Morus nigra L. has flavonoids in its chemical composition with recognized anti-oxidant activity and often associated with anti-inflammatory activity. Therefore, this study aimed to evaluate the hydroethanolic extract of M. nigra leaves’ ability to activate PPAR and promote anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated murine macrophage cells. The leaf extract was prepared by cold maceration, and the chemical profile was obtained by HPLC-DAD. Activation of PPAR α and γ was evaluated by the luciferase reporter assay. The anti-inflammatory activity was assessed by measuring the reactive oxygen species (ROS), nitric oxide (NO), and Tumor Necrosis Factor-α (TNF-α) in RAW 264.7 cells after stimulation with LPS from Escherichia coli. The HPLC-DAD analysis identified two major compounds: rutin and isoquercitrin. The extract showed agonist activity for the two types of PPAR, α and γ, although its major compounds, rutin and isoquercitrin, did not significantly activate the receptors. In addition, the extract significantly reduced the production of ROS, NO, and TNF-α. Treatment with the specific PPAR-α antagonist, GW 6471, was able to partially block the anti-inflammatory effect caused by the extract.Inhibition of systemic inflammation has been a beneficial strategy in treating several non-communicable diseases, which represent one of the major causes of mortality in the world. The Peroxisome Proliferator-Activated Receptors (PPAR) are interesting pharmacological targets, since they can act both through the metabolic and anti-inflammatory pathways.  Exposure to harmful stimuli, such as pathogens, toxins, and cell damage, triggers a series of complex events that are included in the inflammatory response. This response is essential as a natural protective and restorative measure; however, the prolongation of inflammation and failure to resolve it can cause damage and contribute to the maintenance of chronic diseases ,2.Besides the chronic inflammation that develops after an unresolved acute phase, triggered by classic noxious stimuli, there is also chronic systemic inflammation that, in most cases, is related to changes in homeostatic conditions. This general inflammatory state is associated with several non-communicable diseases (NCD), such as atherosclerosis, type II diabetes, neurodegenerative diseases, and cancer ,4, whichThe NCD often present an increase in oxidative stress, pro-inflammatory cytokines, and acute phase proteins ,4,5. FurPeroxisome proliferator-activated receptors (PPAR) are nuclear receptors that function as transcription factors regulating the expression of several genes. These receptors act mainly on lipid and glucose metabolism. However, in recent years, PPAR, mainly the α and γ subtypes, have been associated with anti-inflammatory activity due to their ability to inhibit the expression of inflammatory cytokines and promote immune cell differentiation. Thus, these receptors constitute relevant pharmacological targets for new anti-inflammatory drugs, especially in the case of chronic diseases related to a general chronic inflammatory state, since PPAR can act both through the metabolic and anti-inflammatory pathways ,10,11.Traditional medicine is a health resource based on the experience and long-term practices of different cultures. The World Health Organization (WHO) recommends the inclusion of traditional and complementary medicine in health systems and believes that carrying out these practices with safety and quality can meet the communities need for sustainable and culturally sensitive primary care. In addition, it is especially important as a tool for preventing and managing lifestyle-related chronic diseases ,13.Medicinal plants and herbal medicines are one of the most used traditional and complementary medicine resources in the world. Increased demand for these products includes accessibility, cultural influence, a greater sense of security, and increased scientific knowledge ,14.Morus nigra L., popularly known as mulberry, are traditionally used as a tea, mainly for the relief of climacteric symptoms -2,5 diphenyl tetrazolium bromide (MTT) reduction method. Extract concentrations were considered cytotoxic when a viability reduction was greater than 30%. Therefore, only concentrations that presented a viability greater than 70% were used for future tests, as recommended by ISO 10993-5 .50) values found were 597.1 ± 103.70 µg/mL for RAW 264.7 without LPS stimulation, 622.2 ± 79.04 µg/mL for RAW264.7 stimulated with LPS  to the non-stimulated cells (p > 0.005).The HEMNL showed cytotoxicity in the tested cell lines only at concentrations greater than 500 µg/mL. The concentration needed to inhibit 50% of cell viability   a. The cep < 0.001) of 48% in relation to the treatment with the extract-only at the same concentration (HEMNL 300 μg/mL) (Despite the activation of PPAR-γ after simultaneous treatment with the HEMNL (300 μg/mL) and the substance GW 9662 (selective irreversible PPAR-γ antagonist), there was a significant reduction (0 μg/mL) a.p < 0.05), corresponding to 50 and 200 μg/mL of the extract, respectively. However, the activation rate remained low even with increasing concentration. In addition, the dose of 0.748 μg/mL led to an activation rate 56% lower than the corresponding concentration of the extract  (p < 0.0001)  c. Additi 0.0001) a.p = 0.9739), and at concentrations of 200, 300, and 400 μg/mL, a higher activation rate than WY 14642 (p < 0.002) was reached (p > 0.05)  b,c.p > 0.99) in relation to the simultaneous treatment of the agonist (WY 14642) and antagonist (GW 6471) (While the simultaneous treatment with the extract (300 μg/mL) and the PPAR-α antagonist, GW 6471, reduced the effect of the extract by 86%, showing no statistical difference (GW 6471) a.M. nigra leaves at 200, 300, and 400 µg/mL reduced ROS production by 25, 39, and 53%, respectively, in LPS-stimulated cells compared to untreated cells. The 50 and 100 µg/mL doses, although showing a reduction , were not significant when analyzed by the one-way ANOVA statistical test followed by the Tukey post-test  reagent method. The extract of ost-test .p < 0.04 concerning LPS-stimulated and untreated cells) in 28.86 to 63.40%. Moreover, there was no significant NO production reduction in LPS-stimulated cells (p = 0.0863 in relation to LPS-stimulated and untreated cells) under concomitant treatment with the extract (200 μg/mL) and the PPAR-α antagonist, GW 6471. Therefore, the simultaneous treatment with the extract (200 μg/mL) and the PPAR-γ antagonist, GW 9662, showed no significant difference in relation to treatment with the extract only (p = 0.9995)  .p < 0.0231) this cytokine by 25 to 68% at concentrations of 50 to 400 μg/mL. After concomitant treatment with the extract and the PPAR-α antagonist, GW 6471, a reduction in TNF-α was observed, but it was lower (p = 0.0469) than that caused by extract alone treatment. Meanwhile, the concomitant treatment with the extract and PPAR-γ antagonist, GW 9662, showed no significant difference in relation to treatment with the extract only (p > 0.999)  .Morus nigra leaves are mainly constituted of phenolic compounds acetic acid}, GW 9662 (2-chloro-5-nitro-N-phenylbenzamide), GW6471 {N-[(2S)-3-[4-[2-ethoxy]phenyl]-2-[[(Z)-4-oxo-4-[4-(trifluoromethyl)phenyl]but-2-en-2-yl]amino]propyl]propanamide}, MTT , lipopolysaccharide from Escherichia coli, 2′,7′-dichlorofluorescin diacetate, Griess reagent, and mouse Tumor Necrosis Factor α Elisa kit were purchased from Sigma-Aldrich . Lipofectamine was purchased from Thermo Fisher, Invitrogen . Dulbecco’s modified Eagle’s medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Gibco—Thermo Fisher Scientific. For HPLC analysis, acetonitrile, phosphoric acid, and methanol were purchased from Tedia .Morus nigra L. were collected in October 2017 (fruiting period), in the afternoon, in Brasilia-DF (Brazil). The botanical material was identified by the botanist Assoc Prof Christopher W. Fagg from the Institute of Biology of the University of Brasília, according to the voucher specimen (Fagg CW 2302) deposited in the Herbarium of the University of Brasília.The leaves of The access to Genetic Heritage was approved by CGEN (Genetic Heritage Management Council), registered at SISGEN  under the number A215A9A.Morus nigra leaves (HEMNL) was prepared by passive cold maceration according to previous extract standardization described by [The hydroethanolic extract of ribed by . The masHPLC-DAD analysis was conducted using the chromatograph LaChrom Elite  equipped with an L2455 DAD detector adjusted in the range of 230 to 400 nm, L2200 injector, L2130 pump, L2300 column oven, and a C18 column . Data were obtained with EZChrom Elite software, version 3.3.2 SP1 . Analysis conditions were according to [®, hyperoside acquired from Hwi Analytik Gmbh® , and rutin from Chromadex® .Samples were obtained by diluting the extract with methanol to the concentration of 4 mg/mL followed by filtration with a 0.45 μm filter . Commercial standards were used in an attempt to identify the compounds present in HEMNL by comparison of retention times and ultraviolet spectra. The standards were caffeic acid, chlorogenic acid, ferulic acid, kaempferol, gallic acid, rosmarinic acid, ellagic acid, isoquercitrin, hesperetin, quercetin, resveratrol, vitexin, isovitexin, coumarin, and myricetin acquired from Sigma-Aldrich2 in a humidified atmosphere. The assays were carried out between the third and tenth passages.Cell lines HeLa and RAW 264.7 were grown in high glucose Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and 1% antibiotics (penicillin-streptomycin). Cells were incubated at 37 °C and 5% of CO4 cells/well for HeLa and 1 × 105 cells/well for RAW 264.7. After 24 h of incubation, the cells were treated with MNEH at concentrations ranging from 1000 µg/mL to 50 µg/mL. Additionally, RAW 264.7 cells were tested with or without LPS (1 µg/mL) stimulation 3 h after the treatment. The negative control consisted of treatment with 2% ethanol (solvent used for extract dilution).Cells were seeded in a 96-well plate at the density of 0.8 × 10Following 24 h after treatment, cell viability was assessed by MTT assay described by Mosmann (1983) with modifications . Cells w4 cells/well in DMEM medium with 10% FBS and without antibiotics. The cells were incubated for 24 h and then submitted to transfection with the Lipofectamine 2000®  (Transfection reagent) according to the manufacturer’s protocol. The transfection occurred with a solution of liposomes formed with 60 ng of a plasmid containing the nuclear receptor (PPAR-γ or PPAR-α), 240 ng of the LUC responsive element, and 0.5 µL of Lipofectamine 2000® in DMEM without FBS and antibiotics. The plasmids used were (i) cDNA for the human PPARγ receptor (pCMV-SPORT6-PPARγ); its responsive element was fused to the luciferase reporter gene; (ii) PPARα LBD fused to the Gal4 DNA-binding domain; (iii) a plasmid containing the luciferase reporter gene under regulation by five Gal4 DNA-binding elements. These plasmids were kindly provided by Dr. Paul Webb from the Methodist Research Institute, TX, United States. After 6 h of incubation at 5% CO2 at 37 °C, the contents of the well were removed and replaced by the treatment with HEMNL, rutin, isoquercitrin, or the positive controls, rosiglitazone (PPAR-γ agonist) or WY 14642 (PPAR-α agonist). Additionally, the concomitant treatment with HEMNL and the specific antagonists, GW 9662 (PPAR-γ antagonist) or GW 6471 (PPAR-α antagonist), was tested.HeLa cells were seeded in a 48-well plate at a concentration of 4 × 105 cells/well density. After 24 h, cells were treated with HEMNL at a concentration of 50, 100, 200, 300, or 400 µg/mL and stimulated with LPS (1 µg/mL) 3 h later. Following 