity and 13,600 M−1·cm−1 cm for CS activity and expressed as a percentage of control activity.The fibroblast cells were grown to 70%–80% confluence for approximately two weeks and experiments were performed on cells pooled from the lines. The fibroblasts were purchased from JCRB Cell Bank . The cells were harvested, collected, washed twice with phosphate buffered saline (PBS), and solubilized with 0.5 mL of the lysis buffer containing 25 mM potassium phosphate (pH 8), 2 mM EDTA, 0.1% (2.2.4w/v) Triton X-100, and 1:1000-diluted Protease Inhibitor Cocktail. The lysate was centrifuged at 14,000 rpm for 20 min at 4 °C, the soluble proteins were isolated from the supernatant, and the protein concentration was determined using the Qubit Protein Assay kit .Whole-cell lysates were prepared in lysis buffer containing 25 mM potassium phosphate (pH 8), 2 mM EDTA, 0.1%  sample loading buffer . Protein samples were separated on a 4%–12% gradient SDS-polyacrylamide mini-gel. Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% PBS and incubated overnight with primary antibodies, rabbit anti-ECHS1 from Abcam , and mouse anti-beta-actin from Sigma-Aldrich . Following three washes with PBS-0.05% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  diluted in 5% PBS. The secondary antibody was detected using the chemiluminescence ECL Plus reagent  and the membranes were visualized using the Image Quant LAS4000 Imaging System .2.2.5DNA was extracted from the whole blood samples using a ReliaPrep-Blood-gDNA-Miniprep-System . Whole exome sequences were obtained from Riken Genetics . For Sanger sequencing, primers were designed for polymerase chain reaction (PCR) amplification  of the ECHS1 gene . PCR was performed using the KOD FX Neo DNA polymerase kit  with 25 μL of the reaction mixture according to the manufacturer's instructions. The PCR conditions were as follows: initial denaturation at 94 °C for 2 min, one cycle; 98 °C for 10 s, 64 °C for 30 s, 40 cycles; and final extension at 72 °C for 7 min, one cycle. The PCR products were electrophoresed on a 2% agarose gel and visualized with ethidium bromide. The amplification products were directly sequenced using an ABI 3730 sequencer .2.2.6Two types of treatment milk (Diet therapy 1 and 2) were prepared to reduce the special amino acids  and enteric nutrients  were equally mixed. The ingredients used are listed in Supplementary Table 2.2.6.2The second diet treatment aimed to limit the intake of valine (70 mg/kg/day), leucine (90 mg/kg/day), and isoleucine (50 mg/kg/day). Valine-leucine-isoleucine-removed milk  used for disorders affecting the valine catabolic pathway and lysine-tryptophan-removed milk used for glutaric acidemia type 1  were equally mixed. The ingredients used are listed in Supplementary Table 33.13.1.1p = 0.002 for SCPC, 0.009 for SCPCM, 0.014 for SCEC, and 0.00008 for SCECM, respectively: In ECHS1 deficiency, valine intermediate metabolites, methacrylyl-CoA and acryloyl-CoA, were reportedly elevated Serum metabolites were analyzed in three controls and two patients. The levels of these four metabolites were not significantly different  presented a high intensity in the caudate and globus pallidus on T2-weighted image (T2WI) at the age of 8 months a and difDiet therapy 1 was administered from 14 to 16 months, 20 to 24 months, and post 32 months; diet therapy 2 was administered from 16 to 20 months and 24 to 32 months . Followi3.2.2Patient 2 was a seven-year-old boy. His parents were healthy and unrelated. He was delivered at 40th week of gestation. His birth weight was 3976 g. He had gained head control at the age of four months and could sit for eight months. At 10th month, he lost consciousness following fever and five days of diarrhea, without biochemical abnormalities such as metabolic acidosis, hypoglycemia, lactic acidosis, and CSF lactate elevation. The urine organic acid analysis results were normal. MRI indicated a low intensity on the bilateral striatum and caudate nucleus on T1-weighted image (T1WI) c and higECHS1 (NM_004092) by WES. Silico analysis predicted p.  as pathogenic and p. (Met1?) as uncertain. Western blotting demonstrated no expression of ECHS1 . Following repeated encephalopathy triggered by viral infections, the patient's muscle hypertonia, spasticity, and dystonia were exacerbated and uncontrolled. These symptoms improved slightly with intrathecal baclofen therapy at four years of age. He was diagnosed with ECHS1 deficiency based on genetic and biochemical analyses. Compound heterozygous variants, c.2 T > C, p. (Met1?) / c.5C > T p.  were identified in of ECHS1 a, and ECof ECHS1 . The patof ECHS1 . During of ECHS1 .Fig. 6Cl4This study aimed to establish the diagnostic methods for ECHS1 deficiency using urine and serum samples of the patients. Therefore, we measured the following four cysteine/cysteamine conjugates: SCEC, SCECM, SCPC, and SCPCM. Several metabolites have been reportedly elevated in patients with ECHS1 deficiency and urine tandem mass screening has been implicated as