The optimized process, under an inert atmosphere of nitrogen to avoid oxidation of the material, was established by systematically exploring the operating parameters of the VFD, along with the concentration of the dispersed starting material and the choice of solvent, which was a 1 : 1 mixture of isopropyl alcohol and water. There is also some fragmentation of the 2D material into nanoparticles ca 68 nm in diameter.MXene (Ti Substituting Al or Si with functional groups, T , either side of the sheets (Mn+1XnTx) imparts greater application of the MXene relative to MAX phases [MXenes are a unique class of two-dimensional 2D) material, first reported in 2011 D materia, batteriX phases . The worX phases . The banX phases ,13. TherX phases .2CTx type MXene in the absence of harsh chemicals or surfactant. The VFD microfluidic platform delivers mechanoenergy in the dynamic film. We hypothesized that it would be effective in exfoliating the 2D material, based on its success in exfoliating graphene and h-BN [The exfoliation (delamination) of MXenes provides unique material with different functional groups, microstructure and morphology, which impacts on electrochemical response of the material. The exfoliation of MXene requires harsh chemicals or surfactant in a number of different methods, as in sonication ,14, heatand h-BN –23. The and h-BN .Figure θ, of −45° to 90° relative to the horizontal position, with +45° being optimal for a large number of applications, including in the present study. The VFD has two types of processing—confined mode and continuous flow. The confined mode is where a finite volume of liquid is placed in the glass tube which is tilted at 45° and spun at high speed for a designated period, figure 1b. This mode of operation of the VFD has proved invaluable in optimizing any processing in translating it into continuous flow where liquid is delivered to the inside of the rotating tube at a controlled flow rate, figure 1c [h-BN [The vortex fluidic device (VFD) ,24–26, figure 1c . The uti1c [h-BN –23, cove1c [h-BN , protein1c [h-BN , enhanci1c [h-BN ,30, prob1c [h-BN , protein1c [h-BN  and more1c [h-BN –37.2.2.1.2AlC powder precursor (MAX) was obtained commercially . The etching of the aluminium from Ti2AlC was conducted in a 20% aqueous hydrofluoric acid (HF) solution (Sigma Aldrich) for 24 h at room temperature. The resulting suspension was filtered and washed with deionized water (DI) to reach a pH > 6 [Tia pH > 6 . This as2.2.−1) was then delivered using a jet feed to the base of the rapidly rotating 20 mm OD glass tube in the VFD with another jet feed delivery a low flow rate of dry nitrogen gas. The tube was inclined at 45° and spun at a pre-determined speed, which was optimized at 4000 r.p.m., with the optimized flow rate at 0.5 ml min−1, resulting in ca 7% exfoliated MXene and fragmentation to nanoparticles, exiting the tube. We refer to the exfoliated MXene and MXene NPs exiting the tube during continuous flow processing as ‘collected’. After 1 h of processing (30 ml passed through the VFD), 3.5 ml remained in the tube which was added to 3 ml of a 1 : 1 mixture of IPA and water. The mixture was centrifuged (700g) for 3 min to separate large MXene particles. The supernatant afforded ca 8% exfoliated MXene sheets and MXene nanoparticles. This material which builds up in the VFD during the continuous flow processing is referred to as ‘retained’ material.MXene was dispersed in distilled IPA and Milli-Q water using sonication . The mixture was then transferred to an in-house developed magnetically stirred syringe, 2.3.λexc = 532 nm), XRD , ATR-FTIR Perkin Elmer Frontier, TEM (FEI Tecnai F20 operated at 120 kV), UV–Vis spectrophotometer Varian Cary 50 EST 70772 and dynamic light scattering (DLS) .MXene sheets and MXene nanoparticles were characterized using scanning electron microscopy (SEM) (Inspect FEI F50 SEM), atomic force microscopy (AFM—Nanoscope 8.10 tapping mode), Raman spectroscopy , IPA, DMF (dimethylformamide), o-xylene, m-xylene and toluene. However, their use resulted in little or no exfoliated MXene or resulted in the formation of nanoparticles. The same was also found for combinations of solvents, in a 1 : 1 ratio, including NMP with water, DMF with o-xylene, DMF with toluene, DMF with IPA, toluene with IPA, m-xylene with IPA, o-xylene with IPA and IPA with water, for different speeds and different concentrations, electronic supplementary material, figures S1 and S2 [in situ in the VFD was explored using a Nd:YAG pulsed laser operating at 1064 nm at different power settings, as another parameter to enhance the exfoliation, using a mixture of IPA and water with a volume ratio of 1 : 1, electronic supplementary material, figure S3. Varying the tilt angle θ was also explored, additional to the common tilt angle of +45°, including 20°, −20° and −45°, while varying the rotational speed of the tube, electronic supplementary material, figure S4. We note that −45° tilt angle was effective in exfoliating h-BN in water [The VFD is a flexible processing platform, with a number of operating parameters  [A colour change was observed for the suspension of MXene (from dark grey to yellow) after processing in the VFD in air, which is not surprising, given that the material is sensitive to air, being readily oxidized ,17. Thuse S14(a) .ca 7% of the starting material. The two materials were recalcitrant towards separation by centrifugation. Rotational speeds of 5000, 6000, 7000 and 8000 r.p.m. gave little exfoliation at the ubiquitous optimal 45° tilt angle, electronic supplementary material, figures S8, S9 and S14(b). The optimal flow rate of MXene in IPA and water was 0.5 ml min−1, with a lower flow rate, 0.25 ml min−1, and a higher flow rate, 0.75 ml min−1, not as effective. For all these flow rates, the concentrations of MXene in IPA and water (1 : 1) were varied, at 0.1, 0.25, 0.5 and 1.0 mg ml−1. The optimal concentration was found to be 0.5 mg ml−1, with other concentrations resulting in lower yields, electronic supplementary material, figure S14(c). Thus, the overall optimum processing parameters for generating exfoliated MXene in a single pass in the VFD are a concentration of the material in a 1 : 1 ratio of IPA and water at 0.5 mg ml−1, flow rate 0.5 ml min−1 and 4000 r.p.m. rotational speed. In addition, after VFD processing under continuous flow for 1 h, an additional ca 8% of both exfoliated MXene and MXene nanoparticles were collected from the tube (retained), electronic supplementary material, figure S15. We initially used SEM images of drop cast material on silicon wafers to ascertain the effect of varying the rotation speed, flow rate and tilt angle of the VFD, along with the choice of solvent, for the highest degree of the exfoliation of MXene. We established the structure of MXene (retained) using ATR-FTIR spectra, Raman spectroscopy, powder X-ray diffraction (PXRD) and UV–Vis spectroscopy, We found that the confined mode of operation of the VFD gave little exfoliation of the MXene, unlike continuous flow, and its use was adhered to for all subsequent experiments, electronic supplementary material, figure S7. This included varying the rotational speed, flow rate and concentration of MXene in the 1 : 1 mixture of IPA and water. We found 4000 r.p.m. gave the highest level of exfoliated MXene, with some fragmentation into small nanoparticles of the material; the combined exfoliated MXene and nanoparticles accounted for The shear stress in the VFD is effective in both exfoliating and fragmenting MXene. This is consistent with other established top-down nanomaterials syntheses in the device, covering exfoliating ,22 and sf shows backscattering SEM to determine the elements present in the product relative to the starting material, for different locations, figure 4g–i (collected) and electronic supplementary material, figure S10(a–d) (retained). TEM images established the presence of some MXene nanoparticles, figure 5a–d and electronic supplementary material, figure S11, with figure 6a,b providing additional TEM, and SEM images, respectively, of nanospheres. AFM images provided additional information on the shape of the nanospheres, in measuring their height. The results are in agreement with those from TEM and SEM . DLS of solutions of MXene nanoparticles in IPA and water (1 : 1) generated in the VFD after processing gave the average diameter of nanospheres as 68 nm, figure 6d. SEM images established the presence of exfoliated MXene, figure 4b–e, electronic supplementary material, figure S10(e–l), with AFM images establishing the mean thickness of VFD-exfoliated MXene sheets as 3 nm, figures 8c, electronic supplementary material, figure S12.Figure 4e–h, along with the size of the spheres, electronic supplementary material, figure S8(a–e). However, the spheres were found to be unstable, with the drop cast spheres collapsing after 3 days. Interestingly, the spheres have holes in them, electronic supplementary material, figure S8, but the origin of these is unclear.The TEM images were used to determine the size and number of MXene sheets generated using the VFD processing, figure 5−1 for Ti–O and C–F, respectively [−1, corresponding to O–H, respectively, of solvent, figure 3a [−1. The material exiting the VFD tube has peaks at 263, 419 and 613 cm−1, and retained in the tube at 250, 405 and 613 cm−1. The slight shift of Raman peaks for exiting and retained material presumably relates to the presence of nanospheres, along with the exfoliated MXene, only for material exiting the tube, figure 3b [2CTx MXenes, which has hexagonal P63/mmc symmetry with a lattice constant a ≈ 0.3 nm [θ peaks  of MXene as prepared were 29.4°, 40.6°, 42.4° and 72.4°, with corresponding values for material exiting the tube 29.4°, 40.6°, 42.4° and 72.4°, figure 3c [figure 3d [ATR-FTIR for MXene has characteristic peaks, for material exiting the tube during processing in the VFD, and material retained in the tube, and as-prepared material, with peaks at 684 and 991 cmectively . Howeverigure 3a –45. Ramaigure 3b ,46. Powd≈ 0.3 nm ,48. The igure 3c ,50. The igure 3d .4.ca 3 nm thick, driven by the mechanoenergy in the dynamic thin film in the VFD. During this processing, we also established that the VFD can fragment the laminar MXene into nanoparticles (average diameter 68 nm). The optimized parameters for processing the MXene under an inert atmosphere of nitrogen gas were for a concentration of MXene at 0.5 mg ml−1 in a 1 : 1 mixture of IPA and water, with a rotational speed of 4000 r.p.m. and flow rate of 0.5 ml min−1. The exfoliated material and nanoparticles of the material have potential in applications such as environmental remediation [We have developed a continuous process for exfoliating 2D-MXene into multi-layered sheets ediation , biologiediation ,10, supeediation  and nextediation  Understa