24 h after the treatment, cells were washed with PBS and incubated for 30 min at 37 °C and 5% CO2 with DCFH-DA 40 µM. Finally, cells were washed, and PBS was added. The fluorescence was measured in a microplate reader set at 485 nm excitation and 535 nm emission. The results were normalized by mg of protein and quantified by a BCA kit as per the manufacturer’s instructions.The RAW 264.7 cells were seeded in a 96-well plate at 1 × 105 cells/well. Following 24 h after incubation, cells were treated with HEMNL at a concentration of 50, 75, 100, 150, or 200 µg/mL  and stimulated with LPS (1 µg/mL) 3 h later. After 48 h, 100 µL of cells supernatant was mixed with the same volume of Griess reagent. The absorbance was measured, after 10 min, at a wavelength of 540 nm. Nitric oxide concentration was calculated using a sodium nitrite standard curve.The nitrite concentration was determined by the Griess method. RAW 264.7 cells were seeded in 96-well plate at a density of 1 × 105 cells/well. Following 24 h after incubation, cells were treated with HEMNL at a concentration of 50, 100, 200, 300, or 400 µg/mL and stimulated with LPS (1 µg/mL) 3 h later. The TNF-α was determined with a Mouse TNF-α ELISA kit, according to the manufacturer’s instructions.RAW 264.7 cells were seeded in 96-well plate at a density of 1 × 10® program. The results obtained were compared by one-way analysis of variance (one-way ANOVA) followed by the Tukey test for multiple comparisons or by the Mann–Whitney test for comparing two groups. A significance level of p < 0.05 was adopted.All results were represented by the average of three independent experiments performed in triplicate. Statistical analyzes were performed using the GraphPad Prism 6M. nigra leaves as an agonist of the two types of PPAR, α and γ, probably due to the synergistic effect of its compounds, since its major compounds, rutin and isoquercitrin, were not able to significantly activate the receptors. In addition, the extract showed good anti-inflammatory activity, significantly reducing the production of ROS, NO, and TNF-α, in LPS-stimulated RAW 264.7 cells.This is the first study to demonstrate the extract of M. nigra.The specific antagonist of PPAR-α, the compound GW 6471, was able to partially block the effect of the extract, demonstrating the need for activation of the PPAR-α to obtain the anti-inflammatory effects of HEMNL. However, further studies are needed to confirm the mechanism of the anti-inflammatory action of


However, despite the different morphologies, all MCs possess a similar hierarchical porous structure, numerous heteroatom groups and good electrical conductivity. Therefore, these low-cost, biomass-derived porous carbons with promising capacitive performance are used for supercapacitors application with high performance, for example, the as-assembled supercapacitor based on MC-5 exhibits a high specific capacitance of 288.3 F g−1 at 0.25 A g−1, an excellent rate performance of 243.5 F g−1 even at 30 A g−1 with 84.5% capacitance retention of its initial specific capacitance, and an outstanding long-term cycling stability with 98.7% capacitance retention after 10 000 cycles at 5 A g−1. Furthermore, the maximum energy density for these supercapacitors with an aqueous electrolyte in a two-electrode system is about 10.0 W h kg−1 at a power density of 70.3 W kg−1. Therefore, this work opens up a whole new field for the applications of moxa floss and this novel concept of moxa floss use is an extremely promising strategy for developing high-performance carbons with porous structures and heteroatom-doping from renewable sources.Two novel carbons (MCs) derived from moxa floss of different storage years have been prepared by two low-cost and facile approaches, which are hydrothermal carbonization at a low temperature (200 °C) and direct pyrolysis at a moderate temperature (500 °C) followed by potassium hydroxide (KOH) activation strategy at a high temperature (800 °C), respectively. The physicochemical properties of MCs are investigated by Raman spectra, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and nitrogen adsorption–desorption isotherms. Results show that MCs derived from moxa floss of different storage years by two facile approaches possess different morphologies: MCs by hydrothermal carbonization  exhibit porous nanosheet structures, the highest specific surface area is about 1788.6 m Two novel carbons (MCs) derived from moxa floss of different storage years have been prepared by two low-cost and facile approaches, which are hydrothermal carbonization and direct pyrolysis followed by KOH activation strategy, respectively. The morphologies and heteroatom doping of carbon materials are strongly related to their electrical conductivity. However, the electrical conductivity and chemical stability of carbon materials greatly influence the power density and cycling stability of carbon-based supercapacitors.25–27 Therefore, there is in dire need to fabricate carbon materials with enhanced features.Supercapacitors, owing to their key advantages of rapid charge–discharge rates, long-term cycling life, superior power densities, excellent safety, minimal maintenance costs, and environmental friendliness, have widespread applications in portable devices, hybrid electric vehicles, power tools, and energy backup systems.28 macromolecules,14 and biomass materials.9,29 Compared to traditional carbon precursors, biomass materials, such as Eulaliopsis binata,30 tobacco rods,27 banana peel,31 silk,32 rice husk,33 tealeaves,34 leaf,35Auricularia,36 coconut shell,37etc. have attracted tremendous interest owning to their large availability, low cost, renewability, and environmental friendliness. Generally, biomass-based carbons are prepared by a two-step procedure, in which carbon precursors are directly pyrolyzed at the temperature range of 600–900 °C in an inert atmosphere,38 or hydrothermally carbonized at a relatively low temperature,27 followed by physical or chemical activation at elevated temperature.30 In recent years, KOH activation has achieved giant success in the generation of carbon materials with the features of good electrical conductivity, abundant porous structure, heteroatom doping, ultrahigh specific surface areas and excellent physicochemical stability.27,30,39 Therefore, most of these carbon-based supercapacitors offer a large number of attractive attributes, such as fast charge–discharge rates, high thermal stability, good operational safety, excellent cycling stability, high power capability.26,40 There is still an urgent need to explore new biomass precursors for the production of carbon materials.For years, carbon materials have been successfully produced from fossil materials,Artemisia argyi Levl. Et Vant and used for moxibustion in Traditional Chinese Medicine.41,42Artemisia argyi is a common herbaceous perennial aromatic grass with a creeping rhizome belonged to the family compositae and widely distributed in Northeastern Asia, such as China, Mongolia, Japan, Korea and the Russian Far East regions.43 It is annually renewable, available in abundance and naturalized in dry and semiarid habitats, such as dry mountain slopes, steep river banks, coastal scrub, wasteland and along road and railway verges, which is beneficial to protect soil and water.44 The chemical composition of Artemisia argyi includes polysaccharides , flavonoids, essential oil, triterpenoids, sesquiterpenoids, steroids, nitrogen compounds , carboxylic ester, and inorganics .45,46 The leaves of Artemisis argyi are usually stored for different years before processing into moxa floss. During storage, a number of physicochemical and physiological changes occur. The aged and dried Artemisia argyi leaves are then subjected to several rounds of pulverization and sifting.47,48 Considering that Artemisia argyi leaves are abundant, cheap, and renewable biological resources,49 they should be a kind of promising carbon precursor for the production of carbon materials. To the best of our knowledge, there is no report on the use of Artemisia argyi leaves of different storage years as biomass precursor to synthesize carbons. Obviously, pursuing new potential applications of these leaves is a meaningful choice for enriching the use of Artemisia argyi.Moxa floss is processed from the dried leaves of 2 g−1, hierarchical porous structure coexisting of micro-, meso-, and macro-pores, numerous heteroatom (O and N) functional groups, long-term cycling stability as well as excellent capacitive performance, for example, the as-assembled supercapacitor based on MC-5 exhibits a high specific capacitance of 288.3 F g−1 at 0.25 A g−1, an excellent rate performance of 243.5 F g−1 even at 30 A g−1 with 84.5% capacitance retention of its initial specific capacitance, and an outstanding long-term cycling stability with 98.7% capacitance retention after 10 000 cycles at 5 A g−1. Therefore, using moax floss as carbon source to prepare biomass-derived carbons for high-power supercapacitors is an extremely promising strategy for developing low-cost and high performance electrode materials and pursuing new application of Artemisia argyi.In this study, we presented a systematic investigation on the differences in electrochemical properties of the biomass-derived carbons by two facile approaches using moxa floss of different storage years as the carbon precursor. The moxa floss of different storage years was directly subjected to hydrothermal carbonization at a low temperature (200 °C) (or pyrolysis at a moderate temperature (500 °C)) followed by potassium hydroxide (KOH) activation at a high temperature (800 °C), giving moxa floss-derived porous carbons (MCs). These resulting carbons possess different morphologies but exhibit high specific surface area up to 1788.6 m2.2.1Moxa floss were obtained from a moxa floss production factory in Nanyang, Henan Province, China, which included different storage years  with the production ratio of 3 : 1. Storage year refers to the number of years that the dried mugwort leaves were stored before processing into moxa floss, while production ratio refers to the weight of the starting material (dried mugwort leaves) to the weight of the finished product (moxa floss) after processing, for example the ratio 3 : 1 means 3 kg of dried mugwort leaves is processed into 1 kg of moxa floss. The moxa floss was repeated rinsed with distilled water for about three days and dried. Three types of moxa floss were denoted as MF-1 (0 storage year and 3 : 1 ratio), MF-2 (3 storage years and 3 : 1 ratio) and MF-3 (5 storages year and 3 : 1 ratio), respectively. All of the other chemicals were analytical grade and obtained from commercial suppliers without further treatment.2.22.2.1−1. After cooling down to room temperature, the activated product (MC) was washed several times with 1 mol L−1 HCl solution to remove inorganic salts, and then with distilled water until neutral pH was achieved. Finally, MC was dried in a conventional oven at 100 °C overnight, and then ground to powder in an agate mortar. MCs derived from different moxa floss by hydrothermal carbonization were denoted as MC-1 (MF-1), MC-2 (MF-2), MC-3 (MF-3), respectively.Typically, the dried moxa floss (4 g) and distilled water (60 mL) were added into a 100 mL Teflon lined stainless autoclave. The autoclave was kept at 200 °C for 20 h and then cooled to room temperature naturally. The resulting solid product was collected by filtration and washed abundantly with distilled water, and then dried at 100 °C overnight in a conventional oven. After that, the solid product was impregnated with KOH in an aqueous solution (the weight ratio of the solid product to KOH was 1 : 3) and the mixture was stirred for 2 h at room temperature, which