a screening method S)-3-hydroxyisobutyryl-CoA and have been considered to induce the cell toxicity and relate to the clinical severity SCPC and SCPCM are valine metabolites that accumulate from the defects of methacrylyl-CoA to  and valine-leucine-isoleucine-lysine-tryptophan (diet 2). Diet therapy 1 improved some symptoms, such as dystonia in patient 1 and hypotonia in patient 2. Diet therapy 2 did not improve the symptoms or level of metabolites. The reasons for the difference in the effects of these two diets are not completely understood, since the amount of valine in diet 1 (129 mg/kg/day) was higher than in diet 2 (75 mg/kg/day). The other amino acid composition may have contributed to these effects. Timing and the age of therapy may be related to these effects. As we have checked only one urine sample at pre-diet, we cannot exclude the possibility that the metabolite response in Case 1 is within normal variation. Case 3 was also administered protein-restricted milk and presented a decrease in hypertonicity. The further diet therapy and cell toxicity studies for valine metabolites and other metabolites will contribute to the development of an optimal diet for ECHS1.In conclusion, we presented that the urinary levels of SCPC, SCPCM, and SCECM can be used to diagnose ECHS1 deficiency. An early diagnosis of ECHS and protein restriction therapy may alleviate the symptoms of ECHS1 deficiency.ECHS1Supplementary Fig. 1. Position of mutations in Positions of new mutations found in patients 1 and 2 UTR, untranslated regions; CDS, coding sequences.Supplementary Fig. 2. Serum level of cysteine/cysteamine conjugatesLevels of four cysteine/cysteamine conjugates. We measured four cysteine/cysteamine conjugates to valine metabolites; S-(2-carboxypropyl) cysteine (SCPC) and S-(2-carboxypropyl) cysteamine (SCPCM) from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine (SCEC), and S-(2-carboxyethyl) cysteamine (SCECM) from acryloyl-CoA in urine and serum from 4 controls and 2 patients.Supplementary Fig. 3. MR spectroscopy in patient 2MR spectroscopy shows lactate peak reflecting high lactate level in the CNS.Image 1Ingredients of various diets.Supplementary Table 1Urine levels of SCPC, SCPCM, SCEC, and SCECM.Supplementary Table 2Serum levels of SCPC, SCPCM, SCEC, and SCECM.Supplementary Table 3In silico analysis of variants.Supplementary Table 4The following are the supplementary data related to this article.This study was performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the Ethics Committee of Jichi Medical School 2017.Informed consent was obtained from the parents of all the patients for the investigatory work.None.Authors report no conflicts of interest.b) Activity of ECHS1 is measured by the level of crotonyl-CoA following the addition of lysine.The ECHS1 enzyme activities of skin fibroblasts are 30.2% (patient 1) and 15.2% (patient 2) of the control (100%).


Chamaerops humilis L. Besides crude ethanolic extract, two extracts obtained by sonication using two solvents with increased polarity, namely ethyl acetate (EtOAc) and methanol-water (MeOH-H2O) 80:20 (v/v), were investigated by both spectroscopy and chromatography methods. Between the two extracts, the MeOH-H2O one showed the highest total polyphenolic content equal to 32.7 ± 0.1 mg GAE/g DM with respect to the EtOAc extract (3.6 ± 0.5 mg GAE/g DM). Concerning the antioxidant activity of the two extracts, the EtOAc one yielded the highest value (1.9 ± 0.1 mg/mL) with respect to MeOH-H2O (0.4 ± 0.1 mg/mL). The C. humilisn-hexane fraction, analyzed by GC–MS, exhibited 69 compounds belonging to different chemical classes, with n-Hexadecanoic acid as a major compound (21.75%), whereas the polyphenolic profile, elucidated by HPLC–PDA/MS, led to the identification of a total of sixteen and thirteen different compounds in both EtOAc (major component: ferulic acid: 104.7 ± 2.52 µg/g) and MeOH-H2O extracts (major component: chlorogenic acid: 45.4 ± 1.59 µg/g), respectively. The attained results clearly highlight the potential of C. humilis as an important source of bioactive components, making it a valuable candidate to be advantageously added to the daily diet. Furthermore, this study provides the scientific basis for the exploitation of the Doum in the food, pharmaceutical and nutraceutical industries.The aim of this study was to characterize the phytochemical content as well as the antioxidant ability of the Moroccan species  Chamaerops humilis L.), widely called “Doum”, is found in six cities of the eastern region of Morocco, namely Oujda, Berkane, Ahfir, Saidia, Nador and Jerrada [The Moroccan wild palm tree  have been commonly used for the treatment of diabetes, digestive disorders, spasms, tone and gastrointestinal disorders [Some components of this plant have been used as food as an important source of nutritional energy , or in tisorders ,5. Moreoisorders .Other studies have shown the beneficial effects of these fruits against hyperlipidemia in an animal model of obesity and hyperglycemia . Thanks C. humilis are so far only little characterized. The analysis of the grain’s oil showed higher levels of oleic and linoleic acids than other seed oils, as well as a significant amount of tocopherols and tocotrienols [The phytochemical properties of trienols .C. humilis L., which may explain the pharmacological effects mentioned above [Several biologically important secondary metabolites such as flavonoids, phenols, saponins, gallic tannins and terpenoids have been detected in the leaves and fruits of ed above ,7,9,12.C. humilis. Further, no work has been so far devoted to the analysis of the volatile content of such a species.With regard to flavonoids, they have been previously reported as constituents of the Arecaceae family of plants, even though the literature lacks detailed information on the phytochemical composition of C. humilis L.) by GC–MS and HPLC–PDA/MS. In addition, the evaluation of the physico-chemical properties, and the antioxidant activities of the fruit extracts, was performed as well.The aim of this work was to determine the volatile and polyphenolic content of Moroccan Doum fruits  [The percentage of dry matter attained was equal to 69.6 ± 0.5, approximately indicating the presence of 30.4% water in these fruits. The latter value is twice as high than the one reported by Bouhafsoun et al. (17.4 ± 0.12%) [ecaceae) .The ash content revealed interesting amounts of minerals (3.0 ± 0.3). Such a value coincides with the mean value of ash content (2.4 to 5.0%) recommended by FAO , even thPhoenix dactylifera L.) (5.3 ± 0.0) [Hyphaene thebaica) (4.8 ± 0.0) [Concerning the pH measurement, a value of 3 ± 0.06 was attained. This value is lower than the one found by Bouhafsoun et al. (5.0 ± 0.0)  and, in 3 ± 0.0)  and doum8 ± 0.0) .C. humilis L. fruit revealed a percentage of 1.5 ± 0.3%. This value is slightly different from Algerian fruits (0.2 ± 0.0%) [Hyphaene thebaica (0.22%) [The titratable acidity of fruits 0. ± 0.0% [ (0.22%) .Butia odorata fruits (13.1–14.6%) [C. humilis L. [The TSS results showed a mean value of 15.2 ± 0.7%. Similar values were found in 1–14.6%) , despitea fruits .1–14.6% Butia odorata fruits (4.42–14.20%) [C. humulis L. fruits investigated in this work showed higher values compared to those of B. capitata reported in the literature, 4.7–5.8% [The S/A ratio was 10.3 ± 0.5%. Such a ratio is an important biochemical parameter that influences the taste and acceptability of the fruits. The high values of this ratio indicate good technological properties and consumer acceptance of these fruits ,19. The –14.20%) . On the Results of RS and TS were equal to 18.1 ± 0.7% and 23.7 ± 0.9%, respectively. Vitamin C contents in Doum extracts were determined to be 31.5 ± 0.5 mg/g, which is slightly higher than other research (20.1 ± 0.5 mg/g) .C. humilis L. in each extract were 1.3 ± 0.0 and 1.34 ± 0.0 for ethyl acetate (EtOAc) and methanol–water (MeOH-H2O), respectively. The ANOVA test (p > 0.05) showed that the difference between the fruits in IR was not significant.The refractive index values for the 2O extract, whereas they were absent in the EtOAc fraction. The low levels of protein content can be caused by the ultrasonic extraction, which leads to protein denaturation, as proven by some researchers [With regard to lipid and protein contents, values of 0.7 ± 0.0% and 5.3 ± 1.5% were attained, respectively. A value of 0.6 ± 0.0 mg/g was attained for the MeOH-Hearchers .C. humilis was carried out, for the first time for a Moroccan species. The phytochemical tests revealed the presence of different chemical families, distributed for the studied species according to the solvent concentration used. Anthocyanins were not detected in any of the samples investigated. In the EtOAc extract, unsaturated sterols, terpenes and glycosides, which were absent in the crude extract, were revealed. On the other hand, in the crude extract, catechic tannins, anthracenosides, sterols and steroids were detected in high concentrations.The phytochemical screening of C. humilis are not well characterized, although several studies have reported the presence of tannins, flavonoids, saponins, sterols and terpenoids [C. humilis. From the results achieved, such a species does contain important phytochemical constituents that may contribute to its anti-inflammatory and antioxidant activities  content, equal to 32.7 ± 0.1 mg GAE/g DM, with respect to the EtOAc extract, 3.6 ± 0.5 mg GAE/g DM  between the different solvent concentrations, thus indicating an effect of the solvent concentration on the extraction of these compounds [Statistical analysis (ANOVA) showed that there was a highly significant difference in results (ompounds .2O), the values attained are higher than those found by other authors, e.g., Belhaoues et al. (2017) [C. humilis reported IC50 values of 0.024 mg/mL [Concerning the antioxidant activity of the two fractions   and Gonç. (2017)  , followed by oleic acid (14.66%) (C. humilis works [The  classes . Similar(14.66%) . Such fiis works ,32.2O