Cu–Cr-based alloys exhibit excellent electrical conductivity and strength, but their poor thermal stability limits their application in industry. In this paper, Cu–0.2Cr (at. %) and Cu–0.2Cr–0.12Ag (at. %) alloys were prepared to study the effect of Ag on the properties, microstructure, and thermal stability of the Cu–Cr alloy. Microstructure and precipitation were observed by an optical microscope (OM) and a transmission–electron microscope (TEM). After cold-drawing by 99.9% and aging at 450 °C for 2 h, the peak hardness and electric conductivity of the Cu–Cr alloy were 120.3 HV and 99.5% IACS, respectively, and those of the Cu–Cr–Ag alloy were 135.8 HV and 98.3% IACS, respectively. The softening temperature of the Cu–Cr alloy was 500~525 °C, and that of the Cu–Cr–Ag alloy was about 550 °C. The creep strains of the Cu–Cr and Cu–Cr–Ag alloys at 40 MPa and 400 ℃ for 50 h were 0.18% and 0.05%, respectively. Ag elements improved the thermal stability of the Cu–Cr alloy. Recovery and recrystallization occurred before the coarsening of precipitates during the softening process. Ag atoms mainly improved the softening resistance of the alloy by delaying recrystallization, and mainly increased creep resistance by preventing the increase in mobile-dislocation density. Cu–Cr-based alloys are typical precipitation-strengthening alloys with good electrical conductivity and mechanical properties, and they are used in integrated–circuit lead frames, connectors, and electrical equipment ,2,3,4,5.Liu et al.  reportedIn this paper, the mechanical properties, softening properties, and creep properties of Cu–0.2Cr (at. %) and Cu–0.2Cr–0.12Ag (at. %) were tested. Microstructure evolution during the softening and creep process was analyzed, and the mechanism of Ag on the mechanical properties and thermal stability of the Cu–Cr alloy was explored.2 F20  electronic microscope. The samples for transmission-electron-microscope (TEM) observation were prepared by electrolytic polishing; the ratio of nitric acid to methanol was 1:4.Hardness was measured by a 430 SVD-type Vickers hardness tester  with 5 kg loading and a holding time of 15 s. Electrical conductivity was tested by a Sigma 2008 digital eddy current conductivity meter with 500 KHz. Each value was measured 5 times, and the average value was taken. Creep properties were measured by an RD-50 high-temperature creep-testing machine. The microstructure and precipitates of the alloys were observed by an Axiovert 200MAT  optical microscope (OM) and FEI Tecnai GThe creep curves of both alloys at 400 and 500 °C are shown in Cu direction, respectively. Two sets of diffraction spots occurred near the Cu matrix spots. One set was the diffraction of 0°, 30°, and 60° to the <011>Cu direction of the matrix, and the other was a circle of diffraction spots near the center. According to previous studies [Cu in the matrix were the Cr phase with a bcc structure. Therefore, precipitates with Moiré fringe contrast were bcc Cr, according to the morphology characteristics and distribution orientation of precipitates in Cr‖<110>Cu existed between Cr phase and matrix, namely, an N–W orientation relationship. Many intertwined dislocations were found in the Cu–Cr–Ag alloy, as shown in  studies ,24, diffThe TEM microstructures of both alloys at different annealing temperature levels for 1 h are shown in The hardness of the Cu–0.2Cr alloy was improved by 0.12 at. % Ag, shown in The recovery, recrystallization, and coarsening of precipitates occurred in the softening process of the test alloys, as shown in The addition of 0.12 at. % Ag elements also improved the creep resistance of Cu–0.2Cr . The creThe addition of 0.12 at. % Ag increased the hardness of the Cu–0.2Cr alloy and slightly decreased electrical conductivity. The peak hardness of the Cu–0.2Cr alloy was 120.3 Hv after aging at 450 °C for 2 h, while that of the Cu–Cr–Ag alloy reached 135.8 Hv after aging at 450 °C for 4 h. Electrical conductivity corresponding to the peak hardness of the Cu–0.2Cr and Cu–0.2Cr–0.12Ag alloys was 99.5% and 98.3% IACS, respectively.The softening resistance of the Cu–0.2Cr alloy was improved by 0.12 at. % Ag elements. The softening temperature of the Cu–0.2Cr alloy was 500~525 °C, and that of the Cu–0.2Cr–0.12Ag alloy was about 550 °C. The softening process was accompanied by the recovery, recrystallization, and coarsening of precipitates. Recovery and recrystallization occurred prior to the coarsening of precipitates. Ag elements inhibited the recrystallization of the Cu–Cr alloy and had little effect on the precipitates.The creep strains of the Cu–0.2Cr and Cu–0.2Cr–0.12Ag alloys at 40 MPa and 400 °C for 50 h were 0.18% and 0.05%, respectively. The creep strain of the Cu–0.2Cr alloy was significantly greater than that of the Cu–0.2Cr–0.12Ag alloy at 500 °C. Alloy creep was accompanied by secondary precipitation of the Cr phase, dislocation slip, and recrystallization. The addition of 0.12 at. % Ag increased the creep resistance of the Cu–0.2Cr alloy mainly by hindering the increase in mobile dislocation density.This work studied the mechanical properties, thermal stability, and microstructure of the Cu–0.2Cr (at. %) and Cu–0.2Cr–0.12–Ag (at. %) alloys. Results are summarized as follows:


Acanthopanax trifoliatus (L.) Merr. (A. trifoliatus) belongs to the family Araliaceae, which is called “Le Cai”, and is an indigenous plant to Guangdong Province that has been prevalently planted for years. A. trifoliatus is used in folk medicine and has ginseng-like activity. Kaurenoic acid ((−)-kaur-16-en-19-oic acid, KA) is a kaurane-type diterpenoid that is regarded as a major compound in A. trifoliatus. Early studies have reported the determination of KA by HPLC capillary electrophoresis. However, KA could not be completely separated from other components in the plant extract by HPLC because of their similar molecular structures and physical and chemical properties. UHPLC-MS/MS could be a useful tool to identify and quantify KA. In the present work, a UHPLC-ESI-MS/MS method for determining KA in A. trifoliatus was developed and validated. KA was extracted from lyophilized A. trifoliatus leaves by ultrasound-assisted extraction and further purified by solid phase extraction (SPE). KA was quantified and separated on an Accucore C18 LC column. Mass spectrometry with multi-reaction monitoring (MRM) and quantitative fragment ion/product ion (m/z: 301.3/301.3) in ESI negative mode was used for quantification. The intra-assay and inter-assay relative standard deviation (R.S.D.) were 2.8% and 3.2%, respectively. The inter-person R.S.D. on the same day was 3.6%. The inter-instrument R.S.D. with the same model on the same day was 2.9%. The recoveries evaluated upon spiking three different concentrations of KA were above 97%. A minor matrix effect of 94% was observed. This method has been applied successfully for the determination of KA in A. trifoliatus leaves. Acanthopanax is a plant genus that embraces 18 plant species that are mainly distributed in Asia and the far-eastern region of Russia2. One of the Acanthopanax species is A. trifoliatus, which is called “Le Cai” in Chinese, and is an indigenous plant in Enping, Guangdong Province that has been prevalently planted for years. The planting area has reached 420 hectares, and the total value is worth approximately 120 million. A. trifoliatus, with ginseng-like activity, has been used in folk medicine in southern China since old time to treat sinew, bone pains, rheumatism, bruises, neuralgia, impotence, gout, hepatitis and diabetes5. In Cambodia, Laos, and Vietnam, A. trifoliatus has been used to treat nervous affections and improve memory7. It has also been shown to have a good curative effect on the common cold, jaundice, gastric pain, diarrhea and ulcers8. A decoction of leaves, young shoots or the bark of A. trifoliatus has been used to treat tuberculosis and lung hemorrhages9. Some studies have indicated that extracts of A. trifoliatus possess several beneficial biological effects, including anti-inflammatory, immunostimulatory, and antioxidant properties, protein tyrosine phosphatase inhibitory activity and cytotoxic activities towards some types of cancer cells12.A. trifoliatus are popularly consumed as vegetables in traditional southern Chinese cuisine that can be stir-fried or cooked in a soup13. In addition, the local people of southern China have been using the leaves of this plant to make tea for daily consumption for at least 10 years for daily health management. This plant is also an ingredient in ‘leicha’, a traditional Chinese herbal tea that is believed to have a powerful tonic effect14. A. trifoliatus is usually planted from March to October and is a rigid shrub 1 ± 0.4 m high. It is spiny with alternate palmate leaves and toothed margins, and it has a typical crisp flavor with some bitterness15.Moreover, the young leaves and shoots of 17. Of these components, KA has been reported as an important ingredient in A. trifoliatus for its potent biological activities20. KA is a kaurane-type diterpenoid and is regarded as a key intermediate in the biogenesis of gibberellins and other phytohormones that regulate the growth and development of higher plants and some fungal metabolites22. The structure of KA is shown in Fig. The leaves of this plant contain a diverse range of components, including diterpenoids, lignans, triterpenoids, polyacetylenes, phenylpropanoids, and flavonoidsA. trifoliatus could be very important. Several reports have determined KA contents by HPLC or GC. Early studies found that KA could not be completely separated from other components in plant extracts by HPLC because of its similar molecular structure and physical and chemical properties24.Therefore, for the effective utilization and quality control of natural resources, analysis of KA in A. trifoliatus to use the leaves as an effective medical resource. Although the determination of KA by LC-MS/MS had been developed in early reports, this is the first fully validated method in terms of extraction, UHPLC-MS/MS conditions, linearity, precision, accuracy, limit of detection (LOD) and limit of quantitation (LOQ).UHPLC-MS/MS could be a useful tool to identify and quantify KA. In this study, a UHPLC-MS/MS method was developed for the quantitative determination of KA in the leaves of A. trifoliatus leaves were extracted in 100 mL of 100% methanol by ultrasonic extraction for 15, 30 or 60 min, and the determined concentrations were 4.1 ± 0.3, 4.2 ± 0.3 and 4.2 ± 0.0 μg/mg solid, respectively. From these results, increasing the ultrasound treatment time slightly increased the extracted components but there was no significant difference after applying the ANOVA post-hoc test. Additionally, the R.S.D. of these three treatment times was 2.8–3.4%. Therefore, a 15 min ultrasound time was chosen for further analysis.To investigate the influence on ultrasound time on the recovery of KA, 0.1 g of post-hoc test, there was no significant difference between the four exaction amounts. Therefore, to decrease the matrix effect, 0.1 g of sample was chosen in this study.Different sample extraction amounts were also optimized, including 0.1, 0.2, 0.3 and 0.5 g, and the corresponding results were 4.0 ± 0.8, 4.1 ± 0.0, 4.2 ± 0.1 and 4.1 ± 0.1 μg/mg solid, respectively. The content of KA by weighting different sample extraction amounts were shown in Supplement 18 UHPLC column  and a Zorbax Eclipse Plus C18 column . The retention times of KA on the Accucore and Zorbax columns were 5.467 and 4.166 min, respectively. Standard curves were constructed by using these two columns. The obtained standard curves were Y = 136.7X + 8.1  and Y = 226.7X + 86.8 , where X: concentration (ng/mL) and Y: peak area. Thus, a much better linear relationship was obtained by using the Accucore column. This is probably because this column is longer than the Zorbax column, which gave good separation from the interfering substances to decrease the matrix effect and achieve higher reproducibility. Therefore, the Accucore column was chosen for further study.In this study, UHPLC conditions were mainly optimized in terms of the type of column, column temperature, flow rate and solvent effect. Two different UHPLC columns were tested with regards to method reproducibility, including an Accucore Cpost-hoc test. Therefore, from our results, 0.4 or 0.45 mL/min could be chosen as the flow rate, and we used 0.45 mL/min as the flow rate. The base peak width was reduced to 0.12 min when using 0.45 mL/min as the flow rate. By reducing the peak width, this could be utilized to enable faster analysis times while maintaining resolution.Three flow rates were tested for their method reproducibility, including 0.35, 0.4 and 0.45 mL/min. Three UHPLC-MS/MS chromatograms were obtained as shown in Fig. Two different column temperatures were tested on method reproducibility, including 35 and 40 °C, and their corresponding UHPLC-MS/MS chromatograms are shown in Fig. 25. In this study, KA was extracted from A. trifoliatus leaves with 100% methanol. However, to improve the precision of the standard curve, 50% methanol was applied as a dilution solvent to prepare the standard solutions, and their corresponding UHPLC-MS/MS chromatograms are shown in Fig. As indicated previously, electrospray solvent has a significant impact on the chromatogram and mass spectra of tandem mass spectrometry26. Different collision energies were tested on KA from 0 eV to 40 eV and there was no unique fragments observed. Also the product ion with m/z 301.2 was the only ion detected when charging the collision energy from 0 to 40 eV on the Q2 which were shown in Supplement In this study, we applied MRM as our MS/MS function. Very uniquely, we selected ion m/z 301.3 on Q1 by applying the appropriate Q2 , and Q3 was obtained, but the product ion remained 301.3. This phenomenon indicated that KA lacked fragmentation under collision. Therefore, we chose the MRM transition as 301.3 > 301.3. These unique characteristics were also observed by another studyKA was detected in negative ion mode and was well-separated on the reversed phase column used. The KA molecule exhibited an adequately separate chromatographic peak that was easily distinguished from the baseline. Quantitation was performed based on retention time, which was compared with the authentic standard MRM transition.Method selectivity was assessed by analyzing a blank, a positive sample and a KA standard solution. Selectivity met the criterion because there were no interfering peaks observed at the same retention times as the target analytes.2. For the equation Y = 136.7X + 8.1, X is in