Specifically, using lithiumbis(fluorosulfonyl)imide (LiFSI) in N-propyl-N-methylpyrrolidinium FSI, its “apparent”diffusion coefficient, lithium transference number, thermodynamicfactor, ionic conductivity, and resistance of charge transfer againstlithium metal were isolated. Furthermore, the analysis of these concentrationgradients led to insights into the bulk structure of ILEs, which wepropose are composed of large, ordered aggregates.Ionicliquid electrolytes (ILEs) have become popular in variousadvanced Li-ion battery chemistries because of their high electrochemicaland thermal stability and low volatility. However, because of theirrelatively high viscosity and poor Li Furthermore, in lithiummetal batteries (LMBs), the depletion of Li+ at the lithiummetal surface has been proven to induce lithium dendrite growth andshort-circuiting.10As lithium-ion batteries (LIBs)approach their theoretical energy limit, high-energy alternativesare required for the increasingly high-energy applications societynow depends on. Popular strategies to improve energy density includeutilizing high-voltage cathodes,+ often being present in low concentrations, the transferencenumber of Li+11Common ILEs usedfor battery applications contain 3 or 4 ionicspecies, and because of the lithium diffusion coefficient Di). Without an electric field, the transference number can be estimated by calculatingthe fraction of current carried by Li+ using the productof Di and ci of each component .By applying an electric field, pfg-eNMR can measure the mobility ofthe ions in solution and subsequently isolates the true transferencenumber. These pfg-(e)NMR studies have shown + was moving in the “wrong direction”.13 Others have measured Wd).15 However, thismethod includes an electrolyte ideality assumption, which is especiallyproblematic in a concentrated electrolyte or ILE. To the best of ourknowledge, t+Li values of promisingILEs used in battery applications have not been measured electrochemicallyvia the most defined method of measuring transference, namely, theHittorf method, nor has the “apparent” diffusion coefficient Dapp been measured via the restricted diffusionmethod, a common method that measures Dapp by monitoring the semilog decay of open-circuit voltage (OCV) overtime, after an arbitrarily formed concentration gradient has beenformed.20 Although the mentioned pfg-NMR and Warburg methods for determining t+Li are equivalent in “ideal”conditions, using electrochemical methods like Hittorf provides themost rigorous definition of transference in nonideal or concentrationelectrolytes; therefore, measuring t+Li via electrochemical means provides the best predictor of performancein nonideal electrolyte conditions. Although the pfg-(e)NMR and EIStechniques have shed some light on the complexities and intricaciesof ILE transport, there is yet to be a complete experimental studythat monitors both Dapp and M).The most popular method for determining 24 These studies have not yet been extended to ternary systems or ionicliquid systems. Herein, we use operando Raman microspectroscopy tomeasure Li+ concentration gradients in an IL-based electrolytesystem. We focus on 0.5, 1, and 2 m LiFSI in Pyr1,3FSI,a three-component, common electrolyte system used in high-energy cells.25 To be explicit, this is the firsttime ILE concentration gradients have been visualized. Concentrationgradient formation is regarded as ILEs’ primary weakness inLIBs and LMBs, so the visualization of the gradient is of particularimportance for the understanding and progression of ILEs. Moreover,key electrolyte properties including the “apparent”diffusion coefficient (Dapp), lithiumtransference number (t+), thermodynamicfactor (χM), ionic conductivity (κ), and resistanceof charge transfer (Rct) are isolated.This is also the first time a full suite of electrolyte propertieshas been measured for a promising ILE for use in battery applications.For a comprehensive understanding of binary electrolytetransport,researchers have utilized operando magnetic resonance imagining (MRI)and Raman techniques to visualize concentration gradients.Concentration Gradient Visualization. Concentrationgradients were visualized using operando Raman microspectroscopy  to increase reliability andaccuracy of preparation .troscopy 1, specifc+Li was calculated by correlatingit with the 730 cm–1 FSI– peakshift. Representing the S–N–S bending mode,26 the 730 cm–1 peak shifts tohigher wavenumbers monotonically with increasing 27 The calibration curve used is shown in – 730 cm–1 and –1 peaks. Becauseof the increased spectral noise using this method, we selected theformer method; further analysis is shown in Supporting Discussion 2.1.–1 peak shiftmethod, we checkedthe mass-balance of the system by integrating each concentration–distanceprofile, noting if there was any change in the measured profiles overtime. Each measurement was within 1.2% of the highest and lowest profileintegral over the 36 h experiment. We therefore concluded this isa valid method for calibrating concentration in ILEs.Using the 730 cmAsymmetry and Structural Implications. + in 1 m LiFSI in Pyr1,3FSI at100 μA cm–2 after 12 h. Surprisingly, theprofile had an asymmetry, with both bulk concentration change anddc/dz being larger on the strippingside compared to the plating side. This seemed unique to ILE systems,with other systems not showing this phenomenon.22+-containing species of higher densitywere falling because of gravity. To investigate this further, we measuredthe open-circuit voltage (OCV) of the cell while changing the cell’sorientation. Prior to the application of current, the cell restedfor 4 h anda line-scan was recorded. We noticed an increase in concentrationat the bottom of the cell, which indicated an accumulation of liedsee 3a. ThisSupporting Figure 3, we also saw this phenomenonwith stainless-steel blocking electrodes, albeit to a lesser extent.This suggested that the reactive electrodes increased bulk flow, perhapsbecause of volume changes caused by the interfacial reaction.28It was clear that the OCV was dependent on the orientation of thecell, with it rising from 0 V to ∼4 mV after the first fewhours and dropping to approximately −5 mV when the cell wasinverted. The cell was then placed horizontally, which led to theOCV reverting back to 0 V. Using 4 mV as the OCV, the thermodynamicactivity ratio calculated from the Nernst equation was 1.16, in goodagreement with the 1.11 measured by Raman spectroscopy 3a. As sh30 Past experimental studies usingsmall-angle X-ray scattering (SAXS) experiments have also predictedthe formation of mesoscopic aggregates or domains in neat ILs andwith lithium salt in IL solutions.33 NMR measurements haveshown similar results.34 However, thereis little consensus on the overall size and structure of the aggregatespresent in IL and Li-salt solutions. Using Stokes’ Law35 we estimated the size of aggregates to be 3–8μm, which is larger than others hypothesized.32 (See Supporting Discussion 2.2 for furtherdiscussion.) However, further studies are required to confirm thisvalue.Several molecular dynamic (MD) studieshave proposed the formationof long-range ordered structures in ILEs, with some suggesting mesoscopicaggregate formation.–1 represents the S=O stretching mode of FSI–. In the neat IL there was a single peak at 1215 cm–1, and with increasing LiFSI addition a new, definedpeak appeared at 1225 cm–1 2]−]). WithFSI– experiencing two separate environments, itis at ∼1.1 m that [FSI–] < [Li(FSI)2]−. As the concentration increased past1.25 m, the 1225 cm–1 peak became less defined andmore broad  gradient of 5.3 ± 0.20 × 106 mol m–4 at 100 μA cm–2. We also performed the measurementat 50 μA cm–2 showing dc/dzz=0 as 2.8 ± 0.10 ×106 mol m–4. As is expected, dc/dzz=0 wasdirectly proportional to the current applied, with dc/dzz=0 being almostexactly double when a 100 μA cm–2 was appliedcompared to 50 μA cm–2. The plating interfacialgradient (dc/dzz=L) at 100 μA cm–2 was lower at 3.5 ± 0.60 × 106 mol m–4, presumably because of the accumulated aggregates at the bottomof the cell.Diffusion. By monitoring the diffusion length(b and d) over time, one can calculate Dapp on both sides of the cell. b and d versus time1/2, with the slope being proportionalto Dapp. Also plotted is the 95% confidenceband, which highlights the uncertainty especially on the plating sideof the cell. The fitting on the stripping side is much more accurate,which is reflected in the error of the Dapp calculation. On the stripping side, Dsalt = 1.77 ± 0.06 × 10–11 m2 s–1, and on the plating side Dapp = 2.5 ± 0.50 × 10–11 m2 s–1, with an inverse-variance weighted average of 1.78± 0.09 × 10–11 m2 s–1. Pulsed field gradient (pfg)-NMR measurements wereperformed to compare against these values: using the harmonic mean, Dsalt was calculated as 1.77 × 10–11 m2 s–1 (see Supporting Discussion 2.5), very similar to Dapp calculated using concentration visualization. Themagnitude of the diffusivities would suggest transport is occurringprimarily via an ion-hopping mechanism as opposed to sedimentationof the aggregates identified in the previous sections. However, sedimentationis proposed to be the reason for the asymmetric concentration gradientthat was visualized.Transference Number. 38 This was particularly straightforward when utilizingconcentration gradient visualization techniques, as one can monitorthe concentration on each side of the cell by integrating under theconcentration curve. Moreover, those using a conventional Hittorfsetup would not notice the initial gradient from the settling aggregates.To the best of our knowledge, the Hittorf method has not been utilizedfor studying 44 Like Gouverneur, who used pfg-eNMR, we used an “external”reference, namely, the center-of-mass reference. ncharge isthe number of moles of charge passed; t; Q is the charge passed over time; and A is the area of the electrode.By using the pfg-NMR diffusivities and measuredconcentrations, Thermodynamic Factor. χM correlatesthe electrolytes’ thermodynamic activity with concentration.45 To our knowledge, no room-temperature Li-ionILEs’ χM values have been reported, but theactivity of various LiNO3–AgNO3 meltcompositions (at 260 °C) were measured by Richtera few decades ago.46 χM was calculated asf± isthe molar activity coefficient. Using the potentiostatic electrochemicalimpedance spectroscopy (PEIS) data prior to each line scan, ηc was calculated by ηc = ηtotal – I(Rbulk + Rct), whereηtotal is measured from the chronopotentiometry dataand the resistances are from PEIS. χM was measuredas 0.906 ± 0.064  and resistance of charge transfer (Rct) were calculated from fitted Nyquist plots 2]− structures; χM then began to rise.There was no noticeable trend in Rct,with a clear anomaly for one of the 0.5 m samples, which did not appearto affect the other bulk electrolyte property values described.For 2 m, there was anoticeable change in many of the transportand thermodynamic properties. For example, there was a marginal increasein d2]− > (FSI)−, ordered networks form, increasing This reported data suggested there was a transport mechanism changemoving from 1 to 2 m. We also speculated above that between theseconcentrations there was a structural change, as illustrated fromthe Raman data. We speculate that when [Li(FSI)+ concentration gradient in ILEs has been visualizedfor the first time, along with the isolation of key transport andthermodynamic properties. With ILEs’ main weakness being theirtransport properties, understanding fully the origin of this is paramountfor their continuing development. Moreover, this is the first timea thermodynamic understanding of promising battery ILEs has been measuredthrough χM. Additionally, the sedimentation of clusteredaggregates have been detected, which has not yet been experimentallymeasured in