was followed by evaporation step at 100 °C in a conventional oven. The dried mixture was ground to powder in an agate mortar, and then heated in a tubular furnace at 800 °C for 1 h under a nitrogen gas flow with the heating ramp rate of 4 °C min2.2.22 flow for 2 h. The final temperature and heating ramp rate of the furnace were 500 °C and 4 °C min−1. After cooling down to room temperature, the resulting pre-carbonized product was impregnated with KOH in an aqueous solution (the weight ratio of the solid product to KOH was 1 : 3) and the mixture was stirred for 2 h at room temperature, which was followed by evaporation step at 100 °C in a conventional oven. The dried mixture was ground to powder in an agate mortar, and then heated in a tubular furnace at 800 °C for 1 h under a nitrogen gas flow with the heating ramp rate of 4 °C min−1. After cooling down to room temperature, the activated product (MC) was washed several times with 1 mol L−1 HCl solution to remove inorganic salts, and then with distilled water until neutral pH was achieved. Finally, MC was dried in a conventional oven at 100 °C overnight, and then ground to powder in an agate mortar. MCs derived from different moxa floss by direct pyrolysis were denoted as MC-4 (MF-1), MC-5 (MF-2), MC-6 (MF-3), respectively.Typically, the dried moxa floss (7 g) was directly pyrolyzed in a tubular furnace under N2.32 adsorption analyses (BELSORP-Mini II) at 77 K. The samples were outgassed at 150 °C for 12 h under a vacuum. The specific surface area (SSA) was obtained by using the Brunauer–Emmett–Teller (BET) model. The pore size distribution (PSD) was calculated from the adsorption branches of the isotherms using the Barrett–Joyner–Halenda (BJH) model.The morphologies of the obtained porous carbons (MCs) were observed with field-emission scanning electron microscope  and transmission electron microscopy . The structure of MC was investigated by X-ray diffraction . Raman spectra were collected on a HORIBA Scientific LabRAM HR Evolution Raman Spectrometer system with 514 nm laser. The surface elemental characterization of MC was performed by X-ray photoelectron spectroscopy  with a monochromatic Al Kα source. All binding energies were referenced to the C1s neutral carbon peak at 284.8 eV. The textural properties were characterized by N2.4−1 KOH or EMIMBF4) in a 2016 stainless steel coin cell. In the case of ionic liquid electrolyte, the preparation process of working electrodes was the same as that for the aqueous electrolyte, but the assembly process was carried out in a nitrogen-filled glove box. All the electrochemical measurements except for cycling stability tests were carried out on a CHI 660E electrochemical workstation. The cycling stability tests were studied on a Land cell tester. The electrochemical properties of MC were characterized by cyclic voltammetry (CV), galvanostatic charge–discharge (GCD), and electrochemical impedance spectroscopy (EIS). CV tests were carried out between 0 and 1.0 V for the aqueous electrolyte and between 0 and 3.5 V for the ionic liquid electrolyte. GCD tests were performed at different current density varying from 0.25 to 30 A g−1 in the same potential range as the CV test. EIS tests were carried out with the amplitude of 5 mV in the frequency range of 105 to 10−3 Hz referring to open circuit potential. The specific capacitance (Cs) of the two-electrode device based on GCD was calculated according to the equation:30Cs = 4I/(m × ΔV/Δt), where I (A) is the constant charge–discharge current, m (g) is the total mass of active materials on the two electrodes for MC supercapacitor, Δt (s) is the discharge time, ΔV (V) is the voltage change excluding the IR drop during the discharge process, and ΔV/Δt is calculated from the slope of the discharge curve after the voltage drop. The energy density  and power density  of MC supercapacitors were calculated using the equations:10,40,50E = Cs × V2/(8 × 3.6), P = E/Δt, where Cs (F g−1) is the specific capacitance of the MC supercapacitors, V (V) is the voltage after IR drop, and Δt (s) is the discharge time.The electrochemical performances of MC were evaluated in a symmetrical two-electrode system (supercapacitors device). The working electrodes were typically fabricated by mixing MC as the active material (85 wt%), acetylene black (10 wt%), and polytetrafluoroethylene  binder in ethanol. The obtained electrode materials were coated onto the nickel foam current collectors (1 cm × 1 cm) with a spatula. The pre-made electrodes were dried at 100 °C for 12 h in a vacuum oven. After drying, the electrodes were pressed under a pressure of 10 MPa for 1 min. The two nearly identical (by weight and size) MC electrodes were separated using a polypropylene membrane separator soaked with electrolytes (6 mol L3.3.1The morphologies of MC were characterized by FESEM observations . RegardlThe detailed porous nanostructure of MCs has been further studied by TEM technique . The TEM51 The broad (002) diffraction peaks usually located at about 23° suggests a disturbed structure in the amorphous state of all the samples of MC due to the randomly oriented aromatic carbon sheets, which is beneficial for creating high specific surface area (SSA).52 It is noted that the broad (002) diffraction peak positions of all the samples of MC show slightly different. The difference of the (002) diffraction peak positions may be attributed to KOH activation process and the different surface doping amount of N and O heteroatoms of MC derived from different moxa floss  and 1598 cm−1 (G band). The D band is associated with the defects and impurities in disordered carbon structure, while the G band arising from the bond stretching of all sp2-bonded pairs is should be attributed to graphite in-plane vibrations.55 The intensity ratio of the D and G band (ID/IG) is well known to be an indicator of the disorder degree of MC. The ID/IG peak ratios of all the samples of MC are around 1.00, exhibiting a relatively low degree of graphitization and containing a large number of disordered carbon structures.56The structures of MC were well investigated by X-ray diffraction (XRD) patterns and Raman spectra. The XRD patterns of MC were collected and the results are shown in  spectra  of MC cl51 and then keep almost horizontal with increasing the relative pressure. The distinct hysteresis loops (the inset of P/P0 = 0.4–0.8) prove the presence of abundant mesopores in all the samples of MC.52 Moreover, the isotherms rise slightly near the saturated vapor pressure, which demonstrates the existence of macropores.51 The features of these isotherms indicate that the samples of MC exhibit a well-developed hierarchical porous structures including the micro-, meso-, and macro-pores. The pore size distributions (PSD) of all the MC samples (estimated by the BJH method) are clearly shown in 2 g−1 with the total pore volume ranging from 0.6476 to 0.8170 cm3 g−1 and the average pore sizes are 1.8271 nm, 1.9391 nm and 1.7591 nm, respectively, while the samples  by direct pyrolysis possess high SSA in the range 1323.0–1628.0 m2 g−1 with the total pore volume ranging from 0.5761 to 0.7058 cm3 g−1 and the average pore sizes are 1.7342 nm, 1.8520 nm and 1.7418 nm, respectively. Clearly, as the storage years of moxa floss increase from 0 year to 5 years, the BET SSA and pore volume of MCs decrease. Furthermore, the MC by hydrothermal carbonization possesses the higher BET SSA, larger pore volume and the longer average pore size than that of MC derived from the same storage years by direct pyrolysis.To further evaluate the pore properties of MC, nitrogen absorption–desorption isothermal analysis were employed. As shown in 3.2 PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C (centered at 284.8 eV), C–N (centered at 285.3 eV), C–O (centered at 286.3 eV), and CO (centered at 288.4 eV). The high-resolution O1s spectra of MC clearly present the existence of several oxygen-based groups including three peaks centered at 532.5, 533.3 and 535.6 eV, representing CO quinone type groups (O–I), C–OH phenol groups and/or C–O–C ether groups (O-II), and chemisorbed oxygen (COOH carboxylic groups) and/or water (O-III), respectively. Moreover, the high-resolution N1s spectra of MC can be deconvoluted into three peaks, which are assigned to amine nitrogen (amine N) located at around 399.6 eV, pyrrolic/pyridine (N-5) located at around 400.3 eV, and quaternary (N-Q) located at around 401.3 eV. These results indicate that there are numerous heteroatoms doped in the surface of MC and these N and O functional groups can improve pseudocapacitance through faradaic reactions and enhance electrical conductivity. Therefore, with controllable heteroatoms (N and O) doping state, two different morphologies, high specific surface areas, and hierarchical porous structure, all the samples of MC are expected to exhibit excellent electrical conductivity.X-Ray photoelectron spectroscopy (XPS) was performed to investigate the surface chemical compositions and analyze the surface functional groups of MC. As shown in 3.3−1 KOH aqueous solution electrolyte at room temperature , galvanostatic charge–discharge (GCD), and electrochemical impedance spectroscopy (EIS) tests for a traditional symmetric two-electrode system in the 6 mol Lperature .−1 in the potential range from 0 to 1 V show a nearly symmetrical rectangular shape in their CV curves, indicating an ideal double-layer capacitance behavior based on ionic adsorption and exchange, although their morphologies are different; and with the scan rate increasing from 5 to 200 mV s−1, they still remain a rectangular-like shape  of MC. These results demonstrate that the MC with two different morphologies (the porous nanosheet morphology and the blocky and rod-like morphology), both can exhibit excellent capacitive performance.The typical CV curves  of all s Fig. S2a, suggest−1 are liner and show a nearly perfect isosceles triangle shape with negligible IR drop. Similarly, the GCD curves of these MC electrodes at different current densities also exhibit a nearly perfect isosceles triangle shape shown in Fig. S2b.