extracts of C. humilis, is reported in 2O extracts . Interelucoside . Notably humilis , whereas species . Cinnamo2O extract, chlorogenic acid (45.4 µg/g) was predominant, along with quinic acid (37.0 µg/g).The quantification was determined for three repetitions of different extracts of the same sample. As far as quantification is concerned , ferulic2O extracts of C. humilis, respectively, were attained. Such results are comparable with other Moroccan fruits, e.g., Ziziphus lotus, at least for the EtOAc extract (298.5 µg/g) [In total, 276.7 µg/g and 262.2 µg/g of polyphenolic compounds for the EtOAc and MeOH-HChamaerops humilis L. fruits were harvested in Tangier-Tetouan-Al Hoceima, an area located in the extreme north-west of Morocco. The samples were collected for 4 months . All of the harvest areas were between the longitudes 5°94’84106 and the latitudes 35°44’701. The fruit harvesting was carried out at their physiological maturity in the early morning, transported in well-closed boxes and stored at −10 °C in the Materials and Resources Valorization Laboratory, Faculty of Sciences and Technology of Tangier. The extraction method employed was previously described by El Cadi et al. (2020) [n-hexane; afterwards, it was dried and homogenized with 50 mL of two solvents with increased polarity, namely EtOAc and MeOH-H2O 80:20 (v/v). Each fraction was extracted by using an ultrasound bath (130 kHz) for 45 min. After centrifugation at 5000 g for 5 min, the supernatant was filtered through a paper filter, dried, reconstituted with MeOH-H2O and then filtered through a 0.45 μm Acrodisc nylon membrane  prior to HPLC–PDA-ESI/MS analysis.. (2020) . BrieflyFolin-Ciocalteu phenol reagent was obtained from Fluka. Standards  were obtained from Merck Life Science . In addition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and butylated hydroxytoluene (BHT) were purchased from Sigma . LC-MS grade methanol, acetonitrile, acetic acid, EtOAc, acetone and water were purchased from Merck Life Science . All of the other chemicals were of analytical grade and obtained from Sigma .Physico-chemical analyses and phytochemical screening were carried out according to a previously published work .TPP content was estimated using Folin-Ciocalteu method  and was The DPPH method followed the method described by Braca et al. . Butylat50 of the DPPH radical was calculated from linear regression .The ICGC analyses of the volatile fraction were performed on a GC–MS-QP2020 system  with an “AOC-20i” system auto-injector. The analyses were realized on an SLB-5ms column . The initial temperature was set at 50 °C, and afterwards increased up to 350 °C .GC–MS parameters were as follows: injection temperature: 280 °C; injection volume: 1.0 µL (split ratio: 10:1); pure helium gas (99.9%); linear velocity: 30.0 cm/s; inlet pressure: 26.7 KPa; EI source temperature: 220 °C; interface temperature: 250 °C. The acquisition of MS spectra was realized in full scan mode, in the mass range of 40–660 m/z, with an event time of 0.2 s.Relative quantity of the chemical compounds present in each sample was expressed as a percentage based on peak area produced in the GC chromatogram.Compounds were identified by using the “FFNSC 4.0”  and “W11N17” . Each compound was identified applying a MS similarity match and an LRI filter. Linear retention indices (LRI) were calculated by using a C7–C40 saturated alkanes reference mixture .Data files were collected and processed by using “GCMS Solution” software, ver. 4.50  .LC analyses were performed on a Shimadzu liquid chromatography system , consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a CTO-20AC column oven, a SIL-30AC autosampler, an SPD-M30A photo diode array detector and an LCMS-8050 triple quadrupole mass spectrometer, through an ESI source .v/v, solvent A) and acetonitrile/acetic acid . The flow rate was set at 1 mL/min under gradient elution: 0–5 min, 5% B, 5–15 min, 10% B, 15–30 min, 20% B, 30–60 min, 50% B, 60 min, 100% B. PDA detection was: λ = 200–400 nm (λ = 280 nm) . MS conditions were as follows: scan range and the scan speed were set at m/z 100–800 and 2500 amu sec −1, respectively, event time: 0.3 sec, nebulizing gas (N2) flow rate: 1.5 L min−1, drying gas (N2) flow rate: 15 L min−1, interface temperature: 350 °C, heat block temperature: 300 °C, DL (desolvation line) temperature: 300 °C, DL voltage: 1 V, interface voltage: −4.5 kV [Chromatographic separations were attained on 150 × 4.6 mm; 2.7 µm Ascentis Express RP C18 columns . The mobile phase was composed of two solvents: water/acetic acid . The experiments were carried out in triplicate and the results were expressed as the average of the three measurements ± SD. The comparison of means between groups was performed with one-way analysis of variance (ANOVA) followed by a Tukey test. Differences were considered significant when Chamaerops humilis L. fruits. Considering the two extracts tested, in terms of the antioxidant activity, the EtOAc one turned out to be the most active with respect to the MeOH-H2O. A total of 69 compounds belonging to different chemical classes were positively identified by GC coupled to MS, whereas sixteen and thirteen polyphenolic compounds were detected by HPLC–PDA/MS in both EtOAc and MeOH-H2O extracts, respectively. Such results demonstrate that this fruit can be used for industrial applications in food preparations. In addition, the data attained emphasize an interesting functional composition of the Chamaerops humilis L. fruits, which could be considered a valuable new co-product with commercial importance in the food industry.The present study aimed to elucidate the bioactive content of


Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA.Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h.Leaves from plants of A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation.High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.This is the first report on development of hairy root cultures of  Lepechinia caulescens (Ortega) Epling (Lamiaceae), an herbaceous plant used in Mexican folk medicine has been proposed as an alternative source for the production of UA and OA. This plant has elevated triterpene contents (UA: 16.58 mg/g DW and OA: 1.94 mg/g DW), and its callus cultures also accumulate these bioactive compounds  and oleanolic acid (OA), together with rosmarinic acid (RA), an ester of caffeic acid, are secondary metabolites that have gained considerable importance over the last few decades in the pharmaceutical, cosmetic, food and agrochemical industries, due to their wide range of biological activities which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties . These compounds . Howeverompounds . In contabolites . Hairy rabolites . There aabolites . The welabolites . Althougabolites , few stucultures , have beL. caulescens were collected from Morelos State, Mexico. The plant was identified in the HUMO Herbarium, Autonomous University of Morelos, where a voucher specimen (32444) was deposited. Mature seeds were germinated and grown in vitro on MS medium, solidified with 3.0 g/L phytagel , harboring agropine-type plasmid pRi15834 was cultivated on liquid YM medium (λ = 600 nm (OD600) reached 0.5.Wild plants of phytagel . Hairy rM medium  in an orA. rhizogenes by immersing them in bacterial suspension for 1 min, and then blotted on filter paper. Control explants were treated identically, using YM medium without bacteria. Thereafter, the infected explants and controls were placed for 24 h on MSB5 medium containing MS salts  were harvested from 4-week old hairy root cultures and subcultures were commenced at 4-week intervals, in the same media and in dark conditions.L. caulescens hairy roots and seedlings were extracted using the ZR Plant/Seed DNA MicroPrepTM kit (Zymo Research), following manufacturer’s instructions. The quality and quantity of DNA were analyzed using a Nanodrop . Specific primers for rolB were used to detect PCR of transgenes in DNA extracts from roots, present in the T-DNA region of pRi15834 from A. rhizogenes. The primer pair for amplifying the rolB region was 5′ATGGATCCCAAATTGCTATTCCCCCACGA3′ and 3′TTAGGCTTCTTTCATTCGGTTTACTGCAGC5′  of virD2 gene, located outside the T-DNA  2.5 µL, MgCl2 (25 mM) 1.6 µL, dTNPs (2 mM) 2 µL, primers 3 µL, Taq polymerase 0.5 µl  9.4 µL ultrapure water and 1 µL DNA (50 ng/ µL) template, at a final volume of 25 µL. Conditions for cycles consisted of an initial denaturation at 94 °C for 5 min; followed by 42 cycles of 1 min denaturation at 94 °C, annealing at 52.5 °C for 1.5 min, and extension at 72 °C for 2 min. A final extension cycle of 10 min at 72 °C, completed the PCR. PCR products were resolved by electrophoresis, using 0.8% (w/v) agarose gels in TAE buffer , stained with ethidium bromide (0.5 µg/mL) and observed under UV light.Total DNA of TGCAGC5′ . These yhe T-DNA . The genP < 0.05). The coefficient of determination (R2) was calculated to establish the strength of linear association between the values.One-month old hairy roots (∼2 g fresh weight) of L1 and L5 were inoculated into 250 mL jars, containing 20 mL modified MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose. All other components of MSB5 medium remained unchanged. The hairy roots were incubated in darkness at 25 °C for 4 weeks. Subsequently, roots were dried to a constant weight, in an oven set at 30 °C, to determine dry weight (DW). Each treatment consisted of six replicates. Biomass data were analyzed by linear regressions using STATISTICA 7 software; possibility of significance . Extracts were filtered using filter paper and then air dried in a fume hood.3PO4) 83:17 (v/v), at a flow rate of 0.9 mL/min. Separation of RA present in the methanolic extracts was performed by elution, using a linear gradient of solvents A (acetonitrile) and B (0.01% phosphoric acid solution). The gradient was as follows: 5 min 90:10% A:B, 20 min 74:26% A:B and 10 min 90:10% A:B. Elution was performed at a solvent flow rate of one mL/min. Waters 996 photodiode array detector system was