the academic literature until now. We anticipate thiswork to further promote concentration visualization’s uniqueability to fully understand electrolyte properties, and specifically,we hope our findings regarding ILEs’ properties and structurewill inform their ongoing progress.In summary, by combiningspectroscopic and electrochemical techniqueswith concentration visualization we have presented particularly valuablefindings not yet reported in the ILE literature. Specifically, theLiElectrolyte Description. Lithium bis(fluorosulfonyl)imide(LiFSI)  was purchased from Fluorochem Ltd. N-Propyl-N-methylpyrrolidinium bis(fluorosulfonyl)imide (99.9%) was purchased from Solvionic. Handlingof LiFSI and Pyr1,3FSI was always performed in an argon-filledglovebox (MBraun) with low H2O content (<1 ppm) andlow O2 content (<1 ppm). LiFSI was dried further underhigh vacuum at 70 °C for 48 h. Pyr1,3FSI was driedunder high vacuum at 70 °C for 24 h, with a stirrer bar. TheH2O content of the electrolyte solutions was determinedby Karl Fischer titration, which was also performed in an argon-filledglovebox, and recorded to be below 15 ppm of H2O.Calibration. Using a confocal Renishaw inVia Reflexlaser confocal Raman microscope equipped with a near-IR 785 nm laser,a 5× magnification objective , leadingto a 4.8 μm spot size, along with a 90° mirror (Renishaw) was used to collect Raman spectra of the preparedsolution. An even distribution of calibration electrolytes was prepared,between 0.1 and 3 M, inside an Ar-filled glovebox. For each calibrationsolution, the spectra were recorded with an 800 cm–1 center, at 5% laser power, one second exposure, and 20 acquiredspectra. Using Renishaw WiRE 5.5 software, the background of eachspectrum was removed and normalized. Each 730 cm–1 peak was fitted with an exponentially modified Gaussian (EMG) function,and the wavenumber number (x-axis) at maximum heightwas calculated. The calibration curve is shown in Cell Construction. The custom-designed cell wasconstructed in an Ar-filled glovebox. Two Li discs of 8 mm diameterwere cut and placed onto two stainless steel pistons of the same diameter.One piston was placed into a fused quartz tube; electrolyte was added,and the second piston was added to seal the cell, being careful notto introduce any bubbles into the system. Once sealed, the cell wasplaced onto the Raman stage vertically and connected to a BiologicSP150 potentiostat.Line Scan. 100 μAcm–2 was applied to the cell, and a 1D line scanin the z-direction was performed every 4 h for 36h. The same laser settingsused for calibration were used for the line scan too. A point-by-pointline scan was taken, with equal spacing between the 1.5 cm interelectrodedistance. The confocality of the instrument allowed us to measuredthe line scan in a precise plane of focus; each measurement was 0.5cm inside the ID of the quartz tube. Using Renishaw’s WiRE5.5software, the background was removed, and each spectrum was comparedto the calibration using a Python script. The resulting concentrationgradient was fitted with github.com/JFawd.PEIS. Before any line scan, potentiostatic electrochemicalimpedance spectroscopy (PEIS) was performed on the cell. It was alsoperformed between line scans to provide an estimation of ηc. A voltage amplitude (Va) of 100 mV was used, which allowed for linearity. Thefrequency was scanned from 100 kHz and 1 Hz. The Nyquist plot wasfitted with the equivalent circuit (Qbulk/Rbulk) + (Qdl/Rct), with Rbulk and Rct representing the bulk resistanceof the electrolyte and charge transfer, respectively, and Qbulk and Qdl arethe constant phase element of electrolyte and double layer, respectively.Each spectrum was fitted using Biologic EC-lab V11.26 software.pfg-NMR. All pulsed field gradient (PFG) nuclearmagnetic resonance (NMR) measurements were completed at 9.45 T (0(1H) = 400.20, 0(19F) = 376.58,and 0(7Li) = 155.53 MHz) on a Bruker AvanceIII HD spectrometer using a 5 mm single-axis diffusion probe withexchangeable 1H, 19F, and 7Li ceramicheads. A stimulated echo pulse sequence was utilized with an effectivegradient pulse duration (δ) of between 1 and 2 ms, a diffusiontime (Δ) of 43–46 ms, with the gradient amplitude variedbetween 0.1 and 5 T/m. All samples were sealed in a J-Young valveNMR tube; the temperature was stabilized at 298.1 K, and a 5 s recycledelay was used throughout.


By MS/MS analysis, BVYA1 was identified as sufactin with the precise amino acid sequence Glu–Leu/Ile–Leu–Val–Asp–Leu–Leu/Ile and hydroxyl fatty acids with 11–13 carbons. [M + H]+ at m/z 980.62 was detected for the first time in B. velezensis, which demonstrates that the strain corresponds to a new surfactin variant. In particular, BVYA1 showed antibacterial activity with the minimum inhibitory concentration (MIC) values of 7.5–15 μg/ml. Finally, the preliminary mechanism of inhibiting E. coli treated with BVYA1 showed that BVYA1 effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. In conclusion, this study suggests that the YA215LE from B. velezensis YA215 might be a potential candidate for a bactericide.The increasing burden and health risks of antimicrobial resistance (AMR) pose a great threat to society overall. Lipopeptides exhibit great potential as novel and safe alternatives to traditional antibiotics. In this study, the strain YA215, which was isolated from the mangrove area in Beibu Gulf, Guangxi, China, was identified as Bacillus velezensis. Then, YA215 lipopeptide extracts (YA215LE) from B. velezensis was found to exhibit a wide spectrum of antibacterial and antifungal activities. Additionally, YA215LE was identified and found to contain three groups of lipopeptides . Furthermore, one separation fraction (BVYA1) with significant antibacterial activity was obtained. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of BVYA1 showed three molecular ion peaks ([M + H] The increasing the burden and health risks of antimicrobial resistance (AMR) pose a great threat to society as a whole. It is estimated that approximately 1,000,000 people die each year worldwide from AMR-related diseases . Key facLipopeptides were produced by different species of microbial genera, which included Bacillus, Streptomyces, and Pseudomonas , 9. HoweE. coli treated with BVYA1 was derived by scanning electron microscopy (SEM) and calcein-AM/PI double-staining assays. The results will provide valuable information for further screening a novel alternative to traditional antibiotics from the B. velezensis YA215 strain.Therefore, in this study, antibacterial lipopeptides were isolated and identified as novel alternatives to traditional antibiotics; specifically, this study was designed to isolate, purify, and characterize antibacterial lipopeptides from the YA215 strain, which was isolated from the mangrove area in Beibu Gulf, Guangxi, China. First, the YA215 strain, which was isolated from the mangrove area in Beibu Gulf, Guangxi, China, was identified as B. velezensis based on morphological and 16S rRNA gene sequences. Second, the antimicrobial activity of YA215LE, which were obtained by the combining hydrochloric acid precipitation and methanol extraction, was evaluated by the agar well diffusion method. In addition, the composition of YA215LE was identified using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Furthermore, YA215LE was isolated and purified by gel column chromatography and semiprepared reverse-phase high-performance liquid chromatography (RP-HPLC), and one separation fraction (BVYA1) with significant antibacterial activity was obtained. BVYA1 was identified by MS/MS analysis, its MIC was determined. Finally, the preliminary mechanism of inhibition of Lysogeny broth (LB) medium, potato dextrose agar (PDA) medium and agar was obtained from Guangdong Huan Kai Biotechnology Co., Ltd. . Trifluoroacetic acid (TFA), methanol and acetonitrile were chromatography grade, hydrochloric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, glutaraldehyde, ethanol, and sodium hydroxide were analytical pure and these were purchased from Shanghai Macklin Biochemical Co., Ltd. .Escherichia coli (CGMCC 112252), Bacillus nanotuberculosis, Bacillus subtilis CGMCC 1.14985, Bacillus subtilis CGMCC 1.15792, Staphylococcus aureus CGMCC26003, Salmonella typhimurium CGMCC11190, Shigella flexneri CGMCC110599, and Rhizopus stolonifer CGMCC3.31 were obtained from the National Center for Medical Culture Collections , and Bacillus licheniformis, Bacillus cereus, Fusarium oxysporum f.sp.cubense, and Penicillium expansum CICC 40658 were stored in a 40% glycerol solution at –80°C in this team.The YA215 strain, which was isolated by our research team from the mangrove area in Beibu Gulf, Guangxi, China, was used in the current study , 25. Esc°C, and then the colony morphology of the YA215 strain was observed, in addition a single colony was picked for Gram staining. Subsequently, strain confirmation was performed by 16S rRNA gene amplification with the following regular pair of forward and reverse primers; 27F (5′-GGTTACCTTGTTACGACTT-3′) and 1492R (5′-AGAGTTTGATTTGATCCTGGCTAG-3′). The amplification was carried out in a polymerase chain reaction (PCR) instrument  programmed using the following protocol: 94°C for 5 min; 35 cycles of 94°C for 30 s, 64°C for 1 min, and 72°C for 2 min and then a final elongation step of 72°C for 5 min. The analyzed PCR products were sent to Sangon Biotech Co., Ltd.  for sequencing. Then, the 16S rDNA sequences were compared with the GenBank database using BLAST in NCBI data, along with homology analysis, and a phylogenetic tree was constructed using MEGA 7.0 software.YA215 strain was grown on LB agar medium for 24 h at 37°C and 220 rpm for 12 h. Then, a 4% (v/v) inoculum was fermented in pill bottles at 37°C and 220 rpm for 48 h. The cell-free supernatant was obtained after centrifugation at 4°C and 10,000 g. The YA215LE was obtained by a combination of acid precipitation and methanol extraction method  with a sterile punch at a distance of 2 cm from the mycelium, and 100 μl of YA215LE solution was added to the holes. The culture dish was continued to be incubated at 28°C for 4 days, and then the diameter of the inhibition zone was measured.Antifungal activity was evaluated by the agar well diffusion method with minor modifications . MyceliaThe YA215LE obtained from the fermentation broth of the YA215 strain was first was purified on a Sephadex G-25  gel filtration column (1.6 × 100 cm). The samples were eluted with phosphate buffered saline (PBS)  at a flow rate of 0.3 ml/min, and the absorbance was monitored at 280 nm. An automatic collection device was used to collect the eluent for 10 min per tube. The antibacterial activity of each canal was determined by using the agar well diffusion method as described before. The active eluent collected was further purified by semipreparative RP-HPLC using the method previously described  with modThe purity of the elution peak with the highest antibacterial activity obtained by semipreparative RP-HPLC was measured by an analytical RP-HPLC system equipped with a C18 analytical column  with the same elution procedure. The injection volume was 20 μl, and the flow rate of the mobile phase was set to 0.5 ml/min.The identification of the eluted fraction with antibacterial activity obtained by RP-HPLC was performed by LC-MS/MS analysis as described previously , 31. Mas8 cfu/ml) was added to each well. The MIC value of BVYA1 was determined as the lowest concentration at which growth did not occur. The MIC was investigated in duplicate wells and triplicate experiments.The MIC of BVYA1 was determined by the 2-fold dilution method using a sterile 96-well plate . 