the range of 8 ng/mL~160 ng/mL, the weight is 1/x, and R2 = 0.9995. No unacceptable carryover was observed. Homoscedasticity should be tested in any linear regression analysis, especially for an analytical method in which the concentration range is broad27. By constructing the residual vs. concentration plot and applying the F-test, our calibration data did not meet the homoscedasticity requirement because the residuals are not randomly distributed. Thus, a weighted calibration curve should be constructed. Two different weighting factors (1/x and 1/x2) are compared, and the data are shown in Table 2, and thus, the 1/x weighting factor was chosen.The calibration curve showed an acceptable linearity range and R(S/N) ratio was over 10 and bias (%) in accuracy and precision were below 20%. Lower concentration (6 ng/mL) was tested and found the precision was 35% and S/N < 10. Therefore LOQ was considered as the lowest calibration standard and be quantified reliably. The LOD was determined by S/N ratio of 3 and found LOD was 1.3 pg. These results were lower than those from an early report26.The LOQ was determined as 4 pg which was obtained by injection 0.5 μL of 8 ng/mL KA solution. At this concentration, signal to baseline noise 28.Intra-day, inter-day and inter-person method repeatability were evaluated for 9 replicates. The obtained KA in the extract for the intra-day analysis by the same operator was 4.1 ± 0.1 μg/mg solid, and the R.S.D. was 2.8%. The obtained KA in the extract for the inter-day analysis by the same operator was 4.0 ± 0.1 μg/mg solid, and the R.S.D. was 3.2%. The values of KA concentration from operator 1 and operator 2 were 4.0 ± 0.1 μg/mg solid and 4.2 ± 0.1 μg/mg solid, respectively. The R.S.D. of inter-person was 3.6%. The KA in the extract obtained by different instruments of the same model (Agilent 6460) on the same day was 4.1 ± 0.1 μg/mg solid, and the R.S.D. was 2.9%. Therefore, all precision values were below 15%, which met the EMA guideline requirements28. A minor matrix effect of 94% was observed with the extract as the matrix. MAX SPE purification significantly decreased the matrix effect. The accuracy and matrix effect results were shown in Table Accuracy was expressed as the recovery, determined by analyzing 3 replicates of three quality controls (QCs) spiked in the sample for extract. The three quality controls (QCs) were prepared from the KA standard stock solutions corresponding to 3 points, a low QC, medium QC and high QC. The final concentrations of three QCs were 1.81, 3.62 and 6.03 μg/mg solid. The recoveries (%) were 99.2 ± 8.0, 99.7 ± 2.7 and 97 ± 5.9. The detailed results about accuracy test were shown in Supplement A. trifoliatus leaves after purification by a SPE cartridge. After the implementation of an ultrasound-assisted extraction, this UHPLC-MS/MS method was validated in-house with the plant extract demonstrating good recovery rates above 97% and good R.S.D. This method showed very good selectivity and sensitivity with a LOD of 1.3 pg and a LOQ of 4 pg. The method has been applied successfully in the determination of KA content in A. trifoliatus leaves, which can reach 0.4% (4 μg/mg solid).In this study, a UHPLC-MS/MS method was developed as a new assay procedure and was evaluated and applied for specific quantitation of KA content in A. trifoliatus leaves were picked in April 2018 from a farm in Enping, Guangdong Province and shipped to Jinan University on the same day under refrigerated conditions. Upon arriving at the lab, the fresh leaves of A. trifoliatus were washed, dried in an air flow, ground in liquid nitrogen, lyophilized for 2 days and then powdered with an electronic mill (20-mesh sieve). The powder was kept in centrifuge tubes sealed with parafilm and reserved in dry and cool conditions until analysis.LC-MS grade water , acetonitrile , methanol  and formic acid  were obtained from Sigma-Aldrich . The KA standard with a purity over 98% was obtained from the National Drug Reference Standards Center . A. trifoliatus Merr. was optimized for plant leaf powder in terms of sample mass and ultrasound-assisted extraction time. A set of three replicates of plant leaf powder were weighed into a volumetric flask with a cap, and 100 mL methanol was added as the extraction solvent. The mixture was first vortexed and then sonicated for a certain amount of time, including 15, 30 and 60 min. An ultrasonic water bath  of 500 × 300 × 150 mm) was utilized for this purpose. After sonication, the mixture was cooled to room temperature and then passed through vacuum filter paper , and the supernatant was recovered. Different amounts of powder were tested for extraction, including 0.1, 0.2, 0.3 and 0.5 g. Ultrasound-assisted extraction times included 15, 30 and 60 min. The 5 mL of extract was evaporated completely using a vacuum evaporator centrifuge , dissolved in 10 mL of water and cleaned up by solid phase extraction (SPE).The extraction procedure of KA from 4OH in water and dried for 5 min under a stream of air. The analytes were then eluted with 10 mL of 2% FA in methanol and further filtered through a 0.22 μm nylon filter before injection into the UHPLC-MS/MS. The extract was diluted 80-fold to obtain an extract concentration in the range of the linear standard curve.The KA extract was further cleaned up by an Oasis MAX  cartridge column for acidic compounds. The Oasis MAX cartridge was first activated and conditioned with 10 mL of methanol and 10 mL of Mill-Q water. Then, 0.25 mL of the extract was applied to the cartridge. Next, the column was washed with 10 mL of 5% NH18 LC column  and a Zorbax Eclipse Plus C18 column . Different flow rates for KA recovery and method precision were tested, including 0.35, 0.4 and 0.45 mL/min. KA chromatographic separation was performed using the following gradient elution: 0–1 min, 90% A; 1–3 min, 90% A to 60% A; 3–4 min, 60% A-10% A; 4–6.5 min, 10%, then return to the initial conditions and equilibrate the column for 1 min. The injection volume was 0.5 μL, the autosampler was kept at 20 °C and column temperature was investigated at 35 °C and 40 °C.Chromatographic separation of KA from the other matrix components was carried out by reversed phase chromatography on an Agilent 1290 Infinity UHPLC equipped with a binary pump, autosampler, column oven and degasser . The mobile phase was constituted as follows: 0.1% FA in water (solution A) and acetonitrile (solution B). For the determination of KA, different chromatographic columns were tested in this study, including an Accucore CThe UHPLC eluate was interfaced with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Corp.) operating in negative ion electrospray mode. An Agilent Mass Hunter workstation was used for control of the equipment, data acquisition and analysis. For optimization of the MS/MS parameters, a KA standard solution prepared in methanol was infused into the mobile phase (0.5 mg/mL) at a flow rate of 20 μL/min using a syringe pump  to tune the instrument. Nitrogen was used both as the drying gas at a flow rate of 11 L/min and as the nebulizing gas at a pressure of 45 psi. The drying gas temperature was 300 °C, and a potential of 4 kV was applied across the capillary. Finally, the instrument was operated with a fragmentor voltage of 140 V. Acquisition was performed by MRM, wherein the respective pseudomolecular anion of KA was fragmented by collision-induced dissociation into a fragment for detection. The MRM transition and the collision voltage were optimized, and the value of the collision energy for the qualitative and quantitative ion (301.1) was 30 eV. The dwell time was 100 ms.28.The developed method was validated based on the EMA guidelines, including selectivity, carryover, limit of detection and limit of quantification, linearity, repeatability, precision, accuracy and matrix effectA. trifoliatus leaves. A 1/x weighted linear regression was applied so that the back-calculated concentration should be within 15% of the theoretical concentration.(1) KA stock solutions were prepared by fully dissolving 1 mg of KA into a 10 mL brown volumetric flask in methanol. The concentration of the stock solution was 0.1 mg/mL. The stock solution was completely mixed and stored at −80 °C. (2) KA intermediate standard solutions were prepared by pipetting 1 mL of the stock solution into a 25 mL brown volumetric flask and bringing up to volume with 50% methanol. The final concentration of the intermediate standard solution was 4 μg/mL. The intermediate standard solutions were also stored at −80 °C. (3) The working solutions of KA were prepared by pipetting 20 μL, 40 μL, 100 μL, 200 μL and 400 μL of the intermediate solutions into a 10 mL brown volumetric flask and bringing up to volume with 50% methanol. The final concentrations of the working solutions were 8, 16, 40, 80 and 160 ng/mL. The ranges of the calibration curves were determined from the preliminary analysis of KA in 25. In this study, KA was extracted from A. trifoliatus leaves with methanol as the extraction solvent. However, to improve the precision of the standard curve, 50% methanol was applied as the dilution solvent to prepare the standard solutions. Therefore, the chromatograms of KA in methanol and 50% methanol were compared in terms of peak shape, peak area and retention time.An early study discovered that chromatograms and mass spectra may not only be dependent on careful control of instrumental parameters such as tuning conditions, but also on the electrospray solventS/N ratio reached to 10 and 3, respectively.The LOQ was considered as the lowest concentration of analytes that can be quantified reliably so that the bias (%) in accuracy and precision were below 20%. A signal-to-ratio of 3 and 10 is used for estimating LOD and LOQ, respectively. The LOQ and LOD were determined by diluting the KA standard solution until the The selectivity refers to the method that can differentiate the target analyte from other matrix components. This was assessed by analyzing negative samples, positive samples and KA standard solutions. The presence of possible endogenous or exogenous interferences was verified by monitoring the MRM chromatogram. The MRM chromatogram was specific for KA at its expected retention time. The acceptance criterion for the selectivity was that the peak area of interference should be below 20% of the peak area of the LOQ of KA.Carryover was evaluated by injecting blank samples after the run of the highest calibration concentration. The acceptance criteria for carryover were the same as for the evaluation of selectivity.To assess the robustness of the method, 9 replicates of the sample extraction were prepared by the same operator under the same instrument on the same day.The precision was evaluated by the precision of 2 different operators on 3 nonconsecutive days, and by two instruments of the same model with 9 replicates of sample extraction.The accuracy was expressed as the recoveries, determined by analyzing 3 replicates of three quality controls (QCs) spiked in the sample for extract. Three quality controls (QCs) were prepared from the KA standard stock solutions with 3 points, low QC, medium QC and high QC. The final three QCs were 1.81, 3.62 and 6.03 μg/mg solid, which corresponded to 0.5-, 1- and 1.5-fold the KA concentration in the plant extract. 0.5 mL individual QC stock solution  was spiked in the same amounts of samples and gone through ultrasound-assisted extraction, concentration and SPE clean-up. The final extracts were determined in the same day. The accuracy (recovery) was calculated with the following equations:A: KA concentration in sample extracts spiked with individual QC standard solution; B: KA concentration in sample extracts; C: nominal value of KA in QC standard solution. The KA concentration obtained from A and B were calculated against the calibration curve.The matrix effect was also determined for the method. The sample matrix, such as coeluting compounds, can contribute to alterations in the analyte ionization and overall response. Therefore, complete separation between the analyte and the coeluting matrix could help to decrease or increase the ionization of the target analyte. The matrix effect was determined with the following method. The volume of 10 μL QCs  were spiked into 1 mL post sample extract. The peak area obtained in the matrix was compared with the corresponding peak area in the extraction solution with the same concentration. Each sample was analyzed three times. The matrix effect was calculated as follows:A: mean peak area in the matrix after spiking the QCs; B: endogenous KA peak area in the matrix before spiking the QCs; and C: mean peak area of three QCs in the extraction solution.Supplementary information.