used for both triterpene (250 nm) and RA (230 nm) detection. Calibration curves were plotted at six concentrations for each of the standard solutions in methanol, in order to undertake a linear regression analysis of the peak area for each concentration. The corresponding retention time (Rt) and ultraviolet spectrum of the standard and the sample, confirmed the presence of UA (Rt 10.67 min), OA (Rt 10.36 min), and RA (Rt 14.10 min) (P < 0.05).Separation of UA, OA and RA was achieved, using an analytical high performance liquid chromatography system (Waters HPLC System) and the Waters XBridge™ C18 column (4.6 mm Å∼75 mm). The triterpenes from dichloromethane extracts were separated by applying a mobile phase of methanol: acidic water (H.10 min) . Hairy rP < 0.05).Two grams of hairy roots were inoculated into 20 mL MSB5 medium supplemented with 45 g/L in 250 mL jars and maintained in the dark at 25 °C. After 15 days of incubation , MeJA was added individually to each semisolid media to reach a final concentration of 0, 100, 200 and 300 µM. Effects of elicitation on the production of UA, OA and RA in hairy roots were measured after 24, 48 and 72 h grown in these media. The stock solutions of MeJA  were prepared by dissolving in 1:99 ethanol:water (v:v) and filtering through a 0.22 µm membrane filter. Ethanol and distilled water were added to control media. Each treatment consisted of five repetitions. All data were analyzed by two-way ANOVA, using STATISTICA 7 software. Significance between means was tested by Tukey’s test ; alternatively, the PCR product from uninfected L. caulescens seedlings could not be amplified . After 4 subcultures, only L1 and L5 showed satisfactory growth without callus formation and were selected for further study. The other nine hairy root lines formed multiples callus and ceased growth.In this study, hairy roots of CC 15834 . Four weCC 15834 –1C. AfteCC 15834 . The tramplified . Ten of rol genes) and constant growth rate during 160 days were selected to evaluate the effect of the variation of sucrose concentration in the MSB5 medium on the accumulation of biomass and morphology. As shown in L. caulescens elicited by MeJA.Two lines characterized by hairy root morphology, transgenic nature  between the interaction of MeJA concentration and elicitation time. The highest concentration of UA was obtained by eliciting the cultures for 24 h with 300 µ M of MeJA with a production of 0.22 ± 0.00 mg/g DW. Alternatively, the lowest concentration of UA (0.08 ± 0.01 mg/g DW) was obtained in the control treatment (48 h elicitation without MeJA) , the highest yield of OA (0.24 ± 0.00 mg/g DW) was obtained by eliciting the hairy roots cultures with a concentration of 300 µ M of MeJA, for 24 h; while the lowest concentration of OA (0.07 ± 0.00 mg/g DW) was obtained in the control treatment  . The highest accumulation of RA (60.27 ± 0.88 mg/g DW) was obtained by eliciting the cultures with 200 µM of MeJA for 24 h; and the lowest concentration (10.71 ± 0.34 mg/g DW) was obtained in the control treatment   . Similarut MeJA) . The accut MeJA) .F3,24 = 23.127, F3,24 = 43.374 and F3,24 = 695.78 for UA, OA and RA respectively, P < 0.001 for all cases) and the concentration of MeJA  have a positive effect on the production of UA, OA and RA; with treatments elicited for 24 h with a concentration of 300 µM of MeJA, showing greatest accumulation.According to statistical analyses, the yield means of the elicitation time factor , encoding a key enzyme to provide mevalonate for biosynthesis of triterpene acids. Likewise, β-amyrin synthase (AS) and two cytochrome P450 monooxygenases genes, increased their expression levels in Ocimum basilicum, when elicited with 250 µM MeJA, thus contributing to the accumulation of OA and UA. It has been observed that MeJA can also elevate the expression of the key genes involved in the biosynthesis of RA, such as phenylalanine ammonia-lyase gene (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: coenzyme-A (CoA) ligase (4CL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR) and RA synthase  and exposure times . This effect on the part of MeJA was also observed in terms of OA yield from the cell cultures of Achyranthes bidentata, treated with 0.2 mM MeJA for 24, 48 and 72 h  for between 1 and 7 days ; Sample L1and L15, genomic DNA of hairy root lines; C+, plasmid DNAClick here for additional data file.10.7717/peerj.11279/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11279/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11279/supp-4Supplemental Information 4Lepechinia caulescens hairy roots (1) and wild plants (2) samples for detecting ursolic and oleanolic acid.HPLC chromatogram of Click here for additional data file.10.7717/peerj.11279/supp-5Supplemental Information 5Lepechinia caulescens hairy roots (1), wild plants (2) and standard (3) samples of rosmarinic acid.HPLC chromatogram of Click here for additional data file.10.7717/peerj.11279/supp-6Supplemental Information 6Click here for additional data file.