100 μl E. coli in the log phase of growth was incubated with BVYA1 (2 × MIC) at 37°C for 2 h. The bacterial cells treated with PBS (pH 7.4) served as a control. Bacterial cells were collected by centrifugation at 5,000 rpm for 15 min. Bacterial cells were washed three times with 20 mM PBS (pH 7.4). The bacterial cells were then fixed with 2.5% glutaraldehyde at 4°C overnight. After that, the bacterial cells were washed three times with 20 mM PBS. The bacterial cells were then dehydrated in a series of graded ethanol (30–100%) for 10 min at each concentration. Afterward, the bacterial cells were freeze-dried and sputtered for gold plating prior to the assay (The indicator strain he assay . The preE. coli cells were suspended in 100 μl of 10 mM PBS (pH 7.4) at a concentration of 2 × 106 cells/ml and then treated with 50 μl BVYA1 (2 × MIC) for 30 min at 37°C. Cells were then collected by centrifugation at 8,000 × g for 10 min and resuspended in 100 μl of PBS containing 0.1 μl of calcein-AM stock solution. After incubation at 37°C for 25 min, 5 μl of PI stock solution was added to the reaction system. After continued incubation at 37°C for 5 min, the cells were washed three times with PBS and then fixed on microscope glass slides by air-drying. Imaging was performed using a fluorescence microscope . The control sample was treated with PBS, and observed under the same conditions.The effect of BVYA1 on bacterial membrane integrity was measured using a calcein-AM/propidium iodide (PI) double staining kit  performed according to a previously described method  with somEach assay was performed in triplicate. The experimental data are presented as the mean ± standard deviation.°C , C13 , C14 , C15 , and C16  (+), C16 (m/z 1463.82 [M + H]+), C17 (m/z 1477.82 [M + H]+), C18 (m/z 1491.83 [M + H]+), C19 (m/z 1505.85 [M + H]+), and C20 (m/z 1519.86 [M + H]+) (+), C15 (m/z 1447.80 [M + H]+), C16 (m/z 1461.82 [M + H]+), C17 (m/z 1475.84 [M + H]+), and C18 (m/z 1489.85 [M + H]+) (Identification of the composition of YA215LE was assessed using liquid chromatography-mass spectrometry (LC-MS) analysis. The strain produced molecules with m/z corresponding to cyclic lipopeptides from different families that were previously described in the Bacillus genera, namely, surfactins, iturins, and fengycins . Iturin d chains . Mycosubd chains . For the + Na]+) . These mM + H]+) . FengyciM + H]+) .E. coli  when it enters the live cells. PI is an impermeable nucleic acid dye that only enters cells with damaged membranes. PI shows red fluorescence under laser excitation at 488 nm. Compared with the E. coli cells treated with PBS, the BVYA1 treatment group showed a large number of red PI in the E. coli cells, indicating that BVYA1 could penetrate the E. coli cell membrane   . Among t1 to C13 . InteresE. coli  produced by B. velezensis YA215 were identified, and the BVYA1 lipopeptide showed remarkable antibacterial activity, this activity may be associated with the effective permeabilization of the cytoplasmic membrane and disruption of the morphology of targeted bacterial cells. Our results also suggested that B. velezensis YA215 and its lipopeptides could be used as promising antibacterial agents against gram-positive bacteria, gram-negative bacteria, and fungi. In addition, YA215LE may become a novel alternative to traditional antibiotics from the B. velezensis YA215 strain, and these lipopeptides may have good prospects for application in food and agriculture.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.XL was the guarantor of the overall content, conceived the idea, and assessed each study. FY and XL drafted the study. YS and YQ performed the statistical analysis. HF and XP selected and retrieved relevant manuscript. All authors revised and approved the final manuscript.


Organoids are increasingly used to investigate patient-specific drug responsiveness since they are thought to be more representative of a patient’s tumor than two-dimensional primary cell cultures. Furthermore, cell culture media that mimic physiological nutrient concentrations have been suggested to improve chemotherapy screens of cultured cells. As both come with increased costs and complexity, we investigated the response of two patient-derived pancreatic cancer organoids  growing as 3D organoids versus 2D transformed cell cultures in either conventional or physiological media towards five chemotherapeutics . Both patient-derived pancreatic cancer cell cultures showed similar drug-responses when cultured in 3D compared to 2D, as well as upon culture in physiological versus conventional culture media, except for higher sensitivity towards SN-38 when PANCO11b was cultured in 2D or in physiological media. These data show that drug-responsiveness of primary pancreatic cancer cells is not majorly impacted by culture conditions.−3 ± 2.3 ×10−3 vs. 3D: 1.1 ×10−3 ± 0.6 ×10−3, p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media . Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10 Pancreatic cancer is highly lethal, with a five-year survival rate of only 9% . As surgIn recent years, researchers have made important progress in predicting the patients’ response to chemotherapeutics by using patient derived organoids (PDOs). Organoids are primary cell cultures that can be generated from resected tumors. They grow in a basement membrane extract (BME) which allows them to expand 3-dimensionally in an organ-like structure, and they maintain heterogeneity in terms of cellular and molecular composition ex vivo ,5,6. PreWhile organoids are superior in mimicking in vivo conditions as compared to conventional 2-dimensional cell cultures, their culturing is more time-consuming, highly expensive, and reproducing results is more difficult . As a reAnother recent development in cancer cell culturing is the use of physiological cell culture media like Plasmax and human-plasma like medium (HPLM). These physiological media aim to mimic the nutrient concentrations of human plasma, which are substantially different from the concentrations found in conventional cell culture media like DMEM or RPMI ,13 Supp. Since nHere, we investigated the chemosensitivity of pancreatic tumor PDOs grown in 3D and their corresponding 2D transformed cell cultures in physiological media and conventional culture media.For this study, we used PANCO09b and PANCO11b organoids from patients with pancreatic ductal adenocarcinoma that were previously established in our laboratory .Organoids were maintained in Tumor 2 (T2) medium consisting of adv.DMEM/F-12 , cat. no. 12634010),  supplemeg at 4 °C. Subsequently, supernatant was aspirated and the organoids were mechanically sheared through a narrowed glass Pasteur pipette. After adding cold adv.DMEM/F12+++, organoids were centrifuged for 5 min at 300× g at 4 °C. Supernatant was aspirated and ice-cold Geltrex was added. Organoids were seeded in 50 µL/well Geltrex separated in 3 domes in a NTC 24-well plate. After domes were solidified in the incubator at 37 °C and 5% CO2, 500 µL T2 media was added. Images were taken with an Incucyte Live-Cell Imaging system  at 4× magnification.Basement Membrane Matrix  in 24-wells non-tissue culture (NTC) treated plates , cat. no. 0030722116) and passaged every 7–10 days. For passaging, domes were dissolved in ice-cold adv.DMEM/F12+++, and the suspension was collected and centrifuged for 5 min at 300× If cells were used for RNA isolation, supernatant was aspirated from the well and TripLE , cat. no. 12605010) was added, followed by homogenization via pipetting, and incubation at room temperature for 5 min. The cell homogenate was stored at −80 °C until RNA isolation. All experiments were performed before passage 30.g for 5 min. Next, supernatant was aspirated and the pellet was resuspended in 2D Tumor 2 (2DT2) medium, consisting of 50:50 (v/v) T2 and adv.DMEM/F12+++  or experimental use. If cells were needed for RNA isolation, 1 mL of TRI Reagent was added per well. Lysed cell solution was stored at −80 °C until RNA isolation.2D transformed cell cultures were maintained in 2DT2 media in 12-wells tissue-culture treated plates in an incubator at 37 °C and 5% COTo grow 2D transformed cell cultures in physiological medium, 2DT2 medium was prepared with Plasmax  2DPlxT2. Cells gg for 5 min at 4 °C. After supernatant was aspirated, the pellet was resuspended in 1–2 mL of TriplE + RhoKinase Inhibitor (1:1000) and incubated on a shaker at 37 °C until organoids were dissociated into single cells. 3 mL of adv.DMEM/F12+++ was added and the cell suspension was centrifuged at 200× g for 5 min at 4 °C. Subsequently, supernatant was aspirated, 1ml adv.DMEM/F12+++ added, and cells were counted using a counting chamber under the brightfield microscope. Single cells were plated on a 384-wells plate  at densities of 500 cells/well  or 3000 cells/well (only 2D culture derived single cells) in 30 µL medium/well. After cells settled, plates were cultured at 37 °C and 5% CO2 in the Incucyte to assess morphology and confluency.Organoid-derived 2D cell cultures were trypsinized and counted as described above. 3D organoids were collected in ice cold adv.DMEM/F12+++ and centrifuged at 200× −5–2 µM, whilst 5-FU and oxaliplatin titrations ranged from 0.013–50 µM. The negative control was exposed to 0.5% dimethyl sulfoxide (DMSO), and all wells were normalized to this condition. Plates were cultured at 37 °C and 5% CO2. After 5 days of treatment, cell viability was assessed using CellTiterGlo  following the manufacturer’s instructions. Luminescence was measured with the Tecan Spark M10 multimode plate reader.After one day, chemotherapeutics were added using a Tecan D300e digital dispenser  in triplicate. Cells were treated with 14 different concentrations where gemcitabine, paclitaxel, and SN-38 titrations ranged from 1.08 × 10SSMD) and Z-scores were calculated using Formulas (1) and (2), respectively.To assess the quality of the chemo-sensitivity assay, strictly standardized mean difference . The mean luminescence of the control (0.5% DMSO) was set to 100% cell viability. The logarithm of administered drug concentration was plotted against the corresponding cell viability. The graph was fitted using a nonlinear regression curve described by Formula (3) (in Graphpad: log(inhibitor) vs. response—variable slope (four parameter)).IC50) value was obtained using logarithmic regression and determined as drug concentration at which half-maximal effect was obtained.The relative inhibitory concentration 50 .Growth rate/day was calculated using Formula (5).50) values were calculated using the online tool grcalculator.org, as explained by Clark et al. [Dose–response growth rate 50  in 0.3 mL medium. After cells settled, the plate was put in the Incucyte Live-Cell Imaging system at 37 °C and 5% CORNA isolation was performed according to the TRI Reagent® Solution manufacturer‘s protocol (ThermoFisher). Cells were homogenized with TRI Reagent and transferred to a 1.5 mL Eppendorf tube. After 5 min. incubation time at room temperature, 200 µL chloroform per 1 mL TRI Reagent was added. Samples were incubated for 15 min. followed by a 15 min. centrifugation at 15,000 rpm at 4 °C. After transferring the aqueous phase (300–400 µL) to a new tube, isopropanol was added at a 1:1 ratio. To initiate precipitation, samples were vortexed and incubated for 10 min. at room temperature, followed by centrifugation at 15,000 rpm for 15 min. at 4 °C. Subsequently, the supernatant was discarded and the RNA pellet was washed twice in 75% ethanol in nuclease free water, followed by 15 min. centrifugation at 15,000 rpm. After decanting, the pellet was dried at 40 °C for 5 min. and resuspended in 10 µL nuclease free-water. RNA was stored at −80 °C until further use.RNA was isolated from 3D organoids and 2D transformed cell cultures as described above. RNA was sent to Novogene Co., Ltd.  for quality control and mRNA sequencing. Quality and quantity were checked by Novogene using gel electrophoresis  and