Pseudomonas aeruginosa can produce various virulence factors and form biofilms. These processes are controlled by the quorum sensing (QS) system. Sodium new houttuyfonate (SNH) is an adduct of houttuyfonate, the main component of the common Chinese medicine plant Houttuynia cordata, which has antibacterial and anti-inflammatory effects. We evaluated the effect of SNH on P. aeruginosa biofilms, virulence factors, and transcription. Transcriptome analysis showed that the key rhlI and pqsA genes of the P. aeruginosa QS system were down-regulated after SNH treatment. SNH reduces proteases and pyocyanin production and inhibits biofilm formation by regulating the P. aeruginosa QS system. SNH also changes the expression of genes related to virulence factors and biofilms . These results suggested that the mechanism of SNH against P. aeruginosa by affecting the expression of biofilm and virulence factors controlled by quorum sensing.As a major opportunistic pathogen,  Pseudomonas aeruginosa is a common clinical opportunistic pathogen  is a cellular communication mechanism that depends on the concentration of signal molecules . There aruginosa , the Lasruginosa , whose tbiofilms . At an aHouttuynia cordata is a traditional Chinese medicine, which is derived from Saururaceae. H. cordata has the functions of antibacterial, antiviral, improving immunity, diuresis and so on. And it is one of the varieties officially identified medicine and food by the Ministry of Health . Sodium new houttuyfonate (SNH) is a sodium bisulfite adduct of the active ingredient of the Chinese herbal medicine H. cordata . PDP medium was acquired from Qingdao Hi-Tech Park Haibo Biotechnology Co., Ltd. . A single ATCC 27853 colony was selected from Luria-Bertani (LB) solid medium  and cultured in 5 ml LB liquid media  at 37°C, with shaking at 220 r/min for 24 h, then by centrifugation at 10,800 g for 1 min. The supernatant was discarded and the pellet was resuspended in sterile saline solution for optic density detection at 600 nm(OD600) in a UV spectrophotometer. The absorbance of cell suspensions was adjusted to 0.08–0.1, and diluted to 10^7 times for later use for further experiments.SNH was purchased from Shanghai Yuanye Biological Technology Co., Ltd. , with the content ≥ 98%. Azithromycin (AZM) was acquired from the Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd. . The minimum inhibitory concentration (MIC)  and mini600 (SNH solutions (100 μl) were prepared to final concentrations of 2,048, 1,024, and 512 μg/ml. Equal volumes of SNH bad bacterial solutions were added to 96-well plates, and the control groups established. Plates were incubated at 37°C, the OD600  measuredSNH (2 ml) and an equal volume of bacterial solution was added to the test tube to obtain a concentration of 1 x MIC SNH and the bacteria was cultured at 37°C for 24 h. The blank control group was the SNH treatment control group and no drugs were added. Centrifuge to remove supernatant, and rinsed three times with sterile water. The collected bacterial sample was placed in a centrifuge tube, sealed with sealing film, and placed directly onto dry ice to perform RNA-seq . For eacP. aeruginosa transcription was performed using the BGISEQ-500 platform (Huada Gene Technology). First, total RNA was processed and purified, and reverse transcription performed to synthesize cDNA which was then used to produce double-stranded DNA. The end of the synthesized double-stranded DNA was flattened and phosphorescence added to the 5′ end. The 3′ end then forms a sticky end protruding from an “a”, and a bubbly joint with a protruding “t” at the 3′ end is connected. PCR amplification is then performed using specific primers, PCR products thermally denatured into single strands, and the single-stranded DNA is circularized to obtain a single-stranded circular DNA library.SNH-induced sequencing of Data are obtained by transcription sequencing was counted using the SOAPnuke  filterinP-values calibrated for multiple tests were performed according to the Benjamini and Hochberg method. False discovery rate (FDR) threshold ≤ 0.05 and | log2fold change (FC)| >1 were defined as significant DEGs  were detected based in the method described by ant DEGs .P-value by FDR, and the Q-value ≤ 0.05 is considered significantly enriched. The major biological functions that DEGs perform can be evaluated by analysis of significant GO and KEGG pathway enrichment.DEGs were subjected to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis . In gene600 measured  to an OD600 of 0.8. The supernatants with different SNH concentrations and S. aureus suspensions were mixed at a ratio of 1:9, and OD600 was measured at different time points to calculate the activity of LasA Staphylococcus protease  was added to the test tube, mixed thoroughly and allowed to stand for 15 min. When the chloroform layer turned green they were transferred to another test tube. Then 1 ml of 1 mol/L HCL was added, mixed, and left to stand for 5 min. The OD520 was measured when the solution turned pink. The pyocyanin content was determined by: OD520 * 17.072  were prepared. The strain cultured for 24 h at 37°C and 220 r/min was taken from the shaker and the supernatant was centrifuged at 12,000 r/min, diluted to 0.5 M, and serially diluted 100 times. Using a pipette gun, 2 μl of bacterial liquid was inoculated onto the surface of the floating motion medium, and was placed at 37°C for 24 h to measure colony diameter  extracted following treatment with different SNH concentrations were incubated with 1 ml of skim milk (1.25%) at 37°C for 20 min, and their ODmeasured . The oveprotease . Bacteri* 17.072 . In a wodiameter .SNH solution (2 ml) with final concentrations of 2,048, 1,024, and 512 μg/ml and an equal volume of diluted bacterial solution were added to a 6-well plate and placed a sterile cover glass. The positive control AZM and blank control group were set up simultaneously. Plates were incubated at 37°C for 24 h, then the planktonic bacteria were washed 3 times on the slide with PBS. The samples were then placed in pre-cooled 2.5% glutaraldehyde solution at least 2 hours in the dark at 4°C, and placed in 30%, 50%, 70%, 90% and 100% ethanol for gradient dehydration for 10 minutes. After drying at room temperature, the biofilm samples were sprayed gold after vacuum drying, and observed and imaged (×5000) with Scanning Electron Microscope . Crystal violet (0.4%) was used for staining, 30% acetic acid was used for dissolution, and absorbance was measured at 540 nm as previously described .rhlI, pqsA, lasA, lasB, lecA, phzM, and pilG genes in the QS system was evaluated using PCR. Preparations of different concentrations of bacteria and drugs were used for coculture. In the control group, Mueller-Hinton Broth (MH (B)) medium was added instead of SNH. After culturing at 37°C for 24 h, the supernatant was removed and total RNA extracted from the bacteria using TIANGEN′s RNAprep Pure Cell/Bacteria Kit and following the manufacturer′s instructions. cDNA was reverse transcribed using TIANGEN′s FastQuant RT Kit. RT-PCR was performed using 2 x EasyTaq PCR SuperMix  and a GENETEST Series Gene Amplifier . The reaction involved 35 amplification cycles of: 94°C 5 min, 94°C 30 s, 56°C 30 s, 72°C 30 s, and 72°C 10 min. The synthesized DNA was electrophoresed on a 1.5% agarose gel, and then imaged on a gel imager. SYBR dye was used for qRT-PCR experiments and the 25 μl reaction system included 12.5 μl SYBR dye, 1 μl upstream primer and 1 μl downstream primer (10 μM), 0.5 μl cDNA, and 11 μl DEPC water. The reaction was performed on an Applied Biosystems 7,500 real-time PCR system (USA) and the reaction steps were: 95°C 15 s, 56°C 30 s, 72°C 30 s, for 40 amplification cycles. Reactions were gradient cooled at 95°C–60°C and the dissolution curve detected. rpoD was used as an internal reference, and the gene expression determined by 2-Δ Δ CT. Primer sequences are shown in Expression of Galleria mellonella larvae were obtained from Tianjin Huiyude Biological Technology Co., Ltd., with size of 2-3 cm and weight of 0.2-0.3 g. The larvae were stored at 4°C for later use. SNH and AZM were dissolved in PBS without any cosolvent to avoid the possible toxic effects of cosolvent against larvae. The treatment groups of larvae were set as follows: PBS group  + PBS group, infection + SNH 2048 μg/ml group, infection + SNH 1024 µg/ml group, infection + SNH 512 µg/ml group, infection + AZM 64 µg/ml group. The vivo assays were carried as previously described . In total, 5940 reference genes (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133428.) were assembled from sequencing data, with 5,937 (99.95%) known genes in the control group and 5,934 (99.9%) in the SNH treated group. Principal component analysis was based on FDR threshold ≤ 0.05 and | log2 fold change (FC) | > 1, and 1,674 significant DEGs were identified. Of these DEGs, 1,454 were down-regulated and the remaining 220 were up-regulated  , “membrane” (GO: 0016020), “binding” (GO: 0005488), “cellular process” (GO: 0009987), “membrane part” (GO: 0044425), “metabolic process” (GO: 0008152), and “cell” (GO: 0005623). There were 664 genes in “catalytic activity”, 445 genes in “membrane”, 442 in “binding”, 396 in “membrane part”, 415 in “cell process”, and 294 in “metabolic process” . These results indicate that SNH can inhibit the synthesis of total protease, LasA staphylococcal protease, LasB elastase, LecA lectin, and pyocyanin, and inhibit and swimming ability in P. aeruginosa. Taken together, these results suggest that SNH may inhibit the activity of P. aeruginosa by inhibiting the expression of P. aeruginosa virulence factors.The total protease, LasA staphylococcus protease, LasB elastase, LecA lectin, and pyocyanin levels and swimming ability were assessed  . Hence, these results suggest that SNH can improve the survival rate of larvae infected by P. aeruginosa, thereby