T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, highest similarity was found with Sphingobacterium alkalisoli Y3L14T (96.72%). Cells were observed to be aerobic, non-motile rods. The isolate was found to be able to grow between 0 and 10% of NaCl concentration. The assembled genome of strain Ka21T has a total length of 5.2 Mb with a G + C content of 41.0 mol%. According to the genome analysis, Ka21T encodes several glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in the soil. Based on phenotypic characteristics and phylogenetic analysis, it is concluded that strain Ka21T represents a novel species in the Sphingobacterium genus for which the name Sphingobacterium pedocola sp. nov. is proposed. The type strain of the species is strain Ka21T (= LMG 31575T = NCAIM B.02636T).A Gram-reaction-negative halotolerant bacterial strain, designated Ka21The online version contains supplementary material available at 10.1007/s10482-021-01623-6. Sphingobacterium was proposed by Yabuuchi et al. , the type species is Sphingobacterium spiritivorum . 100–100 µl of the third to the fifth member of the dilution series was subsequently spread onto xylan containing agar  and incubated at 10 °C for 5 days. Single colonies on the plates were purified on the same medium. The isolate was routinely maintained on LB medium  at 28 °C and pH 7.5.Strain Ka21T was tested on LB agar medium by directly observing single colonies. Cell morphology of strain Ka21T was observed by electron microscopy. The Gram reaction was determined with a non-staining method as described by Buck  according to the manufacturer’s instructions. The API 50 CH and 20 NE tests were read after 24–48 h incubation at 30 °C. Anaerobic and microaerophilic growth was checked on LB medium using the Anaerocult A and C systems (Merck).Biomass for chemical and molecular studies was obtained by cultivation in shaker flasks using LB medium at 30 °C for 32 h. Colony morphology of strain Ka21 by Buck . Oxidase by Buck . Growth T was analysed on active growing cultures on LB agar. According to the DSMZ Identification Service, fatty acid methyl esters (FAMEs) were obtained following the methods of Miller . The fatty acid profiles of strain Ka21f Miller  and Kuykf Miller . FAMEs wThe respiratory quinones were extracted from freeze dried material and purified by a silica-based solid phase extraction. Purified samples were further analysed by HPLC and UHPLC-ESI-qTOF system Tindall ; dsmz.deT liquid culture grown in LB medium. Genomic DNA isolation and 16S rRNA gene amplification were performed according to Tóth et al.         NaCl, at a pH range from 6.5 to 9.0 and at temperatures between 10 and 35 °C. Optimal growth was observed at 30 °C, 1% (w/v) NaCl and pH 8.0. Cells of Ka21T are short rods, the mean cell size is 0.5–0.7 µm in width and 1.5–2.0 µm in length (Online resource 1). According to API 50 CH test, Ka21T produces acid from l-arabinose, d-xylose, d-glucose, d-mannose, esculin, and d-trehalose. Assimilation of d-glucose, l-arabinose, d-mannose, N-Acetyl-Glucosamine and d-maltose, hydrolysis of esculin and β-galactosidase activity were demonstrated by using the API 20 NE test. In the API ZYM test, strain Ka21T showed activities of alkaline phosphatase, esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, β-glucosidase α-glucosidase, and N-acetyl-β-glucosaminidase. Distinctive physiological and biochemical characteristics of the isolate are given in Table LB medium was used for general laboratory cultivation, but the novel strain also grows well on TSA, nutrient and R2A media. After 72 h growth on LB agar at 30 °C, colonies were observed to be 1.0–1.5 mm in diameter, circular, non-mucoid, smooth and yellow. Strain Ka21T are summed feature 3 , iso-C15:0 (32.5%) and iso-C17:0 3OH (20.5%). The fatty acid profile is similar to that of related strains, in accordance with the description of Sphingobacterium genus . Strain Ka21T exhibits a complex polar lipid profile consisting of phosphatidylethanolamine (PE) and phosphoglycolipid (PGL) as dominant elements, one aminoglycolipid (GNL), six phospholipids (PL) and six uncharacterised lipids (L) (Online resource 2).The predominant cellular fatty acids of strain Ka21T are 