−1. These results indicate that the MC electrodes in KOH aqueous solution electrolyte have good charge–discharge property and good capacitive behavior contributed by the pseudocapacitance of the N and O functional groups and abundant micropores, which are corresponding to the CV results. The good charge–discharge property may be associated with the large effective surface areas which can guarantee ion storage space, large numbers of mesopores which can improve easily ion diffusion channels. The specific capacitance (Cs) values of MC from the discharge curves at a constant current density of 0.5 A g−1 shown in −1 for MC-1, 278.0 F g−1 for MC-2, 274.5 F g−1 for MC-3, 263.8 F g−1 for MC-4, 279.3 F g−1 for MC-5, and 260.5 F g−1 for MC-6, respectively.The GCD curves  of theseCs) of the MC electrodes with current densities ranging from 0.25 to 30 A g−1 calculated from the discharge curves after IR drop in the GCD tests  of these MC electrodes are comparable or even higher than that of most recently reported biomass-derived carbons for supercapacitors  tests were carried out with the amplitude of 5 mV in the frequency range of 10−1 at a power density of 70.3 W kg−1, which is higher when compared with that of other recently reported carbon-based supercapacitors.25,27,59,60 In addition, the energy density decreases slowly with the rapid increase of power density. Even if the power density increased up to 12.3 kW kg−1, the energy density can still retain a value of 6.0 W h kg−1, suggesting the superior ability for energy storage of the MC supercapacitors.The Ragone plots of the MC supercapacitors are shown in 4 electrolyte is 3.5 times higher than 6 mol L−1 KOH. As shown in −1, suggesting an ideal electrochemical capacitor. Besides, the GCD curves of MC-2 display a nearly isosceles triangle shape at different current densities as shown in −1, implying typical electrochemical capacitive behavior, the high charge–discharge efficiency and low equivalent series resistance. The specific capacitances (Cs) of MC-2 electrode with current densities ranging from 0.25 to 30 A g−1 are shown in Cs displays a decreasing tendency with the current density increasing from 0.25 to 30 A g−1. It is noteworthy that MC-2 exhibits good performance with a high specific capacitance of 286.8 F g−1 at a current density of 0.25 A g−1 and medium retention rate (152.3 F g−1) at a high current density of 30 A g−1 (53% of the capacitance retention).MC-2 exhibits higher specific surface area, more balanced micropores and mesopores as well as higher N atomic concentration, which may endow it delivering higher capacitance performance in ionic liquid electrolytes. In order to further evaluate the electrochemical performance of MC-2, supercapacitors based on MC-2 was assembled in the organic electrolytes. More practically, the potential window of ionic liquid EMIMBF27 Furthermore, the presence of N and O functional groups makes the surface of MCs more easily infiltrated by electrolytes and enhances their electrical conductivity, and thus further improves the electron and ion transportation efficiency.The electrochemical results indicate that although the morphologies of MCs derived from moxa floss of the same storage year by two different pre-carbonization strategies are different, they present excellent performances for supercapacitors application, which can be attributed to their high specific surface areas (high SSA), unique hierarchical porous structures, numerous heteroatom (O and N) functional groups, and good electrical conductivities. High SSA provides sufficient electrode–electrolyte interface to form more electric double layers for charge accommodation; the hierarchical porous structure ensures the rapid ion diffusion by shortening the diffusion pathways, and good electrical conductivity facilitates efficient transport of the electrons, which are important for achieving high-rate performances.4.2 g−1 providing sufficient active sites, abundant hierarchical pores coexisting of micro-, meso-, and macropores, numerous nitrogen- and oxygen- based functional groups, which facilitate electrolyte diffusion and ion transfer, and endow the MC electrodes with good electrical conductivity. Moreover, the as-assembled MC supercapacitors with aqueous electrolyte in a two-electrode system exhibit an outstanding specific capacitance of 288.3 F g−1 at 0.25 A g−1, an excellent rate performance, and an outstanding long-term cycling stability with 98.7% of initial capacitance retention even suffered from 10 000 charge–discharge cycles at 5 A g−1. Therefore, utilizing moxa floss as carbon precursor to prepare biomass-derived activated carbon for supercapacitors is an extremely promising new application of moxa. Meanwhile, in consideration of its renewability, low-cost and the abundance, moxa floss as a novel biomass source significantly responds to the demand of the sustainable development of the society, achieving low-cost and high-performance electrode materials from renewable sources. Considering the fact that the method, time and temperature of the pre-carbonization, KOH activation time and temperature, and the weight ratio of the pre-carbonized product to KOH, play important roles in the regulation of the capacitance performance of the final porous carbons, we will systematically explore the synthesis conditions for morphology change, specific surface area, heteroatom doping level, and the porous structures as well as the correlation between these factors and electrochemical performances of the prepared MCs in our following research.In summary, we have successfully developed a new concept of moxa floss use, that is, the utilization of moxa floss is to fabricate biomass-derived porous carbon for supercapacitors with high performance. Novel MC has been successfully prepared by two low-cost, safe, and facile approaches: hydrothermal carbonization-KOH activation and pyrolysis-KOH activation. Although moxa floss of different storage years were utilized as carbon precursors, the as-obtained MC samples, despite the different morphologies, possess similar physicochemical properties, such as high specific surface area up to 1788.6 mThere are no conflicts to declare.RA-008-C8RA01672K-s001


Streptomyces ardesiacus 156VN-095 led to the isolation of three hitherto undescribed angucycline glycosides, including urdamycins W and X (1 and 2) and grincamycin U (9), as well as their seven known congeners. The structures of the new compounds were elucidated by means of spectroscopic methods  and comparison of their experimental data with literature values. Compounds 1–3 and 9 were evaluated for their anti-Gram-positive bacterial effect and cytotoxicity against six cancer cell lines. Compound 1 displayed significant cytotoxicity against all the tested cell lines with GI50 values of 0.019–0.104 µM. Collectively, these findings highlight the potential of angucycline glycosides as leading structures for the development of new anti-cancer drugs.Chemical investigation of the ethyl acetate extract from the culture broth of the marine-derived actinobacterium  Streptomyces are renowned as the largest contributor of currently used antibiotics . Th. Th9 was3) + , 1 H NMR  and 13 C NMR  see Urdamycin W (2): dark red powder; IR νmax 3417, 2929, 1632, 1515, 1430, 1299, 1088 cm−1; UV (MeOH) λmax (log ε) 298 (4.9), 470 (4.3) nm; HRESIMS m/z 669.1984, [M + Na]+ , 1H NMR  and 13C NMR  see Urdamycin X (9): orange powder; IR νmax 3396, 2929, 1628, 1430, 1371, 1257, 1070 cm−1; UV (MeOH) λmax (log ε) 230 (4.8), 255 (3.9), 442 (3.2) nm; HRESIMS m/z 753.2734, [M + Na]+ , 1H NMR  and 13C NMR  see Grincamycin U  was used for determination of MIC values. Compounds 1, 2, and 9 were tested against three Gram-positive bacteria including, Staphylococcus aureus (KCTC 1927), Micrococcus luteus (KCTC 1915), and Bacillus subtilis (KCTC 1021). The tested compounds were prepared in the range of 0.5–256 µg/mL in 96-microtiter plates by a serial double dilution. An overnight culture broth of each strain was dispensed in sterilized 0.9% saline to an inoculum density of 5 × 105 cfu by comparison with a McFarland standard [1, 2, and 9, 100 µL), in the plate to yield final concentrations from 0.25 to 128 µg/mL. The plates were maintained for 24 h at 37 °C. The MIC value is the lowest concentration at which the microorganism did not demonstrate visible growth, as indicated by the presence of turbidity. Kanamycin was used as a positive control. All experiments were conducted twice to check reproducibility.The antimicrobial assay of standard . The dil1–3, and 9 was performed as previously described [The SRB cytotoxicity test for escribed . Cancer 50 values were determined by the software of GraphPad Prism 8 .Statistical analysis was evaluated by one-way ANOVA followed by Dunnett’s t-test and the GIStreptomyces ardesiacus 156VN-095 and three of them were new compounds . The structures of the new metabolites were elucidated by spectroscopic analysis and comparison of their experimental data with those reported in the literature. The new compounds showed selective anti-bacterial effects against three Gram-positive bacterial strains and significant cytotoxicity against a panel of cancer cell lines with a different potency. Among them, 1 showed the strongest activities against all the tested cell lines with GI50 values of 0.019–0.104 µM. These results expanded biochemical diversities of naturally occurring angucycline glycosides.In conclusion, we have isolated 10 angucycline glycosides from the culture broth of


The nanoscale MnS particles were highly soldered on the biochar support surface, and this adsorbent displayed the effective removal of Cr(vi) (98.15 mg L−1) via synergistic effect between adsorption and reduction/precipitation under weak acid conditions (pH = 5.0–6.0). The adsorption kinetic data were described well by the pseudo second-order kinetic model, suggesting that the reaction process was a chemisorption process. The adsorption isotherm data were described well by the Redlich–Peterson model, further suggesting that this reaction was a hybrid chemical reaction-sorption process. In addition, the Dubinin–Radushkevich isotherm model with 8.28, 8.57, and 12.91 kJ mol−1 adsorption energy also suggests that it was a chemisorption process. The simple and eco-friendly preparation process, low-cost, and the high removal efficiency could make it a promising material for the purification of Cr(vi)-contaminated wastewater.In order to remove hexavalent chromium (Cr( vi)) efficiently and simplify the adsorbent preparation process, we employed a single step method to prepare a new biochar supported manganese sulfide material.In order to remove hexavalent chromium (Cr( According to the published literatures,7,17 there are four mechanisms for the removal of Cr(vi) from waste water by biochar support materials. First one is the electrostatic adsorption. During the process, Cr(vi) cation could be adsorbed onto the charged biochar surface without reduction reaction. While, this form of Cr(vi) removal is relatively ineffective. Another one is the cationic adsorption. For this pathway, all of Cr(vi) could be reduced to Cr(iii) and then Cr(iii) would be adsorbed onto the biochar surface. The third pathway is complete reduction, which Cr(vi) could be adsorbed onto the biochar material surface and then completely reduced to Cr(iii). The last one is the combination of adsorption and reduction. During the process, a part of Cr(vi) would be adsorbed onto biochar materials surface and the others would be reduced to Cr(iii). In order to meet strict environmental regulations, Cr(vi) should be effectively adsorbed onto the biochar materials and then reduced to Cr(iii) as the combination of adsorption and reduction reveals the excellent removal efficiency.18 For the reduction of Cr(vi) in waste water, the typical reducing agents are iron or iron(ii) ions and the reaction is usually executed under strong acidic conditions (pH = 1 or 2).20,21 Moreover, the reported material preparation methods are usually two-step pathway (including biochar preparation and biochar modification), which requires large amounts of chemical reagents, high energy consumption, and complex preparation process, leading to the high preparation cost and secondary environmental pollution.8,18,22 Especially in the published word by Lyu et al., the dissolution of FeS nanoparticles was near 80% after Cr(vi) adsorption.18 Moreover, during the CMC-FeS@biochar preparation process, the water should be purged with purified N2, and the whole devices needed the N2 protection, resulting a complex procedure and high cost. Wadhawan19 proved that Cr(vi) could be reduced by MnS effectively and the preparation process was very simple via hydrothermal method. However, to our knowledge, there were no published works reported about the preparation of biochar-supported MnS nanoparticles (MnS@biochar), and there was also no detailed investigation into the effectiveness of Cr(vi) removal by MnS@biochar.Many technologies have been investigated to remove or reduce Cr(via one-pot method to adsorb and reduce Cr(vi) in aqueous solution. In the experimental design, MnS loaded dosage, MnS@biochar dosage, solution pH, retention time, and initial Cr(vi) concentrations were executed to test the Cr(vi) removal capacity of MnS@biochar. The characterizations and mechanism were analyzed by transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction (XRD) patterns, Fourier transform infrared spectroscopy (FT-IR), ζ-potential analysis, and X-ray photoelectron spectroscopy (XPS).In this work, the overall goal was to prepare a new biochar support MnS (MnS@biochar) material 2.2.14H14MnO8, AR: 99%), and ethylenediamine  were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. Thioacetamide  was purchased from Sinopharm Chemical Reagent. Potassium dichromate  was purchased from Aladdin Chemistry Co. Ltd. The deionized water with a resistivity of 18.25 MΩ cm was used for all the experiments.Manganese acetate , solution pH value (2–10), retention time (0–1440 min), and initial Cr(vi) concentrations (50–300 mg L−1). For the MnS loaded dosage, 0.1 g of MnS@biochar-(1–5) sample and 50.00 mL of Cr(vi) aqueous solution (CCr(VI) = 150.00 mg L−1) were placed in 100 mL of conical flask for 1440 min, and the pH value was kept at 5.0–6.0 (25 °C) using HCl (0.1 mol L−1) or NaOH (1 mol L−1). For the effect of MnS@biochar dosage, the various amounts of the MnS@biochar and 50.00 mL of Cr(vi) aqueous solution (CCr(VI) = 150.00 mg L−1) were placed in 100 mL of conical flask for 1440 min, and the pH value was kept at 5.0–6.0 (25 °C) using HCl (0.1 mol L−1) or NaOH (1 mol L−1). For the effect of solution pH value, the mixture was adjusted to the range of 2–10 (25 °C) using HCl (0.1 mol L−1) or NaOH (1 mol L−1). To examine the effect of retention time, 75.00 mg of MnS@biochar and 50.00 mL of Cr(vi) aqueous solution (CCr(VI) = 150.00 mg L−1) were placed in 100 mL of conical flask for predetermined time, and the pH was kept at 5.0–6.0 (25 °C) using HCl (0.1 mol L−1) or NaOH (1 mol L−1). To determine the adsorption isotherms, different initial concentrations of Cr(vi) solution  were agitated until equilibrium was achieved with the same adsorbent dosage in 100 mL of conical flask with solution pH value of 5.0–6.0 (25 °C) using HCl (0.1 mol L−1) or NaOH (1 mol L−1). Moreover, the adsorption temperature  was also executed. After the adsorption process, the suspension was filtered through a 0.45 μm cellulose acetate membrane filter paper, and the Cr(vi) concentration in the filtrate was analyzed using an Elan drc-e ICP-MS . The average data were reported after three times repeated experiments, and the blank sample without adsorbent was also carried out to verify the insignificance of Cr(vi) degradation and adsorption to the conical flask.All of the batch experiments were executed in 100 mL of conical flask in an air thermostatic shaker . In detail, a series of parameters were carried out, including MnS loaded dosage, MnS@biochar dosage  equilibrium adsorption amounts (mg) of per gram MnS@biochar (mg g−1), C0 was the initial concentration of Cr(vi) (mg L−1), Ce was the concentration of Cr(vi) at equilibrium stage (mg L−1), V was the volume of Cr(vi) solution (L), m was the weight of MnS@biochar (g).The equation of equilibrium adsorption amount was as follows:2.4.223The equation of pseudo-first-order (PFO) kinetic model was as follows:22where, k1 was equilibrium rate constant of PFO kinetic model (min−1), k2 was the constant of PSO kinetic model (g (mg−1 min−1)), qe was the Cr(vi) adsorption amount (mg g−1) at equilibrium time, qt was the Cr(vi) adsorption amount (mg g−1) at any time t (min), k2 was determined by plotting t/qtversus t.The equation of pseudo-second-order (PSO) kinetic model was as follows:2.4.324The equation of Langmuir adsorption isotherm model was as follows:24The equation of Freundlich adsorption isotherm model was as follows:4The equation of Redlich–Peterson (RP) adsorption isotherm model was as follows:25Ce was the concentration of Cr(vi) at equilibrium stage (mg L−1), qe was the adsorption amount of Cr(vi) on MnS@biochar at equilibrium stage (mg L−1), qm was the monolayer adsorption capacity of MnS@biochar (mg L−1), b was the Langmuir constant related to the affinity of the bending sites and energy of adsorption (L mg−1), KR and α were the constants of DP isotherm model, β was the exponent ranged from 0 to 1, KD was the constant of DR isotherm model [mol2 kJ−2], ε was the Polanyi potential energy (kJ mol−1). ε = 2.48 ln(1 + 1/Ce), qm and b could be obtained from the slope and intercepts of the linear plots of Ce/qeversus Ce, Kf was the Freundlich constant indicted the adsorption capacity and n was another Freundlich constant indicated the adsorption intensity.The equation of Dubinin–Radushkuvich (DR) adsorption isotherm model was as follows:2.5λ = 0.15418 nm) radiation source. The contents of C element, H element, N element, and S element in biochar and F–MnS@biochar were measured by Euro EA3000 Elemental Analyzer , and the average data were reported after twice repeated experiments. For specific surface areas (SSA) analysis, the biochar, MnS, and F–MnS@biochar samples were determined by the ChemiSorb 2720 Analyzer . The samples were outgassed at 105 °C for 16 h and then executed the N2 adsorption at 77 K. The Fourier transform infrared spectroscopy (FT-IR) were collected by the Spectrum One FT-IR spectrometer ranged from 4000 cm−1 to 400 cm−1 with average 32 scans at resolution of 2 cm−1 . The zeta-potential of biochar, MnS, and F–MnS@biochar samples were performed at 25 °C by Zetasizer Nano . The X-ray photoelectron spectroscopy (XPS) analysis was recorded by the XPS system (Kratos AXIS Ultra) at 150 W equipped with a monochromatic Al X-ray source. The analysis of the XPS spectra was executed by XPSPEAK software (version 4.1).26The morphology analysis of MnS@biochar was characterized by scanning electron microscopy (SEM) . The size distributions and morphology of MnS particles on biochar were determined by transmission electron microscopy  at accelerating voltage of 120 kV. The structural characterizations of biochar, MnS, fresh MnS@biochar (F–MnS@biochar) and MnS@biochar after adsorption (R–MnS@biochar) were tested by X-ray diffraction (XRD)  with a Cu-Kα (3.3.13.1.1vi) adsorption capacities of MnS@biochar sample with different MnS loaded dosages, different dosages and solution pH values were shown in vi) adsorption capacity decreased with MnS loaded dosages decreased, and the value of MnS@biochar-1 was closed to that of MnS@biochar-2. Considering the equilibrium Cr(vi) adsorption amount and MnS@biochar yield, the MnS@biochar-2 sample was selected as the final experimental adsorbent, denoted as MnS@biochar. As shown in −1 to 1.5 g L−1 caused an increase in Cr(vi) removal amounts (from 90.18 mg g−1 to 98.15 mg g−1). While, when the adsorbent dosage increased from 1.5 g L−1 to 3.0 g L−1, the equilibrium Cr(vi) removal capacities of MnS@biochar decreased to 49.84 mg g−1, which could meet the maximum contaminant level of 0.5 mg L−1 for Cr(vi) in waste water. Compared to 1.5 g L−1 of MnS@biochar, Cr(vi) removal amounts of MnS (0.3 g L−1) and biochar (1.2 g L−1) were 30.61 mg g−1 and 53.53 mg g−1, suggesting that there was a synergistic effect between MnS particles and biochar support. As shown in vi) removal from aqueous solution, and the optimal solution pH was the acidic environment. On the other hand, based on the CrO42− hydrolysis  and negative charged MnS@biochar would be also contribution to the increased Cr(vi) removal amount during the acidic adsorption solution.18The equilibrium Cr removal amount and adsorption retention time. From the results, it suggested that the adsorption rate was rapid within 60 min, and then it slowed down until the equilibrium state was reached at about 480 min. In addition, as shown in vi) adsorption on MnS@biochar was a chemical process occurring on MnS@biochar surface.16 The fitting parameters of PFO kinetic model and PSO kinetic model were listed in Table S1.R2) of PSO kinetic model was 0.999, which was higher than 0.925 for PFO kinetic model. The qe value calculated by PSO kinetic model was closed to the experimental result. Moreover, the normalized standard deviation (NSD) and average relative error (ARE) values of PSO kinetic model were lower than these of PFO kinetic model, further suggesting that the adsorption data could be matched very well by PSO kinetic model. Compared to the published works  and MnS@biochar. vi) removal amounts of MnS@biochar at different initial Cr(vi) concentration and adsorption temperature, and the models of Langmuir  removal capacity increased with the initial Cr(vi) concentration increased, and the higher adsorption temperature would enhance the Cr(vi) removal capacity of MnS@biochar. The fitting parameters of these adsorption isotherm models were listed in Table S3.R2 (above 0.980) at different adsorption temperature, and RP model had the lowest values of MPSD and HYBRID, suggesting that Cr(vi) removal by MnS@biochar was a combination process of chemical reaction and adsorption.23 The β value of RP model was closed to 1.0, suggesting that the Cr(vi) removal by MnS@biochar process was closed to the ideal Langmuir condition. The bonding energy of Cr(vi) adsorption onto MnS@biochar was estimated by DR model (R2 > 0.88), and the values at different adsorption temperature were about 8.28, 8.57, and 12.91 kJ mol−1. Based on the literature,27 the bonding energies for chemosorption usually ranges from 8.0 kJ mol−1 to 16.0 kJ mol−1. Hence, the conclusion could further prove the chemisorption between Cr(vi) and MnS@biochar.The adsorption isotherm was another important factor in describing the interactive behavior between Cr removal capacity of MnS@biochar.The morphology characterizations of fresh MnS@biochar (F–MnS@biochar) 3.2.2vi) removal. For the specific surface areas and porous structures of MnS, biochar, and F–MnS@biochar  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C, and C–O groups.28 (2) For the pattern of MnS, six characteristic peaks at 2θ = 29.72°, 34.46°, 49.44°, 58.72°, 61.57°, 72.43° were assigned to the indices {1,1,1}, {2,0,0}, {2,2,0}, {3,1,1}, {2,2,2}, {4,0,0} (JCPDS: no. 06-0518).19 (3) For F–MnS@biochar and R–MnS@biochar, the presence of the wide peak near 22.5° indicated the presence of biochar support. (4) For F–MnS@biochar, there were several diffraction peaks at 29.59°, 34.27°, 49.24°, 58.63°, 61.52°, 72.36°, which were assigned to MnS (JCPDS: no. 06-0518). (5) Compared to F–MnS@biochar, the new diffraction peaks at 28.82° and 36.06° presented the existence of hausmannite , which was assigned to the indices {1,1,2} and {2,1,1}. The new substance would suggest that there is a chemical reaction between Cr(vi) and MnS@biochar.The XRD patterns of biochar, MnS, F–MnS@biochar, and MnS@biochar after reaction (R–MnS@biochar) were shown in 3.2.423,29 the peak near 3410 cm−1 was attributed to the stretching vibration of –OH; the peaks ranged from 2900 cm−1 to 2800 cm−1 were assigned to the stretching vibrations of C–H; the peak near 1437 cm−1 was assigned to scissoring vibrations of –CH2–. After adsorption, the intensity of peak near 1600 cm−1 assigned to the CO stretching vibrations increased. Compared to the biochar spectra, the peaks near 1697 cm−1 assigned to CC disappeared in the spectra of F–MnS@biochar and R–MnS@biochar; and the peaks near 805 cm−1 assigned to the Si–O stretching vibrations also disappeared in F–MnS@biochar spectra. According to the above result, it indicated that MnS particles would be soldered on biochar surface via the O-containing functional groups .The surface functional groups of biochar, MnS, F–MnS@biochar, and R–MnS@biochar were investigated and shown in 3.2.522,30,31 the bending energies at 530.3 eV, 532.2 eV, and 533.6 eV were ascribed to C–O, –OH, and CO, respectively. After adsorption–reduction reaction (O band increased from 16.9% to 35.6%. These changes suggested the complex complexation between Cr(vi) and O-contained functional groups on F–MnS@biochar surface, which was in accordance with FT-IR