effectively inhibit P. aeruginosa infection in vivo.After pre-experiment study, we determined 10^4 CFU per one larvae as the applied concentration LDP. aeruginosa QS system to inhibit the synthesis of the virulence factors LasA protease and pyocyanin. SNH is a new generation product of Houttuynia cordata. Compared with SH, SNH has higher safety and effectiveness, is less toxic, has fewer side effects, and has better clinical promotion value , which is the main component of biofilm extracellular polysaccharides and is essential for the formation and stability of biofilms. We further analyzed the effect of SNH on its virulence factors and biofilm formation. In P. aeruginosa biofilm, SNH affects the expression of a large number of genes. SNH reduced the synthesis of total protease, LasA staphylococcal protease, LasB elastase, LecA lectin, pyocyanin, and biofilm in P. aeruginosa. RT-PCR and qRT-PCR revealed that expression of QS regulated genes rhlI, pqsA, lecA, lasA, lasB, phzM, and pilG were down-regulated after SNH treatment.The virulence factors regulated by organism . Elastasproteins . LecA isruginosa . PyocyanP.aeruginosa, SNH can inhibit the expression of some virulence factors and the formation of biofilms. Experimental results show that expression of these virulence genes, biofilms, and related genes is dose-dependent at concentrations of 1 x MIC SNH, ½ x MIC SNH and, ¼ x MIC SNH. Additionally, analysis of virulence factor, biofilm, and related gene expression was consistent with transcriptome sequencing and PCR analysis, further supporting that SNH has an inhibitory effect on P. aeruginosa.In P. aeruginosa infection in vivo. We simulated the larval model, which proved that SNH significantly reduced the mortality of larvae. The difference of virulence between the drug groups and the blank control group proved by previous growth and virulence testing experiments can be confirmed in the larval model, which proves that the larval model is a suitable model for P. aeruginosa infection. The larval model can be used to study the pathogenic mechanism of P. aeruginosa. Once the genome sequence of larvae is available, the model of larvae will become more valuable in the future.Larval model is an important tool to study the pathogenesis of P. aeruginosa was 2,048 μg/ml, which was four times higher than SH. This possible because our research group has been doing drug resistance tests on P. aeruginosa in recent years, which may gradually adapt to drugs and produce resistance. Previous studies found that SH could down-regulate lasI gene and up-regulate rhlI gene after 72 h of treatment by our research group. However, through transcriptome sequencing data, it was found that lasI, rhlI, and pqsA were down-regulated by 2.44, 1.5, and 3.78 times after SH treatment for 24 h in this study, while no change in lasI gene expression was detected after SNH treatment, and rhlI gene was down-regulated by 1.11 times and pqsA was down-regulated by 2.93 times, which was inconsistent with the previous research results, possibly due to the different action time selected in the experiment . And it can be seen that when the action time is 24 h, the ways of SH and SNH on inhibiting P. aeruginosa are not completely the same. SH works together by regulating the Las/Rhl and PQS system, in which Las system plays a major role, while SNH plays a regulatory role by regulating Rhl and PQS system. However, due to the limitations of transcriptome sequencing, we also suspect that the relevant data may not be detected because of the small change multiple of lasI.The MIC of SNH to LasR is considered as the main regulator in P. aeruginosa, the cross-regulation between the Las and Rhl system was observed in this experiment. For example, low but detectable C4-HSL is produced without LasR , the Outstanding Talent Support Program in University (Key project) of Anhui Province under Grant No. gxyqZD2020024, and the Natural Science Foundation (Key project) of Anhui University of Chinese Medicine under Grant No.2020zrzd07. The Funding is used to cover laboratory expenses, sample preparation and sequencing. These funding agencies have no role in research design, data collection, analysis of results, or manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, scanning electron microscopy showed that adding biopolymers in the cellulose matrix made the surface compact, which also influenced the visual appearance. Thus, the performance of the biocomposites was directly influenced by their composition. The performance of the different samples obtained resulted in them having different potentials for application considering the injury type. This provides a solution for the ineffectiveness of traditional dressings, which is one of the great problems of the biomedical sector.One of the major benefits of biomedicine is the use of biocomposites as wound dressings to help improve the treatment of injuries. Therefore, the main objective of this study was to develop and characterize biocomposites based on bacterial cellulose (BC) with different concentrations of collagen and starch and characterize their thermal, morphological, mechanical, physical, and barrier properties. In total, nine samples were produced with fixed amounts of glycerol and BC and variations in the amount of collagen and starch. The water activity (0.400–0.480), water solubility (12.94–69.7%), moisture (10.75–20.60%), thickness (0.04–0.11 mm), water vapor permeability (5.59–14.06 × 10 The last five decades have witnessed tremendous growth in the field of biomaterial science and engineering because of vast investments in the development of new products, including wound dressings . DiffereGluconacetobacter, the most efficient BC producer  was evaluated using the equipment software equation  to obtain the correlation between the produced samples and their properties. As they presented different units of measurement, the data concerning the characterization tests cited were normalized in the range of 0 to 1, and after standardization, the PCA was carried out.The results obtained related to the characterization of biocuratives were analyzed for variance (ANOVA) at 95% significance, and the results that present significant differences between treatments were differentiated by Tukey’s test. Assistat software (Version 7.7 beta) was used to analyze the results . PrincipIn total, eight different biocomposites based on BC, collagen, and starch and the control (pure BC–F1 sample) were produced according to w of the nine samples varied between 0.400 ± 0.01 (F5) and 0.480 ± 0.01 , with significant differences between them (p > 0.05)  and the highest in F9 (69.76 ± 1.43%), which had collagen and starch besides BC (p > 0.05) ( > 0.05) . It is isides BC . F7, F8,sides BC . The TS  > 0.05) . The TS −8 g·mm/m2·h·Pa (F1) to 14.06 ± 0.71 × 10−8 g·mm/m2·h·Pa (F9), with a significant difference (p > 0.05) (The thickness of the pure BC and biocomposites is shown in  > 0.05) . It is i−2, respectively). In addition, the behavior of the samples in relation to the thickness was similar, indicating a correlation between these two properties. However, unlike these properties, the opacity of pure BC (36.67 ± 0.37 Abs 600 nm·mm−1) was greater than that of BC–collagen–starch biocomposites (31.85 ± 1.98–8.37 ± 0.08 Abs 600 nm·mm−1) (The grammage of BC–collagen–starch biocomposites was 15.03–338.83% more than that of pure BC . F4 and nm·mm−1) . These rnm·mm−1) , where, th minute. Similarly, the swelling rate of F7 (a BC–collagen biocomposite) decreased from 51.31% after 1 min to 22.52% after 180 min, a greater loss than that found for the other samples . The water release capacity reduced in all samples, regardless of the analysis time  was primarily opaque, while the BC–collagen–starch biocomposites (F2–F9) were comparatively more transparent. The opacity of F1 was 22.85% higher than that of F8, which had the lowest opacity. The opacity of BC has already been demonstrated to be related to the presence of an ultrafine nanofibrous network ,70, whicw in its structure. The aw of the nine samples analyzed ranged from 0.400 to 0.480, which is within the ideal range (0.2–0.6) proposed by Cázon et al. [w varied according to the composition of the biocomposite, similar to the results found in that study [Aiming at the application as a wound dressing, it is fundamental to understand whether the material formed has a hydrophobic or hydrophilic character as well as to know the an et al. , since tat study . Based oat study . The useat study , which pSaska et al.  showed tVapor exchange through a material is a critical property directly related to the effectiveness of wound dressing. Our results showed that increasing collagen concentration (2.00%) increased the WPV; however, it is important to note that the presence of starch also contributed to the high permeability of F8 and F9. This behavior was similar to that reported by Zhuang et al. , who obsGrammage is a property that is directly related to the mechanical resistance and barrier properties of the material, since they are influenced by the thickness and mechanical properties . F8, witAs shown in In the present study, nine different samples were produced based on BC, collagen, starch, and glycerol as the plasticizing agents, with fixed masses of BC and glycerol. All samples were characterized based on their physical, barrier, morphological, mechanical, and thermal properties. The results obtained showed that the performance of a sample was directly related to the presence and the concentration of its constituent components. The different performances indicate that pure cellulose as well as biocomposites may have different application potentials, mainly depending on the injury type and stage of healing. Thus, the results of this study can contribute to a new perspective in biomedicine  positively impacting the quality of life of patients.