Nanodrop. RIN values had to be >8.0 to continue to library preparation. Preparation of RNA library and transcriptome sequencing was conducted by Novogene Co., Ltd. . In short, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification, and was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution. Quantified libraries were pooled and sequenced on the Illumina NovaSeq 6000 with a 2 × 150 bp high output.Raw data (Fastq format) was obtained and datasets were trimmed using Fastp to remove adapters, poly-N and low-quality reads from the raw data, and aligned in single-end format to the Ensembl reference genome  using STAR and quantified using RSEM.IC50 and GR50 values was done using unpaired t-tests. Statistical significance was assumed when p < 0.05. Statistical analysis of RNA sequencing data was done using R-studio version 2022.07.0 for macOS using the packages BiocManager, DESeq2, ggplot2, dplyr, ggpubr, EnhancedVolcano. Genes with Benjamin-Hochberg adjusted p-value < 0.05 were considered differentially expressed.Graphical presentation and statistical analysis were performed using GraphPad Prism for macOS version 9.0.0. All data are represented as mean or median ± SD. Time-point related statistical analysis of the proliferation curves was done using two-way ANOVA with Sidak’s correction for multiple comparisons. The Mann–Whitney U test was used to compare differences between two independent samples. Comparison of p = 0.786 and p = 0.936, respectively) or PANCO11b cultures .PANCO09b and PANCO11b organoids both grew in evenly round to oval shaped structures with a lumen A. PANCO0p = 2.15 × 10−9), which was upregulated in 3D compared to 2D cultures. Furthermore, two cell-cycle related genes showed differential expression (CDKL3 and MYCN) [To investigate the impact of the 2D versus 3D culture conditions on gene expression patterns, we performed RNA sequencing. There were only 31 differentially expressed genes , with thnd MYCN) ,20,21. TTo compare the chemosensitivity of 3D organoid cultures and their corresponding 2D counterparts, three separate drug-responsiveness assays of PANCO09b and PANCO11b cells grown in adv.DMEM/F-12 were conducted . Dose–reIC50 of the two PDO cultures was determined  than the corresponding 2D culture, whilst no differences were observed for gemcitabine, paclitaxel, 5-FU, and oxaliplatin.None of the drugs reduced cell viability by 100% at maximal dose. Using the dose–response curves, the termined C. The IC50 was calculated. GR50 is an advanced metric that considers the growth rate as a confounder for drug sensitivity calculations and defines the drug concentration needed to inhibit growth by 50% [50 values of gemcitabine, paclitaxel, SN-38, 5-FU, and oxaliplatin did not differ for PANCO09b grown in 2D versus 3D (p = 0.0004), but similar drug responsiveness towards gemcitabine, paclitaxel, 5-FU, and oxaliplatin.All of the administered drugs interfere in DNA-synthesis/replication during cell division. As a consequence, the effectiveness of the drug is associated with the cell division cycle . To corrh by 50% . The GR5ersus 3D . PANCO11Next, the effect of culture medium composition on PANCO organoids grown in 2D was assessed by investigating morphology and proliferation rates in adv.DMEM/F-12 or in Plasmax physiological cell culture medium. After plating the 2D cultures, negligible growth was detected by live cell imaging during the first two days . No differences in growth rate/day or doubling time were observed for PANCO11b grown in Plasmax vs. adv.DMEM/F-12.Cell viability assays using CellTiterGlo revealed that PANCO09b grown in 2D showed a slightly higher growth rate/day and lower doubling time in hours in Plasmax compared to adv.DMEM/F-12 . Thus, growth medium composition did generally not affect the drug-responsiveness of 2D transformed organoids, except for PANCO11b cells exposed to SN-38 when corrected for growth rate.Next, we compared the chemosensitivity of 2D PANCO09b and PANCO11b cell cultures growing in physiological or conventional culture medium. Dose–response curves of PANCO09b and PANCO11b to all five chemotherapeutics can be seen in al media . GR50 caIn the current paper, we have shown that the sensitivity of PDOs to chemotherapeutics commonly used to treat pancreatic cancer is generally not affected by 2D or 3D culturing or by the composition of culture media. The apparent robustness of the chemosensitivity assay facilitates the clinical application of patient-derived primary tumor cell drug screenings by simplifying the workflow and lowering the cost and time required.Cellular characteristics known to be relevant for drug-responsiveness, in particular proliferation rate, were not overtly affected by transformation of regular PDOs cultured in 3D into 2D monolayers, even though two cell cycle related genes (CDKL3 and MYCN) were differentially expressed in 3D vs. 2D cultures. Of note, only 29 additional genes in total were differentially expressed between 2D and 3D cultures, of which 8 were RNA- and pseudogenes. These genes are not known to play a role in the pathways affected by the used chemotherapeutics  growing in 2D monolayer or as 3D spheroids. In contrast to our results, all cultures proliferated faster in 2D compared to 3D . HoweverAfter transformation of the 3D organoids to 2D cell cultures, we investigated their sensitivity to gemcitabine, paclitaxel, 5-FU, SN-38, and oxaliplatin. Both PANCO09b and PANCO11b showed no differences in chemosensitivity between the two culturing dimensions, except for SN-38, to which PANCO11b was more sensitive when cultured in 3D. So far, similar comparisons of drug responsiveness of cells cultured in 2D versus 3D in the literature were mostly done using spheroids from established cell lines. For instance, Minami et al. conducted drug-responsiveness assays to gemcitabine and 5-FU using six pancreatic cancer cell lines growing as spheroids . Cell viIC50 of cells cultured as 2D monolayer was lower than for the spheroids [SN-38 was the only agent to which PANCO11b showed higher sensitivity when cultured in 3D. This finding cannot be explained with the common findings in the literature. In general, when differences in chemosensitivity are observed between cells cultured in 2D versus 3D, the 3D condition is more resistant ,32. Thispheroids . Since Spheroids , a potenpheroids .Next to cell–cell and cell-matrix interactions imposed by 2D vs. 3D culture environments of cells, cellular metabolic status secondary to nutrient composition and concentration in the culture medium has been reported to affect major cellular characteristics such as morphology, proliferation rate, and sensitivity to drugs ,14,15. IImportantly, the use of physiological culture medium instead of conventional adv.DMEM/F12 did not affect the sensitivity of PANCO09b and PANCO11b towards several clinically applied chemotherapeutic drugs. This is not in line with the data of Richards et al.  who showIn conclusion, we have shown that various culture conditions do not majorly affect chemosensitivity of primary human pancreatic tumor cells. This indicates that assessing chemosensitivity of pancreatic cancer cell cultures is robust and reproducible. As it is more difficult to generate a personalized 2D cell culture from a biopsy than to generate a 3D organoid culture, we suggest to first establish 3D organoids and subsequently transform them into organoid-derived primary monolayer cell cultures if the aim is to test for chemosensitivity. In this way, the culture is easier to handle and more cost-efficient.


The extensive useof organophosphorus pesticides in agricultureposes a high risk to human health and has boosted the demands fordeveloping sensitive monitoring methods. Herein, we developed a facileand sensitive method for isocarbophos detection based on the remarkablefluorescence enhancement of pyrene during host–guest interactionof β-cyclodextrin polymer (β-CDP) and pyrene. The 3′-pyrene-labeledisocarbophos aptamer could be cleaved by exonuclease I to obtain freepyrene that was tagged on mononucleotides, which could enter the hydrophobiccavity of β-CDP, resulting in a prominent fluorescence enhancement.While the target isocarbophos was added, aptamer could undergo a conformationalchange into a hairpin complex, which prevented the cleavage and host–guestinteraction because of the steric hindrance, leading to a weak fluorescence.The isocarbophos has been sensitively and selectively analyzed bydetecting the system fluorescence intensity with a detection limitas low as 1.2 μg/L. In addition, we have verified the abilityof our proposed method in real sample detection from fruit extract. More severely, the continualassimilation by plants and enrichment in the food chain mean thateven a very low concentration of organophosphorus pesticides couldthreaten human health, which raises people’s concern abouttheir health effects.5 There have been a large numberof reports about organophosphorus pesticides being related to humandiseases such as attention deficit hyperactivity disorder, Parkinson’s,amyotrophic lateral sclerosis, Alzheimer’s, chronic diseasesof the central nervous system, etc.6 Accordingly,it is urgent to develop highly sensitive methods for organophosphoruspesticide analysis in the field of food safety protection.Organophosphorus pesticideshave been widely applied throughoutthe world for their high efficiency in preventing diseases and pestsfrom harming crops.15 have been widely used in the detection of organophosphorus pesticides.Among them, fluorescence methods have aroused great interest becauseof their simple operation, low sample usage, and easy readout andquantification.17 However, many fluorometric immunoassaysthat are based on antibodies as the recognition department sufferfrom the high cost and instability of antibodies, which limits theirwide application. At present, aptamers have been widely used as artificialantibodies in fluorescence methods because of their high affinity,excellent stability, and prolonged storage life.19 For example, Dou et al. have established a gold-based nanobeaconfluorescence probe for organophosphorus pesticide sensing, with adetection limit of quantification reaching 10 μg/L.16 In order to monitor trace-level organophosphoruspesticide residuals and overcome the current issue of severe pollution,it is urgent to develop highly sensitive fluorescence methods.Upto now, chromatography, ultraviolet–visible spectroscopy,electrochemical technology, capillary electrophoresis, fluorescencemethods, etc.,23 Cyclodextrin polymer is constitutedof monomer cyclodextrin, the most commonly used host molecule, throughpolymerization, graft copolymerization, or molecular cross-linking.It not only retains the characteristics of highly specific host–guestrecognition of monomer cyclodextrin25 but also exhibitssome advantages like good solubility, high stability, a cross-linkingagent effect, and a multivalent binding effect. More importantly,Hollas et al. reported that cyclodextrin polymer has shown a strongerfluorescence enhancement effect for fluorophores than monomer cyclodextrinbecause of the increase in the overall complexation constant by morethan 2 orders.26 Based on these excellentcharacteristics of β-cyclodextrin polymer, we have developedhighly sensitive detection methods for biological small molecules,enzymes, nucleic acids, etc.28Because of their unique properties compared to the properties ofmonomers, polymers such as fluorescent conjugated polymers, molecularlyimprinted polymers, nucleic acid, etc., have had widespread use asfundamental materials in analysis fields.Inspired by thesestrategies, we intend to design a highly sensitivemethod for isocarbophos detection based on a prominent fluorescentenhancement of cyclodextrin polymer combined with the highly selectiverecognition ability of the nucleic acid aptamer. As shown in 29 The results of Fourier transform infrared spectroscopy , the particle size distribution , and the ζ potential diagram proved that β-CDP hadbeen successfully synthesized. The detailed experimental proceduresand characterization are described in the Supporting Information.