analysis. In addition, the new peak appeared at 530.8 eV would be ascribed to the O-contained bands in metallic oxide (e.g. Mn2O3 or Cr2O3).7,19 Considering S 2p spectra (vi)/S(iv), S(−ii) in H2S, and S(−ii) in MnS.32 However, there were no obvious changes in the S 2p3/2 spectra on R–MnS@biochar surface, and only the content of S element decreased from 3.7% to 2.9%. Moreover, the content of S(vi)/S(iv) increased from 7.6% to 15.3%. These changes also further proved the interaction between F–MnS@biochar and Cr(vi). For Mn 2p3/2 spectrum (19 the bending energies at 644.1 eV and 641.5 eV were attributed to Mn(ii) and Mn(iv) on F–MnS@biochar surface. While after adsorption–reduction (iii), and the percentage of Mn(ii) decreased from 91.6% to 62.8%. The presence of Mn(iii) was in accordance with XRD analysis and O 1s spectra. These changes further suggested the reactions between Cr(vi) and MnS on F–MnS@biochar surface. Furthermore, for Cr(vi) 2p3/2 spectrum on R–MnS@biochar , and Cr2S3 respectively. The content of Cr element on MnS@biochar surface was about 8.7%, which was closed to the equilibrium Cr(vi) absorption amount. These data would also prove the redox reaction between Cr(vi) and F–MnS@biochar.The XPS spectra of O 1s, S 2p, Mn 2p, and Cr 2p on F–MnS@biochar, and R–MnS@biochar surface and the relevant parameters were shown in reaction , C–O ban spectra , the benspectrum ,19 the beduction , the new@biochar ,33,34 it3.2.6vi) removal mechanism by MnS@biochar as follows (vi) were chemisorbed onto MnS@biochar surface through O-containing functional groups, including –OH, CO, OC–O, C–O, and the surface multihole structure was also contributed to the high Cr(vi) adsorption amount. The other portion of Cr(vi) were reacted with MnS nanoparticles. Generally, there were two forms of MnS on biochar surface in the aqueous solution, as MnS(s) particles could be dissolved in the water with a reported Ks0 of 10−13.73  and the other was MnS(aq). The increased solubility of MnS particles in acid solution may be caused by the proton-promoted dissolution mechanism, and the sequential reactions could be described by the following equations ((aq) primary existed in the form of MnHS+ at weak acid solution (pH = 5.0–6.0). Hence, it favored the reduction of Cr(vi) via a combination of homogeneous and heterogeneous at weak acid solution. For the heterogeneous pathway, Cr(vi) could be adsorbed on MnS(s) surface sites, and the surface complexes were formed. Then electron transfer between Cr(vi) and biochar surface or MnS(s) caused the formation of Cr(iii) and Mn(ii) change to Mn(iii).19 During this process, S(−ii) played an important role in the Cr(vi) reduction with the formation of S(vi), while Mn element had no change in its valence state. The potential reaction was shown in + on MnS(aq) surface would react with Cr(vi) and the products of Mn2O3 and Cr2S3 formed. In the reaction, Mn(ii) was involved in the Cr(vi) reduction with the formation of Mn(iii), while S(−ii) had no change. Summary, both biochar support and MnS particles were contributed to the removal of Cr(vi): Cr(vi) could be adsorbed onto biochar support surface through O-containing functional groups, and Cr(vi) would also be reduced by MnS particles via homogeneous and heterogeneous pathways at weak acid solution.According to the results of XRD, FT-IR, XPS and log C-pH, we proposed a possible Cr in aqueous solution, and the equilibrium Cr(vi) adsorption amount was about 98.15 mg g−1 at pH 5.0–6.0. A part of Cr(vi) could be adsorbed on biochar support surface with the O-containing function groups, while the others would be reduced by MnS nanoparticles via homogeneous and heterogeneous pathways at weak acid solution. The synergistic effect between adsorption and reduction/precipitation and the simple preparation process would make this adsorbent a promising material for the purification of Cr(vi)-contaminated wastewater.This paper employed a one-pot method to prepare a new biochar support manganese sulfide adsorbent. This adsorbent could effectively adsorb and reduce Cr(There are no conflicts to declare.RA-009-C9RA06028F-s001


In addition, DPPH, ABTS, and FRAP assays were used for determining the antioxidant capability. The results of total flavonoids and phenolics contents showed that ethyl acetate fraction (EAF) possessed the highest flavonoids and phenolics with values of 159.7 ± 2.5 mg rutin equivalents/g and 107.6 ± 2.0 mg galic acid equivalents/g, respectively. The results of in vitro hypoglycemic activity showed that all samples had effective α-glucosidase inhibition capacities, and EAF possessed the best inhibitory effect with IC50 value of 1.58 ± 0.24 μg/mL. In addition, n-butanol fraction (NBF) significantly promoted the glucose uptake rate of 3T3-L1 adipocytes. HPLC-MS analysis and molecular docking results proved the interactions between candidates and α-glucosidase. The results of antioxidation capacities showed that EAF possessed the best antioxidation abilities with DPPH, ABTS, and FRAP. In summary, the hypoglycemic activity of PAH might be related to the inhibition of α-glucosidase (EAF > PEF > NBF) and the promotion of glucose uptake in 3T3-L1 adipocytes (NBF). Simultaneously, the antioxidation capacity of PAH might be related to the abundant contents of flavonoids and other phenolics (EAF > PEF > NBF).Our research focused on the hypoglycemic capability and the possible mechanisms of extract and fractions from Polygoni Avicularis Herba (PAH) based on  Polygonum aviculare L. (family Polygonum). It is used in traditional Chinese medicine to treat dysuria, abdominal pain caused by intestinal parasites, skin eczema, and genital itching )).The kinetic analysis of PAHEE and its fractions were carried out with final substrate concentrations of 1, 2, 3, 4, 5 µmol/L, PAHEE concentrations of 2.5, 5.0, and 10.0 μg/mL, PEF, EAF, and NBF concentrations of 1.25, 2.5, and 5.0 μg/mL, respectively, and 4 cells per well, and the experiment was started 24 h later. The 3T3-L1 adipocytes were divided into model group (blank control), insulin group , and sample groups . After 24 h of administration, 10 μL medium was used to measure the glucose content. Cell viability was detected by CellTiter 96® AQueous One Solution Reagent according to the manufacturer’s instructions after the glucose uptake experiment [® AQueous One Solution Cell Proliferation Assay reagent was added to the wells of the experiment plate, and then incubated at 37 °C for 180 min before the absorbance was measured at 490 nm, and the relative cell viability was presented after being normalized to the model group.  in high glucose DMEM before the experiment.)The differentiation process of 3T3-L1 adipocytes was carried out as shown in periment . An amouAgilant 1290uplc liquid chromatography equipped with Agilant mass spectrometry (MS) qtof6550 was used to detect and analyze the component of EAF. A Shimadzu InertSustain C18 column  was used for HPLC . Scanning mode, data-independent analysis (100–1500 m/z), sheath gas temperature 500 °C, and sheath gas flow rate 12 L/min were used in mass MS conditions. The HPLC analysis was performed by step-gradient method, and the mobile phase with solvent A (acetonitrile) and solvent B (water) was as follows: 10% of eluent A at 0 min, 25% at 15 min, 40% at 30 min, and 55% at 45 min. The HPLC-MS chromatogram was preprocessed and compared with the TCM database, and the data came from the analytical results of TCM database.α-glucosidase [α-glucosidase (PDB ID: 3WY1) was obtained from the Online Protein Data Bank [α-glucosidase were virtually removed by pyMOL Win application . AutoDock Tools  was used to accomplish molecular docking in silico [α-glucosidase were defined as x = 82, y = 86, and z = 126 Å with spacing of 0.681 Å. Finally, the PyMOL molecular graphics system (version 2.2.0) was used to visualize ligand–enzyme interactions.In silico molecular docking was used to investigate the interactions between candidates and cosidase . The strata Bank , and then silico . The cub50 value.The DPPH assay was conducted in accordance with Tsamo et al. . InitialThe ABTS radical cation scavenging assay was conducted in accordance with instructions for ABTS kit . Initially, a 10 µL sample (800 μg/mL) and 190 µL of ABTS working reagent were mixed, and incubated at room temperature for 5 min. The absorbance was then measured by using a microplate reader at 734 nm.The Ferric reducing antioxidation power (FRAP) assay radical scavenging was conducted in accordance with instructions for FRAP kit . Initially, a 10 µL sample (800 μg/mL) and 190 µL of FRAP working reagent were mixed, and incubated at room temperature for 20 min. The absorbance was then measured by using a microplate reader at 593 nm.p < 0.05, ** p < 0.01, *** p < 0.001.IBM SPSS Statistics for Windows, version 21.0  was used to analyze all of the data, and the results were expressed as the average of the three measurements ± SD. Multigroup comparisons of the means were carried out by one-way analysis of variance test with post hoc contrasts by Student–Newman–Keuls test. Differences were considered significant when * α-glucosidase inhibitory activity was EAF > PEF > PAHEE > NBF. In addition, glucose uptake experiment showed that NBF possessed significant promotion ability on glucose uptake rate of 3T3-L1 adipocytes. HPLC-MS analysis and molecular docking results proved the interactions between candidates and α-glucosidase.  Consistent with the results of α-glucosidase inhibitory activity, the antioxidation capacities of PAHEE and its fractions on DPPH, ABTS, and FRAP were EAF > PEF > PAHEE > NBF. In general, the best antioxidation capacity of EAF might be related to its abundant contents of flavonoids and phenolics, and the hypoglycemic activity of PAH might be related to the inhibition of α-glucosidase activity  and the promotion of glucose uptake in 3T3-L1 adipocytes (NBF). Our research indicated that EAF and/or NBF might be used as potential natural hypoglycemic agent and/or antioxidant for further new drug research and development.Our study indicated that PAH contain rich sources of natural hypoglycemic molecules and antioxidants, such as flavonoids and phenolics. The results of total flavonoids content and total phenolics content were EAF > PEF > NBF > PAHEE, and the results of


Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial ω subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing ω subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have ω orthologs.Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. The core enzyme comprises two α subunits, one β subunit, one β′ subunit, and one ω subunit. The ω subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an ω subunit for the obligate intracellular bacterium  RNA synthesis in bacteria is carried by a single RNA polymerase (RNAP). The bacterial RNAP is a multisubunit enzyme . In almo2–2 acclimation, biofilm formation, and antibiotic production, and also affects growth under standard culture conditions  is capable of temporarily surviving in extracellular environments and invading host cells. Following the invasion of host cells and entry into cytoplasmic vacuoles, EBs differentiate into proliferative reticulate bodies (RBs). Following multiple rounds of replication, RBs convert back into EBs, which then exit host cells –68. In a–69–66, σ28, and σ54) as well as the α, β, and β′ subunits of the core enzyme. Surprisingly, it was not previously possible to identify a candidate gene encoding the ω subunit in any chlamydial genome  of 2.2 Å for CTL0286 and an RMSD of 4.0 Å for C. trachomatis RNAP β′ on a crystal structure of the ω-β′ subcomplex of the E. coli RNAP holoenzyme (PDB accession number 6ALH)  (We next used AlphaFold-Multimer  to predier 6ALH) . Signifier 6ALH) , as obseer 6ALH) , 106, wher 6ALH) , 14. We er 6ALH) .Taken together, these findings provide further support for our hypothesis that CTL0286 and its orthologs are bona fide chlamydial ω subunits.Chlamydia-like organisms  (Candidatus Midichloria ω ortholog is available in the UniProt database (www.uniprot.org)   each shorot.org) . Howeverortholog , followeortholog , 98, ideruginosa , Mycobacrculosis , and Xans oryzae , respects oryzae . We concRickettsia, Anaplasma, Ehrlichia, Orientia, Wolbachia, and Candidatus Midichloria are longer than the ω subunits of most free-living bacteria (123 to 138 residues versus 78 to 107 residues)   . AlphaFoesidues) . Consistichloria . We infesubunits .Mycoplasma genitalium, whose 580-kb genome is the smallest known bacterial genome   . AlphaFoo acids) , 98, fairpoZ in the supermajority of bacteria, ctl0286 is located immediately downstream of gmk  also encode ω orthologs  was performed using default settings . PDB90, a nonredundant subset of the PDB at 90% sequence identity, was used.The structural homology search for the AlphaFold model of CTL0286 was performed using the DALI server heuristic PDB search option , 98   was usedgmk-rpoZ synteny was identified by finding COG0194 (gmk) and COG1758 (rpoZ) together within the CSBFinder-S output.DeepNOG (v1.2.3)  was run Prediction of intrinsically disordered regions. The DisMeta server  was used