Oxidative stress that causes neural damages in neurodegenerative disorders has been widely studied for the pathogenesis and diagnostic measures. Zhengtian capsule (ZTC), a type of traditional Chinese medicine for headaches, has been found to have extra effects in recent years, such as promoting the release of serotonin and dopamine in the brain, but its specific mechanism has not been clearly elucidated. In this study, we focus on revealing whether ZTC can regulate key proteins of neurotrophic signaling pathway to alleviate depression-like behavior caused by oxidative stress. Experimental results show that ZTC (M 0.34 and H 0.7 g/kg) can elevate the proliferation of neural stem cells and GABAergic-type neurons in the hippocampus, promote the protein levels of BDNF, phosphorylated ERK1/2, and CREB, and inhibit the expression level of a key inflammation factor NFκB in a dose-dependent manner. These data suggest ZTC acts on multiple pathways to resist excessive oxidative stress, proving it to be a potential neurotrophic drug. With the global acceleration of aging population, neurological diseases have raised to become the main killer of human health . Numerouvia modulation of the expression of GABAC receptors and its mediated ion-channels  and alleviate depression-like behaviors . As beine stress .Meanwhile, many similar studies also reported that upregulation of BDNF expression in brain tissue and promotion of neurogenesis in the hippocampus can alleviate mood disorders. Since the synthetic sites of BDNF in the central region of hippocampus are mainly in the CA1, CA3, and the hilus of dentate gyrus , it is pvia regulation of NFκB related pathway. These data indicate that ZTC plays its role as a neurotrophic drug for depression treatment via induction of neurogenesis and regulation of neurotrophic signaling pathway.In this study, we used a single injection of LPS to increase oxidative stress and established a depression-like behavioral model in mice. It was observed that ZTC could effectively alleviate depression-like behavior caused by oxidative stress and improve the exploration ability of stressed mice. ZTC enhanced the neurogenesis and GABAergic neurons in the hippocampus, and improved the expression levels of key proteins, such as BDNF, ERK1/2, CREB, but reduced the expression level of NFκB in the neurotrophic signaling pathway, which is consistent with previous studies . This suSpatholobus suberectus Dunn (169.17 g/1 kg compound), Angelica sinensis (Oliv.) Diels (56.06 g/1 kg compound), Ligusticum chuanxiong S.H.Qiu, Y.Q.Zeng, K.Y.Pan, Y.C.Tang & J.M.Xu (101.10 g/1 kg compound), Asarum caudigerellum C.Y.Chen & C.S.Yang (56.06 g/1 kg compound), Uncaria rhynchophylla (Miq.) Miq (112.11 g/1 kg compound), Paeonia lactiflora Pall (67.07 g/1 kg compound), Rehmannia glutinosa (Gaertn.) DC (56.06 g/1 kg compound), Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (56.06 g/1 kg compound), Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk (56.06/1 kg compound), Notopterygium incisum K.C.Ting ex H.T.Chang (56.06 g/1 kg compound), Prunus persica (L.) Batsch (34.03 g/1 kg compound), Carthamus tinctorius L. (34.03 g/1 kg compound), Angelica pubescens Maxim (34.03 g/1 kg compound), Ephedra sinica Stapf (56.06 g/1 kg compound), and Aconitum carmichaeli Debeaux (56.06 g/1 kg compound) (The detailed information of these plants used in ZTC is listed in Zhengtian Capsule (ZTC) (batch number: Z20010142) is a Chinese patent medicine, that is comprised of 15 traditional Chinese herbs: The study was performed in male Kunming (KM) mice (20–22 g) obtained from Animal Experimental Center of Peking University  2014-0013). Animals were housed under standard conditions  in cages. The mice were randomly divided into 6 groups (n=10 mice per group). After behavioral tests, mice were anesthetized with 60 mg/kg pentobarbital sodium (i.p), and then sacrificed for molecular experiment. When anesthesia, firstly, the mice were soothed and anesthetized by intraperitoneal injection; then the mice were thoroughly anesthetized and cardiac perfused to obtain brain tissue; later, the animal carcasses were handed over to the Beijing Experimental Animal Carcass Management Organization for unified processing. All experiments were carried out under the guidance of the Beijing animal ethics Association and the ethics committee of Beijing Institute of Technology (SYXK-BIT-school of life science-2017-M03).via intragastric administration, and 5 mg/kg of LPS was administered intraperitoneally 1 h after the last day of administration. The mice were grouped based on the administration of ZTC dosages as follows: Normal group, 0.9% normal saline; model group (LPS), 5 mg/kg  was incubated at 4°C overnight. GAD67 staining: brain sections were blocked with 0.5% Tween in PBS for 2 h, and diluted with 0.05% Tween in PBS for primary antibody  and incubated overnight at 4°C. DCX brain sections were blocked with 0.5% Triton-100 in PBS for 2 h, and diluted with 0.3% Triton-100 in PBS for primary antibody  and incubated overnight at 4°C.After continuous administration of ZTC for 8 days, BrdU was firstly injected twice by 2 h interval; and after 20 days, BrdU was again injected twice by 2 h interval; and after another 24 h, the mice were sacrificed and the brain slices were prepared for immunochemistry experiment. The mouse brain was treated with 4% paraformaldehyde for more than 48 h, and then dehydrated by two steps in 20% and 30% sucrose, separately. Coronal continuous sections, thickness 20 um, each 10 pieces were collected in a small hole in a 24-well plate. Subsequently, different samples were randomly sampled at the same location and stained and the results were counted . BrdU stTotal RNA was extracted from the hippocampus of experimental mice, and the RNAiso Plus kit  was used according to its kit protocol. cDNA extracted from the total RNA obtained in the previous step using a kit . The PCR step was as follows: 1 cycle at 95°C for 3 min; 39 cycles at 95°C for 10 s, and 60°C for 30 s .® Premix Ex Taq™ (Tli RNaseH Plus) , using Applied Biosystems’ StepOnePlus™ Real-Time PCR System. Data were analyzed using the delta-delta Ct method.The primer sequences used were as follows: BDNF ; GAD67 ; GAPDH . RT-qPCR was performed in SYBRThe hippocampal tissues were lysed in RIPA buffer  containing the phosphatase and protease inhibitors. Protein concentrations were determined by the Bradford method. 10% or 12% SDS-PAGE gel were used to separate the proteins, and then which were transferred to the PVDF membrane . The membranes then incubated with primary antibodies diluted in TBST at 4°C overnight, then which were incubated with the HRP-conjugated secondary antibody at room temperature for 1.5–2 h . The priP <0.05 was considered statistically significant.Data were expressed as mean ± standard error of mean (SEM). The one-way analysis of variance (ANOVA) was used for data analysis. Difference associated with ZTC was administered to mice in three dosage groups ; resveratrol group (RES)  was used as a positive control . The mic+ labeled cells in the dentate gyrus of the hippocampus was significantly increased by ZTC treatment in a dose-dependent manner, compared to that by the LPS treatment  in the hippocampal dentate gyrus than RES and other groups .LY, NML, YW, BX, and CXY performed the experiments; ZZQ, LY, and QFYa analyzed the data; LY, NML, and ZZQ prepared the draft; JHD, ZD and QFYu helped data analysis; ZD, and HQ designed the experiments; ZD and ZZQ approved the final version. Both LY and YW contributed equally to this work. All authors have read and approved the manuscript.This study was funded by Shenzhen Collaborative Innovation Program (No. GJHS20140829143704130). At the same time, we had also received funding from other funds, the list was as follows: NSFC Regional Science Foundation Project (No. 31960174), and Yan’an University Research Project , Youth Program of National Natural Science Foundation of China .Authors YW, BX, JD, QFYu, and ZD were employed by the company China Resources Sanjiu Medical & Pharmaceutical Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We observed the junction’s uniform responsivity in the visible range and five absorption peaks at 940 nm, 1180 nm, 1380 nm, 1580 nm, and 1900 nm wavelengths. The temperature showed effective affection to the photocurrents at absorption peaks and the transition point occurred at 216 K, which was also displayed in the temperature dependence of junction resistance. On the basis of the results, we propose a possible model involving the quantum size effect at the junction interface as the mechanism. This understanding of the infrared photodetection properties of oxide heterostructures should open a route for devising future microelectronic devices.In this paper, we investigate the broad spectral photocurrent properties of the La Oxide semiconductor devices based on the perovskite oxide films, whose properties can be controlled by magnetic field, electric field, and light irradiation, have attracted a great deal of interest. Experiments confirmed that manganite-based perovskite-type oxides have excellent ultraviolet (UV) photoresponse characteristics with ultrafast response-time of picosecond and high sensitivity, which makes this class of materials potentially useful for UV sensor applications ,2. FurthSi photodetectors have already found wide acceptance for visible light applications, while it has small absorption coefficient in near-infrared (NIR) wavelength range because of the cut off wavelength of ~1100 nm. Now most NIR photodetectors were composed of PbS, PbSe, or InGaAs. The toxic precursors, such as Pb, Se, and As, was usually used to synthesize these materials. It is a meaningful thing to find non-toxic and pollution-free material for photodetector working at NIR wavelength range.0.67Ca0.33MnO3/Si (LCMO/Si) is reported. The temperature dependence of the photocurrent response of the sample was investigated to reveal more information related to the photoelectric response, and selective absorption peaks were observed. The mechanism about the results is also discussed in the paper.The infrared (IR) spectrum has become an important method to study the lattice distortion and been applied to investigate the photoconductive effect in perovskite manganese oxides, where mid-infrared or far-infrared spectra was used to explain the complex physical process in manganites such as the electronic transition, electron-phonon interaction, coupling between lattice, orbital, and spin, etc., ,20,21,22The LCMO/Si heterojunction was fabricated using the facing target sputtering technique. A 100 nm thickness LCMO layer was grown on a 0.5 mm thick n-type Si (001) wafer. The wafer temperature was kept at 680 °C with the oxygen pressure being 60 mTorr during deposition. Immediately after each deposition, the vacuum chamber was back-filled with 1 atm oxygen gas.The photocurrent of the sample was detected by the spectral response measurement system, as shown in The LCMO/Si heterojunction for the photoelectric measurements was cut into 5 × 5 mm and two colloidal silver electrodes were prepared on the LCMO film and Si wafer. The sample was placed in an airtight holder with a quartz window and connected with the spectral response measurement system a. The tyRj in LCMO/Si junction was measured with the temperature. As shown in Rj strongly depends on the bias, e.g., when the bias was turned from 20 μA to −20 μA Rj changed from 166.0 kΩ, 20.0 kΩ, and 15.1 kΩ to 178.1 kΩ, 98.2 kΩ, and 15.6 kΩ at 95 K, 202 K, and 300 K. In addition, taking the bias of 50 μA as an example, Rj decreased slightly from the beginning 95 K to 172 K and had a sharp change from 78 kΩ at 172 K to 8.2 kΩ at 216 K with a corresponding rate of 1.6 kΩ/K. Subsequently Rj maintained small change of about 0.01 kΩ/K till 300 K.The junction resistance PI) spectrum of the LCMO/Si junction under zero bias in the wavelength range of 200 nm < λ < 2200 nm. The junction’s responsivity was spectrally uniform in the visible range, while five absorption peaks P1, P2, P3, P4, and P5 were observed at λ1 = 1940 nm, λ2 = 1180 nm, λ3 = 1380 nm, λ4 = 1580 nm, and λ5 = 1900 nm wavelengths in each temperature because of the absorption characteristics of the LCMO/Si junction, and the peak value decreased with the increase of the wavelength. The temperature dependences of the photocurrent response PIP at the five absorption wavelengths are shown in PIP monotonically increased from 0.0065 A/W, 0.012 A/W, 0.0115 A/W, 0.004 A/W, and 0.002 A/W at 108 K to 0.0122 A/W, 0.017 A/W, 0.0155 A/W, 0.006 A/W, and 0.0025 A/W at a turning point of 216 K and then dropped 0.004 A/W, 0.005 A/W, 0.005 A/W, 0.002 A/W, and 0.001 A/W at 300 K for selected wavelengths of λ1, λ2, λ3, λ4, and λ5.2 layer of 3.6 nm thick exists in the LCMO/Si heterojunction [nm is the electron effective mass