β-CDPwas synthesized according to the literature.To verify the efficient fluorescence enhancementcapability of β-CDP, we investigated the fluorescence responsewith the concentration of β-CDP changing from 0 to 2.5 g/L.In the presence of β-CDP and isocarbophos, the fluorescenceof the system remained weak because DNA aptamer labeled with pyrenecould specifically recognize the isocarbophos to form a hairpin structure,which prevented DNA aptamer from being digested by Exo I, and it hardlyentered the cavity of β-CDP 1. While Gel electrophoresis was further used to demonstratethat isocarbophos could protect 3′-pyrene-labeled aptamer fromdetachment. As shown in Figure S4 in the Supporting Informationdetails the optimization of experimental conditions). F0 – F)/F0 (F0 and F represent the fluorescence responses of this method without andwith isocarbophos) increased linearly with the concentration of isocarbophosin the range 0–50 μg/L . Inaddition, this method showed a low detection limit . Figure S3). The DNA aptamer was also negatively charged. Themutually repulsive force between β-CDP and the aptamer couldensure the low background of this method and therefore contributedto the high sensitivity.In order to verify the abilityof the strategy for quantitative detection, a concentration gradientexperiment of isocarbophos was carried out under optimal conditions(F0 – F)/F0 was 40 μg/L fortriazophos, 50 μg/L for dimethoate, 50 μg/L for chlorpyrifos,70 μg/L for parathion methyl, 50 μg/L for chlorpyrifosmethyl, and 50 μg/L for methidathion. For these potential interferents,the maximum signals of (F0 – F)/F0 were below 0.10. However,only the addition of isocarbophos could trigger a large signal of(F0 – F)/F0; the maximum signal of (F0 – F)/F0 in the case of isocarbophos was close to 0.9. These resultsverified that our constructed method has excellent selectivity.For a furtherevaluation of the selectivity of this method, 6 potential interferentsof other organophosphorus pesticides were investigated. As depictedin 33 with minor alteration.Then, different standard concentrations of isocarbophos were addedinto the apple extract. n = 3) from the actual apple samples spikedwith our proposed method. While using GC analysis to detect isocarbophos,the recoveries were in the range 79.3–105.3%, with a much higherdetection limit of 28.9 μg/L than our first spiked sample, 5μg/L.15 These results proved theaccuracy and reliability of our established method in food safetyapplications.Our proposed method was usedto analyze residual isocarbophos in apples to demonstrate the strategyin the application of real samples. The apple extract was preparedaccording to a reportIn conclusion, a highly sensitive and facile fluorescence methodfor isocarbophos monitoring has been constructed that combines thesignificant fluorescence enhancement of β-CDP with the high-specificity/affinityaptamer. Highly sensitive detection of isocarbophos was achieved dueto the remarkable fluorescence enhancement ability of β-CDPwith the wide range 5–50 μg/L and a detection limit aslow as 1.2 μg/L. Additionally, the high selectivity was attributedto the ultraselective aptamer with high affinity. Moreover, it wassuccessfully used to determine residual isocarbophos in fruit samples.Altogether, the construction of this method offers a new choice forhighly sensitive and selective organophosphorus pesticide detectionand has great application potential in the field of food safety inspection.2, 670mM glycine–KOH, 10 mM dithiothreitol, pH = 9.5) and Exo I wereprovided by Sangon Biotech Co., Ltd. . The ultrapurewater was provided by a Millipore water purification system (≥18.2MΩ cm).Isocarbophos  was brought fromShanghai Pesticide Research Institute , and chlorpyrifos(98%) was provided by Sinopharm Chemical Reagent Co., Ltd. . Acetone was purchased from Xilong Scientific Co., Ltd. . A 3′-pyrene-labeled isocarbophos DNA aptamer (5′-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3′) was synthesized by TaKaRa Bio. Inc. .β-Cyclodextrin was bought from Sigma-Aldrich Co., Ltd. Trishydroxymethyl aminomethane, toluene, epichlorohydrin, isopropanol,and sodium hydroxide were obtained from China National PharmaceuticalGroup Co., Ltd. 10× Exo I buffer  was added into 208 μL of sterilized ultrapure waterand 30 μL of 4 μM DNA aptamer solution in a centrifugetube. Then, 42 μL of isocarbophos standard solution was addedto the mixed solution. Subsequently, 140 units of Exo I was mixedinto the system and incubated at 37 °C for 30 min in a dry bath,followed by heating for 20 min at 80 °C to inactivate Exo I,and the mixture was then allowed to cool to 25 °C. Afterward,30 μL of 15 g/L β-CDP was added into the centrifuge tube.Lastly, the fluorescence signals of the system were detected withan Agilent G9800A fluorescence spectrophotometer. The excitation wavelengthwas 345 nm, and the scanning range of the fluorescence emission spectrumwas 350–500 nm, with the excitation set at 5 nm and emissionslit at 10 nm.2EDTA, 40 mM Tris-acetate, pH 8) to prepare anagarose gel (2%). A 2 μL portion of Sybr Gold and 2 μLof loading buffer were mixed with 10 μL of the sample, and themixture was then added to the agarose gel for electrophoresis experiments.In the same TAE buffer, electrophoresis was performed at 100 V for30 min, and then, the gel was photographed using gel imaging underultraviolet light.Agarose powder(0.5 g) was mixed into 25 mL of 1× TAE electrophoresis buffer.At first, 20 mL of freshly squeezed apple juice was mixed with 40mL of acetone and 10 mL of water, and the mixture was allowed to sitfor 10 min. Then, the mixture was filtrated with a 0.22 μm filtermembrane after remaining in an ultrasonic bath for 30 min. Afterward,we concentrated the filtrate in rotary evaporators at 40 °C.Finally, the solution product was redissolved in 10× Exo I bufferwith a ratio of 1:100.


Esophageal squamous cell carcinoma (ESCC) is a common digestive tract malignant tumor with high incidence and dismal prognosis worldwide. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of ESCC are not unequivocally understood yet. The serum proteome may provide valuable clues for the early diagnosis of ESCC and the discovery of novel molecular insights.In the current study, an optimized proteomics approach was employed to discover novel serum-based biomarkers for ESCC, and unveil abnormal signal pathways. Gene ontology (GO) enrichment analysis was done by Gene Set Enrichment Analysis (GSEA) and Metascape database, respectively. Pathway analysis was accomplished by GeneCards database. The correlation coefficient was assessed using Pearson and distance correlation analyses. Prioritized candidates were further verified in two independent validation sets by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) staining.α, Cori cycle, TCA cycle, folate metabolism, and platelet degranulation. The latter finding was all the more noteworthy as a strong positive correlation was discovered between activated glycolysis and TLR4 pathways and unfavorable clinicopathological TNM stages in ESCC.A total of 633 non-redundant proteins were identified in the serum of patients with ESCC, of which 59 and 10 proteins displayed a more than 1.5-fold increase or decrease compared with healthy controls. Verification was performed for six candidate biomarkers, including S100A8/A9, SAA1, ENO1, TPI1 and PGAM1. Receiver operating characteristics (ROC) curve plotting showed the high diagnostic sensitivity and specificity of these six protein molecules as a biomarker panel: the area under characteristic curve (AUC) is up to 0.945. Differentially expressed proteins were subjected to functional enrichment analysis, which revealed the dysregulation of signaling pathways mainly involved in glycolysis, TLR4, HIF-1Our findings propose a potential serum biomarker panel for the early detection and diagnosis of ESCC, which could potentially broaden insights into the characteristics of ESCC from the proteomic perspective.The online version contains supplementary material available at 10.1186/s12014-022-09357-x. Esophageal squamous cell carcinoma (ESCC), the major histopathological subtype of esophageal cancer, is one of the most lethal malignancies of digestive tumors worldwide, with a 5-year survival rate of less than 20% , 2. The 3K signaling pathways . Differentially expressed proteins were screened by FC combined with significance levels . Protein abundance was significantly changed if a more than 1.5-fold increase or decrease was observed between two groups with a P < 0.05.Proteins were executed based on the following criteria. Potentially contaminated proteins, including keratin, were excluded from the data. Proteins were required to have at least half of the valid values in both groups, and the remaining missing values were imputed with K-nearest neighbors (K = 10) using the R package (v.3.5.2). Protein abundance was quantified using peak area and normalized by each sample’s total area, which represented standardized value of a protein across samples. The standardized value was multiplied by 10https://metascape.org/gp/index.html) [P-value < 0.05, a minimum count of 3 and an enrichment factor of more than 1.5 were collected and grouped into clusters based on their membership similarities (Kappa scores > 0.3) [https://cn.string-db.org/) [https://www.genecards.org/) [GO enrichment analysis of biological processes was conducted using GSEA software or the Metascape database (ex.html) . Terms ws > 0.3) . The mosdb.org/) . The phyds.org/) .2SO4. Analysis of the optical absorbance was performed by using a plate reader. The absorbance values were determined from the background subtracted from the signal at 450 nm.For validation set, serum S100A8/A9, SAA1, ENO1, TPI1 and PGAM1 of 30 healthy controls and 53 ESCC patients were detected using the Human S100A8/A9 Heterodimer Immunoassay ELISA Kit and Serum Amyloid A1 DuoSet ELISA kit , TPI1 ELISA Kit, ENO1 ELISA Kit and PGAM1 ELISA Kit  according to the manufacturer’s instructions. Plates were coated with the capture antibody at room temperature for 1 h and subsequently at 4 °C overnight. After incubation, wells were washed in triplicate by TBST (TBS with 0.05% Tween). Wells were then blocked with 250 µL TBST containing 1% BSA and incubated for 1 h at 37 °C. Diluted samples (100 µL) were incubated with detection antibody for 1.5 h at 37 °C and finally with the HRP for 1 h at 37 ℃ and substrate solution for 30 min at room temperature. The substrate reaction was halted by adding 2 M H2O2 for 5 min at room temperature. Heat-induced epitope retrieval was carried out using Tris-EDTA buffer in a microwave oven heated on high for 3 min and medium for a further 10 min. Sections were cooled at room temperature and then incubated with ENO1, TPI1, PGAM1, SAA1 and S100A8/A9 primary antibody (dilution 1:100) overnight at 4 °C in a humid chamber, followed by incubation with an appropriate secondary antibody. Images were obtained by scanning the slides using a Zeiss Axio Scan Z1 in brightfield mode . An IHC score was assigned to each specimen according to the staining intensity and the proportion of positive cells. 1+: light brown and stained cells < 30%; 2+: brown and stained cells 30–60%; 3+: deep brown and stained cells > 60%. Comprehensive scores were made according to the sum of the two indexes. 