Searching for biological approaches for effective and safe AFB1 degradation is imminent. In our study, Microbacterium proteolyticum B204 isolated from bovine faeces degraded 77% of AFB1 after 24 h, becoming the first reported bacteria from the Microbacterium family to possess AFB1 degradation characteristics. Temperature variation showed little effect on its degradation ratio, demonstrating high thermostability of 75% and 79% after boiling and sterilization, respectively. We suppose that the components playing a key role during this process were proteins, considering the decreased degradation rate caused by Proteinase K. Cell viability detection on HepG2 cells indicated that the degradation products were much less toxic than pure AFB1. Furthermore, B204 cell-free culture supernatant also degraded AFB1-contaminated food, such as peanuts, corn and cheese. These results suggested that this strain with AFB1 degradation properties could be a prospective candidate for application in the food and feed industries.Aflatoxin B Aspergillus flavus and Aspergillus parasiticus [1 has been classified as a Class IA Danger and a Category I carcinogen by the WHO and IARC’s Human Carcinogen Risk Assessment Working Group due to its high toxicity [Aflatoxins (AFs) are typical mycotoxins ubiquitous in agricultural and sideline commodities that are generated as highly toxic secondary metabolites produced mainly by asiticus ,2. Amongtoxicity ,4,5. AF toxicity ,7. As a toxicity .1 safely and effectively therefore has become a hot topic of scientific studies; there is also an emergent demand for these strategies within national welfare and public health. The application of adsorbent products is one of the most commonly used physical methods to remove AF contamination, including heating and irradiation [1 toxicity to animals [Searching for strategies to detoxify AFBadiation . Acid, aadiation . Moreove animals ,12. Howe animals ,14,15. C animals ,17.Lactobacillaceae [Saccharomyces [Nocardia corynebacterioides was first identified as an AFB1-degrading microorganism in 1966 [Bacillaceae [Staphylocococcus warneri, Sporosarcina sp., Lysinibacillus fusiformis [Enterococcus faecium strains [Pseudomonadaceae [Rhodococcus erythropolis [Armillariella tabescens [1 and converts it to epoxide, which has less toxicity. However, the main AF-degrading substances currently known are secreted as extracellular enzymes, such as laccase [In general, the removal of AFs using biological methods consists of two methods, one of which is microbial adsorption based on the special structure of phosphoric acid and/or peptidoglycan of the microbial cell wall. This process mainly depends on hydrophobic and electrostatic interaction, which is typical of strains belonging to illaceae ,19,20,21aromyces ,23, etc. in 1966 . Active illaceae ,26,27, Ssiformis , Enteroc strains , Pseudomnadaceae ,31, Rhodhropolis , and so hropolis . Aflatoxabescens ,34,35 th laccase ,37, pero laccase , reducta laccase , etc.1 degradation, the species of Microbacterium genera have not been reported to possess this property for the moment. Moreover, few strains are broadly used in commercial applications, considering their narrow operation temperature range, relatively long incubation time and low degradation efficiency. In this study, a strain of Microbacterium genera from cow dung, which is successfully isolated using coumarin as the sole carbon source, demonstrates a high performance of AFB1 degradation at a broad working temperature range. The optimal degradation conditions and the cytotoxic potential of the degradation metabolites are also characterized.While these bacteria show the potential for AFBMicrobacterium proteolyticum strain B204, which showed the highest degradation activity (77.00 ± 1.53%) after 24 h of incubation at 30 °C . Nevertheless, the minimum degradation efficiency fell to 12.77 ± 3.42% at pH 4.The influence of pH and temperature on AFB1 by strain M. proteolyticum B204 was barely affected by temperature  suffered little impact from the addition of EDTA, while this value was markedly affected by Proteinase K and Proteinase K plus SDS in the cell-free supernatant of strain ectively .1 degradation products formed by the strain M. proteolyticum B204 cell-free culture supernatant were more, less or not toxic are not unreasonable. Therefore, it is imperative to detect and analyze the toxicity of degradation products in comparison with AFB1 (the parent compound). The cytotoxicity of AFB1 degradation extracts was performed on human hepatocellular liver carcinoma (HepG2) cells by MTT assay in this study. The cell viability was relatively stable at around 37% with 1, 5 and 10 μg/mL AFB1, until the concentration of AFB1 was raised to 20 μg/mL, which then decreased to 16.16% ± 2.42%  purchased from Yuanye Shengwu  was initially diluted in acetonitrile (HPLC grade)  to a stock solution of 100 mg/L, then filtered through a 0.22 μm filter , sealed in a brown bottle to avoid light preservation for better storage. Proteinase K was purchased from . Metal salts and inorganic salts containing NaCl, MgSO4, FeSO4, FeCl3, KH2PO4, NH4NO3 and SDS powder were purchased from Solarbio .Luria-Bertani (LB) medium containing 10 g/L tryptone, 10 g/L NaCl and 5 g/L yeast extract was used for bacteria cultivation. Coumarin medium composed of 10 g/L coumarin, 0.5 g/L KH−4, 10−5 and 10−6. Aliquots of each diluent (100 μL) were homogeneously spread on coumarin medium plates and incubated at 30 °C for 21 days until visible colonies appeared. Single colonies growing well were picked and then inoculated to fresh new plates, the purification process of which was performed at least three times to obtain the pure isolates.Preliminary Screening: One gram of bovine faeces was dissolved in 10 mL sterilized distilled water after a continuous vortex for 3 min; it was then serially diluted to the final concentration of 101 solution to acquire the desired concentration of 10 μg/μL, then cultured in a rotary shaker incubator at 30 °C for 12 h away from light. The supernatant was collected after centrifugation at 8000 rpm for 20 min, followed by concentration and redissolution in 1 mL acetonitrile (HPLC grade), then filtered through 0.22 μm filter and preserved at 4 °C for the further detection of residual AFB1. AFB1 standard solution mixed with sterile LB medium  was used as negative control.Secondary Screening: Each of the purified strains was further inoculated in 15 mL fresh LB liquid medium at 30 °C under continuous shaking of 180 rpm/min for 12–24 h. Fifty microliters of activated bacterial solution derived from the previous step were transferred to 9.95 mL fresh LB medium mixed with AFBv/v, 1:1) was employed as mobile phase B, with a flow rate of 0.6 mL/min. The concentration of residual AFB1 was quantitatively determined by fluorescence, the excitation wavelength and detection wavelength of which was set as 350 nm and 450 nm, respectively. AFB1 standard solution mixed with sterile LB medium instead of cell-culture supernatant was used as negative control. The AFB1 degradation ratio was evaluated using the following formula: The prepared filtered samples were loaded on Waters C18 Column  equipped with a fluorescence detector according to Guan et al., with some modifications . MillipoM. proteolyticum B204 was extracted by TIANGEN bacterial DNA Kit  based on the manufacturer’s recommended instructions. Then, 16S rRNA gene fragments were amplified with Primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and Primer 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). Afterwards, the sequence was aligned against known species with the NCBI BLAST algorithm  and EzTaxone BLAST analysis was undertaken  [Genomic DNA of 07/2021) ,47. The 07/2021) .1 by M. proteolyticum B204 were achieved independently as follows: incubation time 12 h, initial culture pH 4, 5, 6, 7 and 8, incubation temperature 4, 10, 20, 25, 30, 40, 45 and 50 °C, with other conditions kept constant. The final concentration of AFB1 was consistent (10 μg/μL) and the addition of M. proteolyticum B204 was also maintained in each experimental group. The detection of AFB1 degradation efficiency was performed as described in The influences of different culture conditions on the biodegradation of AFB1 by different components of M. proteolyticum B204 was investigated as described below, referring to Wang et al. with minor modifications [1 degradation assay. The precipitation of bacteria was thoroughly washed by sterilized MilliQ water and re-dissolved in PBS buffer . The resuspending cells were then disrupted by ultrasonic homogenizer  at 28% power with a 4 s pulse on and 6 s pulse off repetition cycle on ice. Cell-free extracts were obtained after centrifugation at 8000 rpm for 20 min at 4 °C and filtration through 0.22 μm filters. AFB1 standard solution was then introduced into cell-free culture supernatant, cell-free extracts and cells with a final concentration of 10 μg/μL, respectively. LB culture medium + PBS buffer with equivalent AFB1 was set as a negative control. After incubation at 30 °C for 24 h, the residual AFB1 was detected by HPLC as described in The degradation of AFB1 degradation by M. proteolyticum B204 cell-free culture supernatant were studied according to Farzaneh et al., with slight adjustments [1 degradation efficiency was investigated by boiling for 20 min. Moreover, autoclave sterilization  was even adopted to evaluate the detoxification of AFB1. Sodium dodecyl sulfate (SDS) and Proteinase K treatments were also performed to evaluate the degradation process. One-milliliter of cell-free culture supernatant was mixed with 0.1 mL 10 mg/mL Proteinase K, 0.1 mL 10% SDS buffer and 0.1 mL 10 mg/mL Proteinase K plus 0.1 mL 10% SDS buffer, respectively. Moreover, EDTA with a final concentration of 0.1 mol/L was also added to 1 mL cell-free broth to analyze the degradation efficiency of AFB1. All the degradation reactions with the addition of SDS, Proteinase K and EDTA were incubated at 30 °C for 1 h in the dark. The degradation products of AFB1 were determined by HPLC as described in The effects of heat treatment, SDS, proteinase and EDTA on AFBustments . The imp1 and B204 cell-free supernatant-induced degradation of AFB1 were detected on HepG2 cells via evaluating cell cytotoxicity using the MTT assay referenced in Wang et al., with some modifications [1 were set as 0, 1, 5, 10 and 20 μg/mL, respectively. The only difference between experimental groups and control groups was the addition of M. proteolyticum B204 cell-free culture supernatant. After degradation for 24 h with the help of B204 cell-free culture supernatant, using equivalent LB culture medium as controls, the mixture was then incubated with HepG2 cells (1 × 104 cells per well) in a 96-well multi-plate at 37 °C under 5% CO2 for 24 h. Then, 20 μL of 5 mg/mL MTT -2,5-diphenyltetrazolium bromide) was added and co-incubated for another 4 h. Finally, 150 μL DMSO was introduced to every well, making the cell crystals sufficiently dissolved. Cell viability was assessed according to the absorption value at 560 nm.The effects of the AFBM. proteolyticum B204 against AFB1 contamination was conducted on peanuts, corn and cheese. One gram of each sample mentioned above was sprayed with 100 μL AFB1 with a concentration of 100 μg/μL. After incubation with 450 μL of M. proteolyticum B204 at 30 °C for 16 h, the residual AFB1 was then detected by HPLC, as described above.The application to food matrices of Student’s t test or Duncan’s test using SPSS software .All experiments were independently repeated in triplicate. The statistical analysis was performed by one-way analysis of variance (ANOVA) within the 95% confidence interval followed by the