and wd is the quantum well width. Thus,  is independent on n andSi cannot produce strong absorption features. Most of heterojunctions of manganite-based perovskite-type oxides exhibit the properties of p-n junction and quantum size effect can be produced when the thickness of the potential well material is about 50 nm thick. If quantum size effect occurs, the energy is quantized in the direction of the vertical interface, which will lead to the quantization of energy absorption in the material. Since a thin SiOjunction , a quantλn ,As for present five special wavelengths Here, the above two similar data suggested that the present model involving the quantum size effect was adopted as the mechanism of IR photocurrent in LCMO/Si.In is about 10−4 A/W in dark and is very low compared to the responsivity PI of the LCMO/Si junction when the light was on. The detectivity D* is determined by the ratio of PI and In, and D* = PI (fS)1/2/In, where f is the amplifier frequency bandwidth (500 MHz) and S is the detector area (~7.065 mm2). Thus D* is estimated to be about 2.38 × 103 Hz1/2m, 2.97 × 103 Hz1/2m, 2.97 × 103 Hz1/2m, 1.19 × 103 Hz1/2m, and 0.59 × 103 Hz1/2m at 300 K for selected wavelengths of λ1, λ2, λ3, λ4, and λ5, suggesting that the LCMO/Si junction could be well-suited as an IR detector.Noise performance is a critical factor for evaluating a detector. The noise current Perovskite-type oxides detectors possess a number of significant characteristics, and are ideally suited to detect small changes in a relatively large background level of incident energy, which can be used over a large spectral bandwidth. Here it has been shown that a specific manganite heterojunction has the ability to be an IR detector since it can produce photocurrent in the IR regime. The devices have a number of important characteristics  which make them ideal for a range of applications from consumer and commercial to military requirements. LCMO/Si junction is a new material for photodetector fabrication compared to traditional materials. It is anticipated that manganite heterojunction IR detectors will assume an ever growing importance in our society over the next few years.PI mechanisms.In conclusion, we fabricated a manganite-based heterojunction by depositing a LCMO thin film on the Si substrate. The broad spectral photocurrent effect of the junction was systematically studied in a temperature range from 95 to 300 K. The responsivity of LCMO/Si heterojunction was spectrally uniform in the visible range. Five absorption peaks occurred at 940 nm, 1180 nm, 1380 nm, 1580 nm, and 1900 nm in the IR range, which is explained in terms of a quantum size effect model since an interface existed in the present photodetector. However, relative contributions from individual interface are still not clear and further studies is needed to clarify the


Chronic obstructive pulmonary disease (COPD) is a major public health problem and cause of mortality worldwide. However, COPD in the early stage is usually not recognized and diagnosed. It is necessary to establish a risk model to predict COPD development.A total of 441 COPD patients and 192 control subjects were recruited, and 101 single-nucleotide polymorphisms (SNPs) were determined using the MassArray assay. With 5 clinical features as well as SNPs, 6 predictive models were established and evaluated in the training set and test set by the confusion matrix AU-ROC, AU-PRC, sensitivity , specificity, accuracy, F1 score, MCC, PPV (precision) and NPV. The selected features were ranked.P = 0.010; rs2910164, OR = 1.416, P < 0.037; rs473892, OR = 1.473, P < 0.044; rs161976, OR = 1.594, P < 0.044; rs159497, OR = 1.445, P < 0.045; and rs9296092, OR = 1.832, P < 0.045) were risk factors for COPD, while 3 SNPs  were protective factors for COPD development. In the training set, KNN, LR, SVM, DT and XGboost obtained AU-ROC values above 0.82 and AU-PRC values above 0.92. Among these models, XGboost obtained the highest AU-ROC (0.94), AU-PRC (0.97), accuracy (0.91), precision (0.95), F1 score (0.94), MCC (0.77) and specificity (0.85), while MLP obtained the highest sensitivity  (0.99) and NPV (0.87). In the validation set, KNN, LR and XGboost obtained AU-ROC and AU-PRC values above 0.80 and 0.85, respectively. KNN had the highest precision (0.82), both KNN and LR obtained the same highest accuracy (0.81), and KNN and LR had the same highest F1 score (0.86). Both DT and MLP obtained sensitivity  and NPV values above 0.94 and 0.84, respectively. In the feature importance analyses, we identified that AQCI, age, and BMI had the greatest impact on the predictive abilities of the models, while SNPs, sex and smoking were less important.Nine SNPs were significantly associated with COPD. Among them, 6 SNPs (rs1007052, OR = 1.671, The KNN, LR and XGboost models showed excellent overall predictive power, and the use of machine learning tools combining both clinical and SNP features was suitable for predicting the risk of COPD development. It has been reported that chronic obstructive pulmonary disease (COPD) is a public health challenge due to its high prevalence and related disability, mortality and socioeconomic burden worldwide –3. ApproThe typical symptoms of COPD include dyspnea, chronic cough, and sputum production, and spirometry is considered the gold-standard method for the diagnosis of COPD . SpiromeRehman et al.  reportedSmoking is a well-known risk factor for COPD development; however, fewer than 20% of smokers develop COPD, and more than 15% of non-smokers have COPD . Recent 2, NO2, PM10, PM2.5, CO and O3 and other pollutants. The higher the AQCI is, the more serious the pollution. In the present study, we used a combination of AQCI and other risk factors, such as age, sex, BMI and smoking, to predict COPD development.Wang et al. reported a national cross-sectional study in China that indicated that COPD was highly prevalent in the Chinese adult population. Cigarette smoking, ambient air pollution, underweight, childhood chronic cough, parental history of respiratory diseases, and low education are major risk factors for COPD in the Chinese population. Among these factors, cigarette smoking and air pollution are major preventable risk factors for this disease . With raHHIP [IL13 [MMP9 [SFTPB [SOD3 [CHRNA3 [RNF150 [COL4A3 [AQP5 [AGPHD1 [IREB2 [In addition, COPD susceptibility- or disease progression-related genes have been reported , and genHHIP , IL13 [2IP [IL13 , MMP9 [213 [MMP9 , SFTPB [9 [SFTPB , SOD3 [2PB [SOD3 , CHRNA3  [CHRNA3 , RNF150  [RNF150 , BICD1 [ [RNF150 , COL4A3  [COL4A3 , AQP5 [3A3 [AQP5 , AGPHD1  [AGPHD1 , IREB2 [1 [IREB2 , etc. MaLung function is the gold standard for the clinical diagnosis of COPD; however, when the FEV1/FVC or FEV1% value is abnormal, lung function is a defective indicator in approximately 30% of patients . COPD inIn the present study, we performed a case–control study including 441 patients with COPD and 192 healthy controls. Then, the odds ratios (ORs) of the genotypes of 101 SNPs and clinical features for COPD development were calculated. We established and compared six prediction models that included susceptible SNPs and clinical features using statistical, machine learning and neural network approaches.A total of 441 COPD patients and 192 control subjects were randomly recruited from seven subcenters in China from January to December 2017, including Linfen People’s Hospital , Jincheng People’s Hospital , Heji Hospital Affiliated with Changzhi Medical College , General Hospital of Tisco  , Hebei General Hospital , General Hospital of Datong Coal Mine Group Co., Ltd. , and Shanghai Zhangjiang Institute of Medical Innovation (Shanghai). COPD was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria , and pat2. AQCI values were derived from the following seven regions according to official government website statistics in China: five cities from Shanxi Province (http://sthjt.shanxi.gov.cn/html/tndt/20180119/58694.html), Shijiazhuang from Hebei Province (http://hbepb.hebei.gov.cn/hjzlzkgb/) and Shanghai (http://www.mee.gov.cn/hjzl/zghjzkgb/lnzghjzkgb/).The basic characteristics of all participants are listed in Additional file We selected 101 SNPs from 76 genes and 9 intergenic regions that were previously reported to be associated with COPD , 35–60. https://agenacx.com website to design multiplex primers for each SNP: 1st PCR primer, 2nd PCR primer, and UEP primer. The primers for the 101 SNPs are shown in Additional file ® Analyzer 384-well Configuration  according to the standard protocol recommended by the manufacturer.We used the PCR amplification was performed in a reaction system with a total volume of 5 μl containing 10 ng genomic DNA, 1 U PCR enzyme (Agena), 0.5 μl 10× PCR buffer, 0.1 μl dNTPs mix and 0.5 μl of each primer under the following program: 2 min denaturation at 95 °C, 45 cycles of 30 s at 95 °C, 30 s at 56 °C and 60 s at 72 °C and a final extension at 72 °C for 5 min. Then, the PCR products were cleaned by 2 μl SAP (Agena) including 1.53 μl nanopure water, 0.17 μl SAP buffer and 0.5 U SAP enzyme with the following steps: 40 min at 37 °C and 5 min at 85 °C. Finally, the single-base extension used 2 μl iPLEX EXTEND mix (Agena) containing 0.619 μl nanopure water, 0.94 μl Extend primer mix, 0.041 μl iPLEX enzyme, 0.2 μl iPLEX buffer and 0.2 μl iPLEX termination mix and was performed with the following steps: initial denaturation at 94 °C for 30 s, followed by 40 cycles of a 3-step amplification profile of 5 s at 94 °C, an additional 5 cycles of 5 s at 52 °C and 5 s at 80 °C and a final extension at 72 °C for 3 min. Data management and analysis were performed using Typer Analyzer 4.0 software (Agena). Several SNP samples were finally excluded because ≥ 10% of the genotyping data were missing.First, 290 COPD patients and 103 control subjects were enrolled as the training set, and the OR values of all genotypes of SNPs were calculated using the PLINK software package (version 1.07) . The genModel selection was based on several currently and frequently adopted predictive model types. For example, the linear LR model  and SVM https://github.com/weilan-yuan/COPD_machine-learning).In the validation set, we no longer calculated the OR values from PLINK software and directly mapped the OR value of each genotype from the training set. However, the genotypes with missing OR values were assigned the average OR value independently in the test set. To validate the training set, we recruited 151 COPD patients and 89 controls in the test set. Six models were selected for validation in the test set. The entire process is shown in Fig. t-test) were used for normally distributed data, and nonparametric statistics (Mann–Whitney) were used for non-normally distributed data. The t-test and nonparametric Mann–Whitney U test or Chi squared test was used to compare parametric and categorical variables, respectively. Statistical calculations were performed in R studio (R.3.51). P ≤ 0.05 was the threshold for statistical significance.For all SNPs, the ORs and 95% confidence intervals (CIs) of the minor alleles were assessed without adjusting for age, sex, BMI, smoking status and AQCI by Chi squared tests using the PLINK package between COPD patients and healthy controls. Furthermore, six predictive models  for COPD risk were used and evaluated by Python (version 3.7.0) and included 9 SNPs with 5 clinical features. The diagnostic values of the 6 models were assessed by ROC and PRC analysis. Parametric statistics (P < 0.0001) in both the training and test sets. Only the BMI levels were similar between the two groups. The results indicated that COPD patients were more likely to be older, male, and smokers, and the FEV1/FVC (%) and FEV1 (%) values were lower in the COPD group than in the healthy controls.The clinical characteristics of the cases and controls are shown in Additional file In the training set, 6 SNPs  were excluded because ≥ 10% of the sample data were missing. Finally, 95 of the 101 SNPs were included in the following analysis. We assumed that the minor allele (A1) of each SNP was a risk factor compared to the main allele (A2) and obtained the OR values. All results are shown in Additional file RNF150 gene. Ding et al. first reported that polymorphisms of RNF150 were significantly associated with COPD risk in the Chinese Han and Li populations [P = 0.010). rs2910164 was reported as a C/G polymorphism in the precursor stem region of pre-miR146a on chromosome 5q33 and was first associated with genetic predisposition towards papillary thyroid cancer [OLIG3, the T allele of rs473892 was associated with a higher FEV1 level in subjects with high exposure to mineral dust than in those without exposure in the LifeLines and Vlagtwedde-Vlaardingen samples [P < 0.045) was a risk factor for COPD development. These findings indicated that the T allele of rs473892 may