1+ = weakly positive; 2+ = moderately positive, 3+ = strongly positive. Staining in non-cancer cells was not accounted for in the IHC scores.ESCC tissues and matched adjacent normal tissues (n = 29) were acquired from the Pathology Department of Affiliated Hospital of North Sichuan Medical College. Patients did not receive chemotherapy or radiation before surgery. Immunochemistry was performed as per the standard procedure. Tissue sections were heated at 60 °C for 1 h and then dewaxed in xylene, rehydrated in a graded series of ethanol and incubated in Ht-test. The correlation coefficient was assessed using Pearson and distance correlation analysis. Data were expressed as mean ± SEM, P < 0.05 was considered statistically significant. Data were displayed using GraphPad Prism 8.0.1  or the R package (version 3.5.2). The diagnostic performance of biomarkers was evaluated by calculating ROC curves, AUC, optimal cutoff, sensitivity, specificity, and positive and negative predictive values using the R package. The optimal cutoff value was defined by maximizing Yoden’s index.Statistical analysis was accomplished using IBM SPSS 23.0 package . Normality of the data distributions was tested via the Shapiro-Wilks test. The data with normal distribution were analyzed by unpaired Student’s A discovery set containing 30 ESCC patients and 30 healthy controls was recruited to characterize the differential proteome. Potential protein biomarkers were further investigated by procuring two independent validation sets, of which validation set_1 included 30 healthy controls and 53 ESCC patients, and validation set_2 included 29 ESCC patients. Serum or tissue samples, including cancerous tissues (CT) and their adjacent normal tissues (ANT), were obtained from each of the subjects. The clinical characteristics of controls and ESCC patients are summarized in Table Serum proteomics-based integrated function validation approach was employed to identify potential ESCC-associated protein biomarkers and uncover functional characteristics in our study. A brief description of the workflow is shown in Fig. http://proteomecentral.proteomexchange.org) via the iProX partner repository with the data identifier PXD031287. A total of 633 non-redundant proteins were identified with at least two unique peptides at 1% peptide level FDR. In this pool, 310 proteins detectable in at least half of all samples were selected for subsequent bioinformatics analysis  of the peak area in each QC sample displayed good repeatability , whereas decreased proteins (FC < 1.0) were involved in lipid transport and complement cascade   were subjected to GO terms enrichment analysis by the Metascape database, and the results were presented in Fig. 10P = 17.22), acute inflammatory response (−log10P = 15.86) and glycolysis (−log10P = 13.94), and many others.   in the ESCC group were moderated associated with cellular metabolism, adhesion and migration process  .The protein expression of glycolysis increased in ESCC patients Fig. B, and amBlood biomarkers are widely applied for the screening and diagnosis of tumor because of their ready availability. Blood contains a range of proteins with concentrations up to 12 to 13 orders of magnitude, while 90% of low concentration proteins are masked by a few high-abundant ones, such as albumin and immunoglobulins. Due to this, although these low-abundance proteins may contain critical information regarding the disease status, they are hard to be detected and quantified in proteomics. In this study, we employed the High Select TOP 14 Abundant Protein Depletion Mini Spin Columns to deplete the 14 highest abundant proteins, which contributes to the in-depth detection of ESCC-related proteins, including low abundance proteins, as well as provides data support for biomarker discovery and signaling pathway research.Traditional biomarkers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9), as well as squamous cell carcinoma antigen (SCCA), showed low sensitivity and specificity to the diagnosis of ESCC . BesidesS100A8 and S100A9, known as calcium-binding proteins, belong to the S100 family. They usually exist in the form of heterodimers for stability. S100A8/A9, being multifunctional, can induce recruitment of leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration . Its intκB signaling pathways resulting in the amplification of the pro-inflammatory cascade [R2 = 0.41, P = 1.0e−4 and R2 = 0.36, P = 4.1e−4, respectively.Nevertheless, other studies suggested that S100A8 and S100A9 were significantly downregulated in the tissues of ESCC patients compared with controls at the mRNA level , 27. Sin cascade . In accoα (TNFα). It has been investigated in various malignancies as a predictor of cancer risk and prognostic factor [SAA1 is recognized as a nonspecific, acute-phase protein secreted in response to inflammation-associated cytokines, such as interleukin-1/6 (IL-1/6) and tumor necrosis factor c factor , 31. A pc factor . SAA1 shSAA1 is produced by the liver and enters the systemic circulation in response to stimulation by inflammatory cytokines, such as IL-6. As a potent pro-inflammatory cytokine, IL-6, stimulates the liver to produce SAA1 . ElevateOur proteomics data showed 5.01, 3.55, 7.18, 3.84, 3.67 and 4.90-fold higher levels of S100A8, S100A9, SAA1, ENO1, TPI1 and PGAM1 in ESCC patients sera compared with controls, respectively. Given the close relationship between S100A8/A9, SAA1, ENO1, TPI1, PGAM1 and tumor, with our present data, we were committed to developing a biomarker panel consisting of these six proteins for early detection of ESCC patients, which have yielded high specificity and sensitivity of 0.906 and 0.967, and AUC up to 0.945, higher than each of them alone, suggesting the synergistic effects and increased diagnostic efficacy of these six biomarkers as a potential biomarker panel.R2 = 0.59, P = 6.53e−7). Overall, these results suggested that ENO1 played different biological roles depending on the types of cancer while the exact mechanisms of action remain unclear.ENO1, a key glycolytic enzyme, which may play a pivotal role in aerobic glycolysis, contributed to solid tumor progress. Evidence showed that ENO1, whose overexpression was associated with multiple tumors, is explained as a key protein in tumorigenesis, proliferation, metastasis and poor outcomes , 36. HowR2 = 0.33, P = 9.1e−4).TPI1 is a metabolic enzyme that catalyzes the interconversion between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glycolysis and gluconeogenesis. Elevated TPI1 expression is associated with high levels of metabolism required for rapid tumor growth. A previous study showed a higher expression of TPI1 in metastatic ovarian tumors than in primary ovarian cancers. However, the expression of TPI1 was lower in metastatic cervical tumors than in primary cervical cancers . It was R2 = 0.46, P = 3.99e−5), thereby potentiating the role of PGAM1 as a diagnostic and prognostic marker for ESCC. Furthermore, several proteins related to glycolysis, such as PKM, PGK1, ALDOA and LDHB, were significantly elevated in the serum of ESCC patients and positively correlated with TNM stages , which suggested activated glycolysis could be a significant characteristic of ESCC.PGAM1 is known as a metabolic enzyme in glycolysis. High expression of PGAM1 is closely correlated with lymphatic metastasis and tumor re-occurrence, which might account for the poor prognosis . We founIt is widely known that PGK1, ALDOA, ENO1, PKM, TPI1, GAPDH, PGAM1 and LDHB are key regulators in glycolysis and are mainly located in the cytoplasm. However, recent studies detected several glycolysis-related proteins in the extracellular region, such as serum –43, of wOur study comprehensively portrayed serum proteomic profiling of ESCC patients, identified 69 differential proteins and systematically elucidated multiple abnormal signaling pathways in ESCC patients. Our data showed a potential biomarker panel composed of six protein molecules, including S100A8/A9, SAA1, ENO1, TPI1 and PGAM1, with high diagnostic sensitivity and specificity and revealed the relevance of activated glycolysis and TLR4 pathways to TNM stages. These results propose potential biomarkers for early screening and diagnosis of ESCC and shed new light on the biological characteristics of ESCC from the perspective of signaling pathway.Additional file 1: Figure S1. Spearman’s correlation coefficients from QCs samples to assess the experiment reproducibility of control and ESCCgroups.Additional file 2: Quantitative proteins in ESCC and Control groups.


Accordingly, the total chlorophyll content was reduced by 47.1%, 47.4%, and 53.8% in the interior, center and edge regions, respectively, and the net photosynthetic rate was significantly decreased in the 9th leaf. Transcriptome analysis revealed that MD induced 9,314, 7,425 and 5,716 differentially expressed genes (DEGs) in the interior, center and edge regions, respectively. Of these, the chlorophyll metabolism pathway was preferentially enriched according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The expression levels of the five candidate genes in leaves were consistent with what is expected during chlorophyll metabolism. Our results suggest that changes in the expression of genes related to chlorophyll synthesis and decomposition result in the yellowing of banana seedling leaves, and these results are helpful for understanding the banana response mechanism to long-term MD.Magnesium (Mg) is an essential macronutrient for plant growth and development. Physiological and transcriptome analyses were conducted to elucidate the adaptive mechanisms to long-term Mg deficiency (MD) in banana seedlings at the 6-leaf stage. Banana seedlings were irrigated with a Mg-free nutrient solution for 42 days, and a mock control was treated with an optimum Mg supply. Leaf edge chlorosis was observed on the 9 As the e (MgCh) . Mg-cheln plants –16. A seproaches .CAB2) gene in a feedback mode, leading to a decreased chlorophyll concentration [Arabidopsis, AtSGR1 and AtSGR2, which encode the enzyme Mg-dechelatase, are responsible for the breakdown of chlorophyll [OsSGR is negatively regulated by ROS, affecting chlorophyll degradation in rice leaves [Arabidopsis, barley and Citrus sinensis [The typical chlorosis symptom of MD appears in the leaf intervein and usually appears first on the lower and older leaves owing to the mobile nature of Mg , 19. At ntration . In Araborophyll . Under Me leaves . miRNAs e leaves . As a resinensis , 24–26.Arabidopsis, rice, maize and soybean, the molecular mechanism of banana leaf chlorosis is unclear [Musa acuminata, AAA, cultivar Cavendish) seedling leaves exposed to long-term MD.Banana is one of the world’s major food crops in the tropical and subtropical regions, where soil acidification has been a serious growing problem , 28. As  unclear , 30, 31.M. acuminata, AAA, cultivar Cavendish) seedlings were purchased from the Danzhou Rapid Propagation of Banana Breeding Base , cultivated in potting soil  until the 6-leaf stage and then used for MD treatment.Banana 2·4H2O, 2 mM NH4H2PO4, 4 mM KCl, 60 μM Fe-EDTA, 25 μM H3BO3, 2 μM MnSO4·H2O, 2 μM ZnSO4·7H2O, 0.5 μM CuSO4·5H2O, and 0.05 μM H2MoO4. For the mock control group, normal Hoagland’s nutrient solution containing 1 mM MgSO4·7H2O and 6 mM KNO3 was used, while 1 mM K2SO4, 4 mM KNO3 and 1 mM NH4NO3 were used to maintain the same K, S and N supply in the MD group. The two kinds of Hoagland’s nutrient solutions were supplied every 2 days. For each group, at least 15 plantlets were used, and 3 biological replicates were set. All seedlings were grown in the greenhouse of Hainan University. After 42 days of treatment, the 9th leaves and the roots were collected, immediately frozen in liquid nitrogen and then stored at -80°C for RNA extraction. The 9th-leaf samples were divided into the edge (LM_E), center (LM_C) and interior (LM_I) regions.For MD treatment, all seedlings were transplanted to sand after rinsing the roots