5,205,271 bp and 41.0 mol%, respectively. According to the annotation, there are 4320 genes, 4260 CDSs and 60 RNA genes in the genome. The coding density is 89.33%.The completeness and contamination values of the genome are 97.2% and 0.9%, respectively. Other quality labels of genome sequencing and assembly are as follows: 270-fold genome coverage, contig N50 = 433,102, number of contigs are 27. The genome size and G + C content of Ka21The RAST analysis revealed the presence of 242 subsystems, the subsystem coverage was 20% (Online resource 3).T contains 5 putative biosynthetic gene clusters  in 4 genomic regions.The genome of Ka21http://pfam.xfam.org/), several glycoside hydrolase (GH) genes in various GH families were found. These enzyme genes may play a role in the breakdown and modification of carbohydrates in soil. Some of the enzymes (in parentheses are the GenBank accession numbers) belonging to the GH1 (MBE8721502), GH2 , GH3 , GH9 (MBE8722726), GH10 , GH16 (MBE8722218), GH26 (MBE8722637), GH43  and GH130 (MBE8722632) families are active on plant cell wall polysaccharides (http://www.cazy.org/)  that potentially play a role in xylan degradation was identified  and 2213 single-copy gene clusters     16S rRNA gene sequences in the EzTaxon database, highest level of sequence similarity occurred with 43 bp 16SSphingobacterium olei HAL-9T (GenBank assembly accession: GCA_005048855) (86.5% ANI) and Sphingobacterium alkalisoli Y3L14T (GenBank assembly accession: GCA_005049105) (84.39% ANI). The p-value of taxonomic novelty at species level is 0.00292.According to genome based analysis, the closely related taxons found by MiGA are T and Sphingobacterium olei HAL-9T and Ka21T and Sphingobacterium alkalisoli Y3L14T are 31.60% and 28.20%, respectively. Whole genome based tree generated by TYGS also confirmed the taxonomic position of Ka21T within Sphingobacterium genus as a novel species  between Ka21T represents a novel species in genus Sphingobacterium. The generally accepted species boundary for 16S rRNA gene similarity, ANI and dDDH values are 98.7%, 95–96% and 70%, respectively  are all lower, confirming the results of phylogenetic treeing.According to the 16S rRNA based and whole genome based phylogenetic analyses, Ka21T exhibit the typical traits of the genus Sphingobacterium: Gram-negative, non-motile and rod shaped cells, positive for catalase and oxidase, MK-7 as respiratory quinone, major amount of phosphatidylethanolamine in the polar lipid profile, fatty acid profile with C16:1ω7c/C16:1ω6c, iso-C15:0, iso-C17:0 3OH as the most dominant compounds and low genomic G + C content. According to 16S rRNA gene and whole genome based phylogenetic trees, strain Ka21T occupies a separate lineage in the genus. The 16S rRNA gene sequence similarities to the closely related taxons and overall genome related indices  also indicate its distance from other species. Phenotypic, biochemical, chemotaxonomic and phylogenetic information of strain Ka21T support its classification as a novel species of Sphingobacterium, for which the name Sphingobacterium pedocola sp. nov. is proposed. The GenBank accession numbers for the 16S rRNA gene sequence and the whole genome of Sphingobacterium pedocola strain Ka21T are MF471355 and PSKQ00000000, respectively.In conclusion, the revealed characteristics of Ka21Sphingobacterium pedocola . Cells are strictly aerobic, Gram-reaction-negative straight rods and non-motile. It grows well on TSA, LB, nutrient and R2A plates. Colonies have yellow pigmentation on LB after 72 h incubation. Cells are 0.5–0.7 µm in width and 1.5–2.0 µm in length. It grows at 10–35 °C  and at NaCl concentrations of 0.0–10.0 w/v % . The major fatty acids are summed feature 3 (C16:1 ω7c/C16:1ω6c), iso-C15:0 and iso-C17:0 3OH. The only respiratory quinone is MK-7. The major polar lipids are phosphatidylethanolamine and phosphoglycolipid. The DNA G + C content of the type strain is 41.0 mol%. The type strain is Ka21T (= LMG 31575 = NCAIM B.02636) isolated from agricultural field in the Great Hungarian Plain, Hungary.Supplementary file1 (PDF 375 KB)Supplementary file2 (PDF 309 KB)Supplementary file3 (PDF 318 KB)Below is the link to the electronic supplementary material.