be a protective locus for COPD, while the C allele is a risk locus for COPD. It has been reported that BICD1 (rs161976) is a potential susceptibility gene in COPD patients. rs161976 was previously demonstrated in a GWAS to be associated with emphysema in patients with COPD with an FEV1 < 80% of the predicted value [P < 0.044) is also a risk locus in COPD. rs159497 is located in intergenic regions near the PDE4D gene. It was reported that PDE4D was not only a susceptibility gene for asthma [P < 0.045) was a risk factor for COPD development. rs9296092 is located in intergenic regions that lie in the gene region between the zinc finger and BTB domain containing 9 and BCL2-antagonist/killer1 (ZBTB9-BAK1) at chromosome 6 p21.32. Ding et al. also reported that in a case–control study, rs9296092 was associated with the greatest increase in the risk of COPD in the Hainan Province of China [P < 0.045) is also a risk factor for COPD development.Among the 6 risk SNPs, the SNP rs10007052 is located in the first intron of the ulations . In our d cancer . In the ed value . In the r asthma  but alsoof China . In our SOD3 gene [P < 0.015) was associated with a reduced risk of COPD. The SNP rs20541 is in the fourth exon of the IL-13 gene [P < 0.018) is a protective factor against COPD. The SNP rs12922394 is in the first intron of the CDH13 gene and is usually removed during the gene-splicing process. Yuan et al. reported that the T allele of rs12922394 was associated with a significantly reduced risk of COPD [P < 0.022) was also associated with a reduced risk of COPD, indicating that this variant may protect against COPD development.In addition, 3 SNPs were protective against COPD; among them, the SNP rs8192288 is located in the first intron of the OD3 gene . In our -13 gene . In the  of COPD . In the In the training set, k-fold cross-validation (k = 5) was used, and various parameter combinations were exhausted by grid search. Six models were established in this study. For each model, the evaluation indicators used were the confusion matrix, AU-ROC, AU-PRC, specificity, sensitivity , PPV (precision), NPV, accuracy, F1 and MCC score. The average AU-ROC, AU-PRC and 95% CI are shown in Fig. To verify the importance of clinical features or SNPs for predicting COPD in all models, we used only 9 SNPs and 5 clinical features and selected the top 5 ranked features, which included 3 clinical features  and 2 SNPs (rs12922394 and rs10007052), to be included in the six models. The results indicated that with only the input of 9 SNPs, the AU-ROC values were only 0.51–0.66, but the AU-PRC values of all models were 0.80–0.86  was a protective factor for COPD in the Chinese population. More data are needed to validate this finding in the Chinese population.In the present study, we employed a dataset with 441 patients with COPD and 192 healthy controls, which satisfied the power analysis. A total of 101 SNPs were identified, and 9 SNPs were significantly associated with COPD development based on PLINK software. Our results showed that 6 SNPs  were risk factors for COPD, while 3 SNPs  were protective factors for COPD development. These results were roughly shown by previous studies. However, there is controversy regarding the role of individual SNPs in COPD. For example, a GWAS found no significant relationship between rs10007052 and COPD in Europeans . HoweverApart from SNPs, we also considered 5 clinical features that may be associated with COPD development, such as smoking history, ambient air quality, BMI, age and sex. In the training set, 6 models  were established to predict COPD risk and included 9 SNPs and 5 clinical features. We evaluated the predictive performance of these 6 models for COPD and found that the XGboost model presented the best AU-ROC and AU-PRC values in both the training and test sets in all features. The XGboost algorithm is a highly effective and widely used machine learning method that can build complex models and make accurate decisions when given adequate data . We usedThere were some limitations in this study. First, the sample size used was relatively small, and the total sample of patients with COPD and healthy controls was unbalanced. Second, the samples from the seven centers were severely unbalanced; in the training set, only control subjects and no COPD patients were collected from the Shanghai area; no control subjects were recruited from Jincheng, and only one COPD patient came from Datong. In the validation set, control subjects and COPD patients from only 5 centers, excluding Jincheng and Shijiazhuang, were enrolled in the test set. Third, we only obtained 9 SNPs for the prediction models, and these SNPs performed worse than the clinical features; there may be other genetic susceptibility SNPs to be discovered. Fourth, we only collected five clinical features for the prediction models, while other risk factors were not collected, such as occupational exposure, childhood chronic cough, parental history of respiratory diseases, and low education in the Chinese population. More work is required before these models can be applied in the clinic for COPD prediction, and these findings should be validated in a larger cohort.In conclusion, we identified 9 genetically susceptible loci for COPD and constructed COPD prediction models that comprised SNPs and clinical factors, including ambient air pollution. The KNN, LR and XGboost models showed excellent overall predictive power. We also identified that clinical features were more important than SNPs in predicting COPD development. Our study also revealed that these machine learning tools showed good performance for COPD risk prediction and could potentially be beneficial for the early diagnosis and treatment of patients with COPD in the Chinese population in the near feature.Additional file 1: Table S1. The Chinese COPD studies with basic information in international journals.Additional file 2: Table S2. Demographics of COPD patients and control subjectsin the training set.Additional file 3: Table S3. Demographics of COPD patients and control subjectsin the test set.Additional file 4: Table S4. The sequence of 101 SNPs and their primers in multiplex PCR.Additional file 5: Table S5. The parameters selection in the predictive models.Additional file 6: Table S6. Allele frequencies in COPD and control subjects for SNPs.Additional file 7: Fig. S1. Evaluation of the predictive models with only the 9 SNPs as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of the 6 models in the training set. Mean AUC values and 95% CIs of different prediction models are shown in the box.Additional file 8: Table S7. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the training set of 9 SNPs features.Additional file 9: Fig. S2. Evaluation of the predictive models with only the 5 clinical features as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of the 6 models in the training set. Mean AUC values and 95% CIs of different prediction models are shown in the box.Additional file 10: Table S8. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the training set of 5 Clinical features.Additional file 11: Fig. S3. Evaluation of the predictive models with the top 5 ranked features as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of the 6 models in the training set. Mean AUC values and 95% CIs of different prediction models are shown in the box.Additional file 12: Table S9. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the training set of top 5 ranked features.Additional file 13: Table S10. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the test set of 9 SNPs features.Additional file 14: Fig. S4. Validation of the models in the training set with only the 9 SNPs as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of all models in the test set.Additional file 15: Fig. S5. Validation of the models in the training set with only the 5 clinical features as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of all models in the test set.Additional file 16: Table S11. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the test set of 5 clinical features.Additional file 17: Fig. S6. Validation of the models in the training set with the top 5 ranked features as inputs. a, b The picture shows the AU-ROC and AU-PRC curves of all models in the test set.Additional file 18: Table S12. The efficacy of KNN, LR, SVM, DT, MLP and XGboost in the test set of top 5 ranked features.


Graphomya rufitibia (Diptera: Muscidae) was distributed worldwide. In this study, the complete mitogenome of G. rufitibia was sequenced and annotated, and the full-length was a 15,374 bp fragment, consisting of A (40.64%), G (8.96%), T (36.80%), and C (13.59%). The mitogenome is composed of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding AT-rich region. Most PCGs used the canonical putative codon to start and stop, 21 tRNAs are folded into the classic clover-leaf structure, with the one exception of tRNA-Ser (AGN). A total length of 11,182 bp encoding 3727 amino acids, and account for 72.7% of the whole mitogenome. Phylogenetic analyses showed that G. rufitibia belongs to family Mydaeinae. Graphomya rufitibia mainly spread over Asia and Australia, the larvae were semi-aquatic , China. All voucher specimens were assigned a unique field code and reserved in the Guo’s Lab (Changsha city). The G. rufitibia mitogenome has been submitted to GenBank with accession number MG735216.Specimens of G. rufitibia was amplified with 11 overlapping short PCR primers. PCR amplicons were conducted with the following procedures: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 30 s at 94 °C, annealed at 50 °C for 30 s, and elongation 1 min at 72 °C, end in a final elongation for 10 min at 72 °C. PCR products were using an ABI PRISM 3730 automated sequencer  to sequence. The primitive sequence files were manually corrected for repeats at the beginning and end of the sequence to authorize a circular mitogenome, and then assembled into contigs with BioEdit 7.0.9.0  and Mydaeinae (Graphomya genus), which was corresponding to Kutty et al. (Stomoxyini+ Muscini), the topological structure was congruent with the previous studies (Kutty et al. Phylogenetic analyses of  methods , and usey et al.  researchy et al. . The cla


In beef cattle production, producers are constantly looking for ways to improve their bottom line. Feed costs can make up to 80% of operation costs for beef producers . ImproviPrevious studies have determined a high correlation between genotypic and phenotypic feed efficiency in growing Charolais bulls . Predictn = 25; 2016–2017, n = 32; 2017–2018, n = 28; 2018–2019, n = 33) for an average of 6 months (December–June). Calves were donated from producers across Montana and were comprised of various breeds; however, the majority were Angus based. Upon arrival, calves were tagged with an RFID . In addition, calves were implanted with Component T-S  at the beginning of the feeding period. Steers were penned in three drylot pens, fenced with wooden-slatted fencing. Calves in each drylot had access to water in the center of the pens and a shelter at the north end. Each pen was equipped with two GrowSafe Intake units  for measuring individual animal intake. Calves were assigned to pens based on initial weight . The number of calves per pen varied between 8 and 12 head (4–6 head per GrowSafe unit). Steers were started on a ration of 50% coarsely chopped hay, 5% 40-20 Beef Finisher supplement , and 45% cracked corn. Through January, corn increased in 5% increments once a week and, by mid-February, the calves were on a full ration of 20% coarsely chopped hay, 5% 40-20 Beef Finisher supplement, and 75% cracked corn. Steers had ad libitum access to feed and were fed twice daily at 0800 and 1700 hours. Sweetlix Bloat Guard  was available to steers on an ad libitum basis.Experimental procedures described herein were approved by the Agriculture Animal Care and Use Committee of Montana State University . Calves were finished at Bozeman Agriculture Research and Teaching Farm (BART Farm) in Bozeman, MT. In the Steer-A-Year program, 118 weaned steer calves ranging from 9 to 11 months of age upon arrival were fed over a 4-yr period  for analysis. For many of the performance traits predictions, Igenity placed the values into a relative index (1–10). For most traits, higher scores are more desirable. Carcass measurements were attained at a local abattoir after slaughter.RFI was calculated for years 2–4 using the method developed by P ≥ 0.14; P ≥ 0.14; P < 0.01; r = −0.58; P < 0.01; ρ = 0.55; P ≥ 0.29; P ≥ 0.31; P ≥ 0.11; P ≥ 0.21; P ≤ 0.08; ρ = 0.34, 0.31, respectively; Igenity scores were not related to actual steer ADG for years 1, 2, and 3 (Similar to our study, weak correlation and no statistical significance for predicted versus actual estimates for RFI have been reported by other researchers . However