Paenibacillus polymyxa remain unclear. In this study, knocking out msmR1 was found to reduce polymyxin synthesis by the SC2-M1 strain. Chromatin immunoprecipitation assay with sequencing (ChIP-seq) revealed that most enriched pathway was that of carbohydrate metabolism. Additionally, electromobility shift assays (EMSA) confirmed the direct interaction between MsmR1 and the promoter regions of oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp. MsmR1 stimulates polymyxin biosynthesis by directly binding to the promoter regions of oppC3 and sdr3, while also directly regulating sucA and influencing the citrate cycle (TCA cycle). In addition, MsmR1 directly activates pepF and was beneficial for spore and biofilm formation. These results indicated that MsmR1 could regulate carbohydrate and amino acid metabolism, and indirectly affect biological processes such as polymyxin synthesis, biofilm formation, and motility. Moreover, MsmR1 could be autoregulated. Hence, this study expand the current knowledge of MsmR1 and will be beneficial for the application of P. polymyxa SC2 in the biological control against the certain pathogens in pepper.The multiple-sugar metabolism regulator (MsmR), a transcription factor belonging to the AraC/XylS family, participates in polysaccharide metabolism and virulence. However, the transcriptional regulatory mechanisms of MsmR1 in  Paenibacillus polymyxa, proposed by Paenibacillus polymyxa is an important strain resource in microbial medicine, microbial environmental remediation, microbial pesticides, and fertilizer research  and a variable ligand-binding domain (LBD) at the N-terminal  region genes, including nra, which inhibit the expression of crucial virulence factors  was performed and the results were verified by electrophoretic mobility shift assay (EMSA). The oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp genes were found to be directly regulated by the global regulator MsmR1; the functions of these genes were further explored to delineate the regulatory mechanism of MsmR1. The results of this study provide a theoretical basis for the improved application of P. polymyxa SC2 in the biological control against the certain pathogens.e, China . Paenibaulations . A previd pepper , indicatPaenibacillus polymyxa SC2-M1 and its mutants, Escherichia coli DH5α, E. coli BL21, and Trans 110 were cultured in Luria-Bertani (LB) broth at 37°C. When plasmids were presented in cells, antibiotics were added to LB medium as follows: chloramphenicol (6 μg/ml for strain SC2-M1 and its mutants), kanamycin (50 μg/ml), tetracycline (20 μg/ml), ampicillin (100 μg/ml), and erythromycin (12.5 μg/ml). To measure carbon source utilization by the strains, basal medium 2SO4 0.8%, K2HPO4 0.368%, KH2PO4 0.132%) with a 1% sole carbon source was used.The strains and plasmids used in this work are presented in E. coli were prepared using the transformation and storage solution (TSS) method and transformed by the heat pulse method , and ligated into pRN5101 to construct the complement vectors. Demethylated complementary vectors were introduced into the mutant strains. Double-crossover recombinants were detected as described above, and the complemented strains were confirmed by PCR using the corresponding primers  were cloned by PCR amplification, and ligated to pRN5101 to construct the plasmid pRN5101-msmR1-Flag. Demethylated pRN5101-msmR1-Flag were introduced into SC2-M1 and double-crossover recombinants were detected as described above, designated as strain SC2-M1-Flag. For overexpression of target genes, fragments carrying complete target genes were cloned from genomic DNA, fused with the promoter fragments, and ligated into pHY300PLK between BglII and SalI sites by ClonExpress MultiS One Step Cloning Kit  for overexpression in SC2-M1. To evaluate the promoter activity of target DNA fragments, the fragments were cloned from genomic DNA, and the promoterless green fluorescent protein (gfp) fragment from pGFP4412 was cloned , and cultured at 37°C with shaking at 180 rpm for 7 h. The two strains were then fixed with 1% formaldehyde for 10 min at 28°C and the reaction was stopped with 125 mM glycine for 5 min. The cells of the two strains were washed thrice with phosphate buffer solution (PBS) at 8000 rpm for 5 min at 4°C and stored at-80°C. ChIP was performed by Wuhan IGENEBOOK Biotechnology Co. Ltd., China. The manufacturer’s instructions of Illumina TruSeq ChIP Sample Prep Set A were followed to construct sequencing libraries using chromatin immunoprecipitated DNA which was then sequenced on an Illumina Xten using the PE 150 method (P. polymyxa SC2 genome using Bwa (v 0.7.15), which allowed for up to two mismatches  was used as an internal control.SteadyPure Universal RNA Extraction Kit  was used to purify the total RNA from the target bacteria. cDNA was obtained from 1 μg of total RNA using the Evo M-MLV RT Kit with gDNA clean for qPCR II . qRT-PCR analysis was performed on a Roter-Gene Q PCR system using the SYBR Green Pro Taq HS qPCR Kit . The relative expression of target genes was calculated using the 2t method . The houmsmR1 was cloned from the genomic DNA of strain SC2-M1 using primers MsmR1F and MsmR1R (msmR1 and plasmid pET-28b(+) were simultaneously cleaved with XhoI and SalI, purified, ligated using T4 DNA ligase, and transferred into E. coli DH5α to obtain pET28b(+)-msmR1.An 828 bp fragment (275 amino acids) of d MsmR1R . The framsmR1 was transferred into E. coli BL21. The transformants were cultured in kanamycin-resistant LB broth at 37°C with shaking at 180 rpm for 10 h. Next, the cells were inoculated into 50 ml of kanamycin-resistant LB broth at 2% (v/v) and cultured at 37°C with shaking at 180 rpm for approximately 1.2 h until the OD600 reached 0.5–0.6. Then the cells were induced by 0.4 mM IPTG at 16°C for 20 h, harvested, washed, resuspended in PBS, and sonicated on ice. MsmR1-His6 protein was purified using BeyoGold™ His-tag Purification Resin  following the manufacturer’s instructions. The purified protein was added to a final concentration of 10% (v/v) glycerol and stored at-80°C. EMSA was performed using a chemiluminescent EMSA kit -E. coli DH5α cells. The strains were inoculated in LB plate containing 20 μg/ml tetracycline and fluorescence was observed with an OLYMPUS fluorescence microscope at 1000× 2SO4 8 g/l, CaCO3 5 g/l, KH2PO4 0.2 g/l, MgSO4 0.2 g/l, NaCl 0.2 g/l) at 2% (v/v) and cultured at 37°C with shaking at 180 rpm for 48 h.An Oxford cup assay was used to evaluate polymyxin production by the SC2-M1 strain, as previously described . Bacteri600 was adjusted to 0.6. Diluted cell solutions (2 μl) were added to the center of the plate containing 0.5% glucose, 0.22% NaCl, 0.13% yeast extract, 0.45% tryptone, and 0.5% agar.A motility assay was performed as previously reported . Bacteri600 of 0.8. Diluted strain solutions were inoculated at 2% in 50 ml of fresh LB medium. The bacteria were cultured at 37°C with shaking at 180 rpm. The OD600 and number of live bacterial cells were measured every 3 h. A total of 100 μl of the strain solution was serially diluted with 900 μl of sterile water; 100 μl of the dilution was then spread on an LB plate. The plates were subsequently incubated for 24 h at 37°C.Bacteria were cultured overnight in liquid LB medium and adjusted to an OD600 value was approximately 0.1. Cultures were incubated at 37°C with shaking at 180 rpm, and growth curves were determined according to OD600 every 3 h.The growth curves of bacteria on the carbon sources were determined using a basal medium. Bacteria were cultured overnight in LB medium. The cultures were centrifuged, washed thrice with basal medium, and resuspended to inoculate 50 ml of fresh basal medium with 1% sole carbon source; the original ODp ≤ 0.05) were performed using statistical software SPSS  and Least Significant Difference (LSD) test  showed no fluorescence  family is important for carbohydrate, amino acid, lipid metabolism, and redox sensor mechanisms , part of the 2-oxoglutarate dehydrogenase complex (sucAB) and succinate dehydrogenase  utilization, the SC2-M1-Δh SC2-M1 . Hence, pepF gene encodes an oligoendopeptidase F. In Bacillus subtilis, the pepF homolog phrA can regulate spore formation  family, Sdr3 can influence fatty acid, carbohydrate, amino acid metabolism, and motility, which in turn reduces energy and precursors for polymyxin synthesis. PepF affects spore and biofilm formation. Due to the fact that spontaneous mutant strain SC2-M1 lost the ability to form endospores and biofilm  promoter; however, no retarded signal was observed in the EMSA experiment, indicating that VjbR and PvirB cannot withstand the electrophoretic conditions of EMSA , and 44 AraC family transcription factors were identified in the genome of P. polymyxa SC2, most of which were related to carbon source metabolism, such as arabinose metabolism and L-rhamnose metabolism  and double mutant . In the tcpA, ctxAB, and tcpI-2 promoters, two “toxboxes” are direct repeats, while in the acfD/acfA, tagA, and tcpI-1 promoters, two “toxboxes” are inverted repeats  of Mga in Streptococcus pyogenes, a 45-bp binding site was identified by DNase I protection experiments, containing three conserved nucleotide regions and 29 nucleotides, separated by several unrelated nucleotides  is primarily involved in importing short-chain peptides, such as dipeptide and tripeptide, from the extracellular environment . The OppP. polymyxa SC2 and pepper, the biosynthetic genes of polymyxin and fusaricidin were upregulated, which induced systemic resistance in pepper and enhanced its resistance to certain pathogens. Simultaneously, the upregulation of genes related to chemotaxis, motility, and biofilm formation is beneficial for the colonization of strains in the rhizosphere of plants  repository, accession number PRJNA885964.DZ performed the laboratory work, analyzed the data, and wrote the manuscript. HL, YC, and ST analyzed the data. CW, BD, and YD provided insights on the manuscript. YD and BD supported the study. All authors contributed to the article and approved the submitted version.This work was funded by the National Natural Science Foundation of China (31770115), Key Research and Development Program of Shandong Province (2021CXGC010804), and Tai-Shan Scholar Program from the Shandong Provincial Government.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.