with double distilled water and then seedlings of uniform growth were randomly divided into two groups, th leaf of the banana seedlings at 9:00–11:00 a.m. on a sunny day. All measurements were carried out using a CIRAS-3 portable photosynthesis system . The photosynthetic photon flux density provided by a red/blue LED light source was amounted to 1200 μmol m-2s-1, the ambient CO2 concentration was adjusted to 390 μmol mol-1 by CO2 injection and the leaf temperature was maintained at 27°C.After 42 days of treatment, photosynthetic parameters were measured in the 9th leaf in each plant was recorded from 31 to 60 days after treatment. The measurements were taken from at least three biological replicates per treatment, and the values were averaged.The relative chlorophyll content of each leaf was measured using a chlorophyll meter , and the SPAD value of the 9After harvest, the plants were washed with deionized water to remove any residual ions. The roots, stems and leaves were heated at 105°C for 15–20 min, and then dried at 80°C until they reached a constant weight. The dry weight was measured and recorded.For Mg content measurement, leaves were dried at 60°C for 3 days and then digested with nitric acid using a microwave digestion system (MILESTONE Ethos UP). After dilution in deionized water, the metal content in the samples was determined by ICP-MS (Agilent 7000 series).Fresh samples were ground into powder in liquid nitrogen, and total RNA (at least 1 μg) was extracted from LM_I, LM_C, LM_E, and control samples using the RNA-prep Pure Plant Plus Kit  according to the manufacturer’s instructions. After pre-processing and rRNA removal, a cDNA library was constructed using the Illumina NovaSeq 6000 platform .Musa acuminate genome by using the HISAT2 program, and the read count for each gene was obtained from mapping results [Clean reads were mapped based on the  results . Gene ex results . DEGs be results . KOBAS w results .MaRPS2 gene was used as an internal control.Five candidate genes related to chlorophyll metabolism were selected, and the primers were designed by Premier Primer 5.0 software . The primer sequences and reference gene are listed in a supplemental table . cDNA syM. acuminata, AAA, cultivar Cavendish) seedlings at the 6-leaf-stage were planted in quartz sand without a Mg supply for 42 days (th leaf (L9) was visible etiolated and showed symptoms of chlorosis  under long-term MD in the LM_I, LM_C and LM_E regions (th leaf (L8) to the 11th leaf (L11) under the MD treatment, but showed the opposite trend in the control group  effect on the fresh and dried biomass of the 9th leaf  decrease after 60 days of MD treatment, with the differences starting at 40 days (p < 0.01) from L9 to L6 in the MD group after 42 days of Mg starvation treatment  was measured. The SPAD value of L9 showed a significant ( 40 days . Moreovereatment . As Mg i2 concentration was significantly (p = 0.02) increased, and the net photosynthetic rate of L9 was significantly (p < 0.01) reduced. Moreover, the contents of chlorophyll a, chlorophyll b and carotenoids in different regions of L9 leaves were significantly  lower under long-term MD treatment in the interior region than in the control group  was used to evaluate the reliability of samples to explore differences within or between groups. There was a significant difference of 74.54% between LM_E and the control group, while the intragroup difference was 10.02% . ReplicaA total of 20,114 transcripts were detected in all samples. In total, 80, 148, 278 and 595 transcripts were specifically expressed in the LM_E, LM_C, LM_I and control, respectively . After uAll DEGs found were classified into the cellular component (CC), molecular function (MF) and biological process (BP) GO categories. For the MF category, the most significant terms included ’binding’, ’catalytic activity’, ’transporter activity’ and ’nucleic acid binding transcription factor activity’. For BP terms, the most significantly overrepresented terms were ’metabolic process’, ’cellular process’, ’single-organism process’, ’biological regulation’ and ’regulation of biological process’. In addition, ’cell’, ’membrane’, ’cell part’, ’membrane part’ and ’organelle’ were significantly overrepresented terms in the CC category .In this study, KEGG pathway enrichment analysis of the DEGs revealed 131 pathways that were enriched, including ’carotenoid biosynthesis’, ’carbon fixation in photosynthetic organisms’, ’glycolysis/gluconeogenesis’, ’carbon metabolism’, and ’porphyrin and chlorophyll metabolism’ . AccordiPhysiological and transcriptome analyses revealed that MD treatment inhibited the photosynthetic capacity and chlorophyll content in banana leaves. A heatmap of 64 candidate genes differentially expressed between treatments (MD and control) was generated to visualize the expression pattern. A total of 21 genes were associated with photosynthesis-antenna proteins, while 17 genes were upregulated in LM_E .MaCHLI), Ma09_g24880 (MaCHLM), Ma10_g13280 (MaCHLG), Ma02_g05320 (MaSGR1) and Ma07_g18910 (MaSGR2)) related to the chlorophyll metabolism pathway were selected to investigate their expression profiles using qRT-PCR. The verification results of these genes showed similar expression patterns in the RNA-seq data  were downregulated, while 2 genes were upregulated under long-term MD in different banana leaf regions.To confirm the accuracy of the RNA-seq data, 5 genes  were simultaneously downregulated in different regions of banana Click here for additional data file.S2 FigA) Chla content in different L10 samples. (B) Chlb content in different L10 samples. C. Car content in different L10 samples.((TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Herein, we demonstrate a case study to efficiently find the herbal medicine(s) with potent hCYP3A4 inhibition in vitro and to accurately assess the potential HDIs risk in vivo. Following screening over 100 herbal medicines, the Chinese herb Styrax was found with the most potent hCYP3A4 inhibition in HLMs. In vitro assays demonstrated that Styrax could potently inhibit mammalian CYP3A in liver and intestinal microsomes from both humans and rats. In vivo pharmacokinetic assays showed that Styrax  significantly elevated the plasma exposure of two CYP3A-substrate drugs (midazolam and felodipine) when midazolam or felodipine was administered orally. By contrast, the plasma exposure of either midazolam or felodipine was hardly affected by Styrax (i.g.) when the victim drug was administered intravenously. Further investigations demonstrated that seven pentacyclic triterpenoid acids (PTAs) in Styrax were key substances responsible for CYP3A inhibition, while these PTAs could be exposed to intestinal tract at relatively high exposure levels but their exposure levels in rat plasma and liver were extremely low. These findings well explained why Styrax (i.g.) could elevate the plasma exposure of victim drugs only when these agents were orally administrated. Collectively, our findings demonstrate that Styrax can modulate the pharmacokinetic behavior of CYP3A-substrate drugs via inhibiting intestinal CYP3A, which is very helpful for the clinical pharmacologists to better assess the HDIs triggered by Styrax or Styrax-related herbal products.Human cytochrome P450 3A4 (hCYP3A4) is a predominant enzyme to trigger clinically relevant drug/herb-drug interactions (DDIs or HDIs). Although a number of herbal medicines have been found with strong anti-hCYP3A4 effects  It has s (DDIs) . TherefoRecently, our group and colleagues developed a novel fluorescence-based high-throughput assay for assessing the inhibitory effects of compounds/herbal extracts on human CYP3A4, which offers a powerful platform for investigating CYP3A4-related HDIs or DDIs . With thin vivo. Therefore, this study aims to solve two key questions: 1) Can Styrax change the pharmacokinetic behavior of co-administrated CYP3A-substrate drugs and trigger HDIs in vivo; 2) Styrax triggers clinically relevant HDIs via inhibiting either hepatic CYP3A4 or intestinal CYP3A4 or both.The major constituents in Styrax have been reported, in which cinnamic acids, phenylpropanoids and pentacyclic triterpenoid acids (PTAs) are major constituents in this herb, while the PTAs in Styrax are reported as naturally occurring CYP3A inhibitors . HoweverTo better solve these two questions, two CYP3A-substrate drugs (midazolam and felodipine) were selected as victim drugs in this work, while two administrated ways  were used to study the pharmacokinetic interactions between Styrax and two CYP3A-substrate drugs (midazolam and felodipine). Briefly, the inhibitory effects of Styrax against CYP3A-catalyzed midazolam 1′-hydroxylation were carefully assessed in liver and intestinal microsomes from both humans and rats. After then, the pharmacokinetic interactions between Styrax  and CYP3A-substrate drugs (midazolam and felodipine) by two administrated ways  were assessed in rats. Furthermore, the inhibitory effects of the PTAs in Styrax on CYP3A were assessed, while the tissue distributions of seven major PTAs in Styrax (the key substances responsible for CYP3A inhibition) were investigated carefully.+ were purchased from Sigma-Aldrich . MgCl2, 0.9% NaCl, and sodium carboxymethyl cellulose (CMC-Na) were provided by Sinopharm Chemical Reagent . The soybean oil was supplied by COFCO Fulinmen Food Marketing Co., Ltd. (China). Midazolam was from J&K Scientific . 1′-Hydroxymidazolam was provided by Shanghai Macklin Biochemical Co., Ltd. . Felodipine was purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Osalmide was ordered from Meilun Bio. Tech . N-ethyl-1,8-naphthalimide (NEN) and N-ethyl-4-hydroxyl-1,8-naphthalimide (NEHN) were synthesized by using the previously reported protocol  was used throughout. LC grade methanol, acetonitrile and formic acid were supplied by Fisher Scientific Co., Ltd. . Other reagents were of the highest grade commercially available. The purities of all compounds used in this study were greater than 98%.The standard extract of Styrax was acquired by Shanghai Hutchison Pharmaceuticals . More than 100 herbal medicines granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. . Lansoprazole was provided by Hairong . D-glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH) and β-NADPprotocol . Betulin2, and HLMs were added to the 96-well plate in turn, then the samples were vortexed and pre-incubated for 3 min at 37 °C. The oxidative reactions were initiated by adding β-NADP+ to yield a final volume of 200 μl, then the formation rates of NEHN (hydroxylated metabolite of NEN) were continuously monitored for 30 min at 37 °C using a 96-well fluorescence microplate reader . The percentage of organic solvent in all incubation is not over 1%. Please refer to the supplementary material  were added to the EP tube in turn and vortexed, then the samples were pre-incubated for 3 min at 37 °C. Next, the reactions were initiated by adding β-NADP+ to yield a final volume of 200 μl. Following incubation for 30 min at 37 °C, and 200 μl ice-cold acetonitrile containing internal standard  was subsequently added to quench the oxidative reaction. The mixture was then centrifuged at 20,000 ×g, 4 °C for 20 min, and the supernatant was quantified by LC-MS/MS analysis as described in The inhibitory effects of Styrax or its constituents against CYP3A were assayed by using midazolam as the substrate . Brieflygroup 1: Styrax + midazolam ;group 2: soybean oil + midazolam ;group 3: Styrax + midazolam (intravenous);group 4: soybean oil + midazolam (intravenous);group 5: Styrax + felodipine ;group 6: soybean oil + felodipine ;group 7: Styrax + felodipine (intravenous);group 8: soybean oil + felodipine (intravenous);Sprague-Dawley rats (weighing 200–220 g) were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.  2018-0006). The animal experiments were carried out in Experimental Animal Center of Shanghai University of Traditional Chinese Medicine  and conducted in accordance with the State Committee of Science and Technology of China. In brief, forty male rats were housed under controlled environmental conditions  for 1 week with free access to food and water. The rats were fasted overnight before dosing but were free to drink water, and provided food after finishing the study. Styrax was suspended in soybean oil. Oral midazolam/felodipine was suspended in 0.5% CMC-Na, and intravenous midazolam/felodipine was suspended in 0.9% NaCl. Styrax or soybean oil was administered orally at a dose of 100 mg/kg. The oral dose of midazolam is 20 mg/kg and the intravenous dose is 5 mg/kg. The oral dose of felodipine is 10 mg/kg and the intravenous dose is 2 mg/kg. The dose of Styrax in rats was referred to the daily recommended oral dose in humans. The dose of midazolam and felodipine in rats was referred to the doses in published literature . Forty rStyrax  or soybean oil  was administered orally. After 30 min, midazolam/felodipine was administered orally and intravenously. For oral midazolam groups, blood samples were collected at 5, 10, 20, 30, 60 min, 2, 3, 4, 6, 9 h. For intravenous midazolam groups, blood samples were collected at 2, 5, 10, 20, 30, 60 min, 2, 3, 4, 6, 9 h. For oral felodipine groups, blood samples were collected at 10, 30, 60 min, 3, 4, 6, 8, 10, 24 h. For intravenous felodipine groups, blood samples were collected at 2, 5, 10, 15, 30, 45, 60 min, 4, 6, 10 h. Then blood samples were centrifuged at 8,000 rpm, 4 °C for 10 min, the supernatant was stored at −80 °C until analysis . 50 μl pNine Male Sprague-Dawley rats (250–300 g) were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd.  and were fed in Experimental Animal Center of SHUTCM. All animals were housed under controlled environmental conditions  for 1 week with free access to food and water. The rats were fasted overnight before dosing but were free to drink water. Nine rats were randomly divided into three groups . Styrax (100 mg/kg) was administered orally, then nine rats were anesthetized by pentobarbital after administrating 1, 2, and 4 h, respectively. Blood samples were collected from the abdominal aorta, and the tissues  were removed rapidly and placed on ice. After then, the blood samples were centrifuged at 8,000 rpm, 4 °C for 10 min, and the supernatant was stored at −80 °C until analysis.Firstly, each tissue  was accurately weighed after sucking the water on tissue surface. Each tissue was put into an EP tube and then cut (3-5 times) by a scissor. After then, PBS (pH 7.4) and magnetic beads were added into EP tube, grind the tissue by a tissue grinder. The tissue grinding fluid was centrifuged at 9,000 g, 4 °C for 20 min. 50 μl tissue supernatant was diluted by adding 250 μl LC grade acetonitrile. The protein was precipitated by centrifuging at 20,000 ×g, 4 °C for 30 min and the supernatant was collected to determine the protein concentration by a BCA Protein Quantification Kit. After then, the concentrations of seven PTAs were determined by using UHPLC-Q-Orbitrap HRMS analysis. Mass spectrometry parameters were described in 50) was determined as the concentration of Styrax/PTAs at the catalytic activity of CYP3A was inhibited by 50% compared to the negative control (solvent only). In this study, all assays were tested in triplicate, while the data were shown as mean ± standard deviation (SD). IC50 values were fitted by GraphPad Prism 6.0 . Meanwhile, the following formula was used to calculate the residual activities of CYP3A:The dose-inhibition curves of Styrax/PTAs against CYP3A were depicted by using different dosages of Styrax/PTAs, while the half maximal inhibition concentration  = /the Peak Area or Fluorescence value of the metabolite in negative control (solvent only) × 100%.Firstly, with the help of fluorescence-based high-throughput CYP3A4 inhibition assay, the inhibitory potentials of more than 100 herbal medicines against CYP3A4-catalyzed NEN 4-hydroxylation were assayed in HLMs by using a single dose . As shown in 50 values of 3.72 μg/ml, 2.84 μg/ml, 2.90 μg/ml and 1.67 μg/ml, for HLMs, HIMs, RLMs, RIMs, respectively. These findings demonstrated that Styrax could potently inhibit mammalian CYP3A in liver and intestinal microsomes from both human and rat in vitro, with similar inhibition tendency and potency. However, it is still unclear that Styrax can inhibit hepatic CYP3A or intestinal CYP3A in vivo. Therefore, the in vivo pharmacokinetic interactions between Styrax and midazolam were carefully investigated by using two administrated ways (i.v. and i.g.).Next, the inhibitory effects of Styrax against mammalian CYP3A were validated by using a substrate-drug (midazolam) in different tissue preparations. To this end, the inhibitory effects of Styrax against CYP3A-catalyzed midazolam 1′-hydroxylation were carefully investigated in different enzyme sources including HLMs, HIMs, RLMs, and RIMs. As shown in in vivo effects of Styrax on the pharmacokinetics of midazolam (i.v. and i.g.) were carefully investigated in rats, by using soybean oil (without Styrax) as control groups. The blood concentration-time profiles of midazolam with two administrated ways  were shown in (0-inf) of midazolam (i.g.) was increased 2.04-fold (from 211.23 ng/mL·h to 431.20 ng/mL·h), while the Cmax value of midazolam (i.g.) in rats plasma was increased 2.28-fold (from 129.16 ng/ml to 294.05 ng/ml). Moreover, the metabolic half-life (t1/2) of midazolam (i.g.) was prolonged 2.11-fold (from 2.14 to 4.52 h). These findings clearly suggested that Styrax strongly elevated the plasma exposure of midazolam when midazolam was co-administered orally. Considering that both intestinal CYP3A and hepatic CYP3A participate in midazolam metabolism when this agent is administrated orally, while Styrax (i.g.) cannot influence hepatic metabolism of midazolam, suggesting that Styrax may modulate the in vivo pharmacokinetic behavior of midazolam (i.g.) via inhibiting intestinal CYP3A but not hepatic CYP3A.Next, the via two administrated ways (i.v. and i.g.). As shown in (0-inf)  and Cmax  of felodipine when felodipine was administered orally in combination with Styrax (i.g.) in rats. These findings further suggest that Styrax can modulate the pharmacokinetic behavior of orally administrated CYP3A-substrate drugs (midazolam and felodipine) by inhibiting intestinal CYP3A but not hepatic CYP3A in vivo.To further confirm that Styrax could influence the pharmacokinetic behavior of orally administrated agent, felodipine  was used as a victim drug to assess the pharmacokinetic interactions between Styrax  and felodipine Next, the chemical constituents in Styrax were analyzed by using UHPLC-Q-Exactive Orbitrap HRMS. The total ion chromatograms (TICs) of Styrax were shown in 50 values of 19.39, 32.20, 14.83, 7.74, 0.64, 2.51, and 0.99 μM, for maslinic acid, corosolic acid, oleanolic acid, betulinic acid, epibetulinic acid, betulonic acid and oleanonic acid, respectively. Meanwhile, all tested PTAs dose-dependently inhibited CYP3A4-catalyzed NEN 4-hydroxylation, with the IC50 values of 3.44, 41.93, 1.10, 4.11, 0.47, 0.96, and 0.80 μM, for maslinic acid, corosolic acid, oleanolic acid, betulinic acid, epibetulinic acid, betulonic acid and oleanonic acid, respectively. These results suggest that the PTAs in Styrax are key substances responsible for CYP3A inhibition, while epibetulinic acid, betulonic acid and oleanonic acid display potent CYP3A inhibitory effects.A previous study revealed that the PTAs in Styrax displayed potent inhibition on CYP3A-catalyzed testosterone 6β-hydroxylation . In this50 value of oleanonic acid against CYP3A4. These results suggested that the major PTAs in Styrax could be distributed in intestinal tract at relatively high exposure levels but their plasma and liver exposure in rats was extremely low. These findings well explain why Styrax (i.g.) significantly elevates the plasma exposure of CYP3A-substrate drugs only when these agents are orally administrated, and why Styrax cannot modulate the pharmacokinetic behavior of CYP3A-substrate drugs when these agents administered intravenously.Next, the tissue distribution  of seven PTAs from Styrax was determined in rats after single oral dose of Styrax (100 mg/kg). As shown in via acting on various therapeutic targets or prolonging the half-lives of active compounds in either Chinese medicines or Western medicines  and molecular mechanisms involved in the pharmacokinetic HDIs triggered by Styrax.As one of the most abundant drug-metabolizing enzymes (DMEs) in human liver and intestine, CYP3A4 plays a crucial role in the metabolic clearance and detoxification of a wide range of Western medicines and DDIs . In thisand DDIs . It is wand DDIs . In thesin vitro assays demonstrated that Styrax and its major constituents (seven PTAs) potently inhibited CYP3A-catalyzed oxidative metabolism of various substrates. In vivo assays showed that Styrax only affected the pharmacokinetic behavior of oral victim CYP3A-substrate drugs (midazolam and felodipine), which prompted us to carefully evaluate the tissue distribution of Styrax/its active constituents (seven PTAs) after oral administration of this herbal extract in rats. These results clearly showed that seven PTAs from Styrax could be exposed to the intestinal tract  at relatively high levels in rats after single oral dose of Styrax (100 mg/kg), but these PTAs were hardly exposed to the liver. The extremely low exposure of these PTAs from Styrax to mammal liver could be attributed to the poor oral bioavailability and the first-pass metabolism of PTAs  and paxlovid (nirmatrelvir and ritonavir) (in vivo. Considering that hepatic CYP3A plays an important role in endogenous metabolism (such as bile acids and hormones) and the metabolic clearance of toxins, potent inhibition of hepatic CYP3A may lead to serious consequences  (It should be noted that some CYP3A4 potent inhibitors (such as ritonavir) have been approved by FDA in combination with CYP3A4-substrate drugs to slow down the metabolic clearance of some important CYP3A4-substrate drugs tonavir) . Thus, tabolism) . In thisin vitro and to accurately assess the potential HDIs risk in vivo. With the help of fluorescence-based high-throughput hCYP3A4 inhibition assay, the Chinese herb Styrax was found with the most potent hCYP3A4 inhibition in HLMs. In vivo pharmacokinetic assays demonstrated that Styrax  only affected the pharmacokinetic behavior of orally administrated CYP3A-substrate drugs (midazolam and felodipine). Further investigations demonstrated that seven PTAs in Styrax were key substances responsible for CYP3A inhibition, while these PTAs could be exposed to intestinal tract at relatively high exposure levels but the exposure levels of these PTAs in rat plasma and liver were extremely low. Collectively, our findings showed that the Chinese herb Styrax (i.g.) could significantly elevate the plasma exposure of orally administrated CYP3A-substrate drugs but hardly modulated the pharmacokinetic profiles of intravenously administrated CYP3A-substrate drugs, which could be attributed to the high exposure levels of the PTAs in Styrax in intestinal tract and the extremely low exposure levels in rat liver. All these findings clearly suggest that the tissue exposure of some key constituents in herbal medicines strongly affect their in vivo inhibitory effects on CYP3A4, which are very helpful for the clinical pharmacologists to better understand the pharmacokinetic HDIs triggered by herbal products that contains naturally occurring CYP3A4 inhibitors.In summary, we demonstrate a case study to efficiently find the herbal medicine(s) with potent hCYP3A4 inhibitory effect