N′-benzylidenepyrazine-2-carbohydrazonamide) were synthesized and characterized by appropriate analytical techniques: elemental analysis (EA), thermogravimetric analysis (TG–DTG), infrared spectroscopy (FTIR), and flame-atomic absorption spectrometry (F-AAS). The biological activity of the obtained compounds was then comprehensively investigated. Rational use of these compounds as potential drugs was proven by ADME analysis. All obtained compounds were screened in vitro for antibacterial, antifungal, and anticancer activities. Some of the studied complexes exhibited significantly higher activity than the ligand alone.New coordination compounds of Mn(II), Fe(III), Co(II), and Ni(II) and the biologically active ligand L ( Cancer is the general name for a large group of diseases that affect every part of the body. The terms “malignant tumors” and “neoplasms” are also used. The development of tumor is called carcinogenesis and refers to the transformation of normal tissue cells into cancerous cells. The fact is that the cells of human body divide not only at a young age during growth and development but throughout life, since live tissues are constantly renewed. According to the World Health Organization (WHO), cancer is the second leading cause of death in the world after cardiovascular disease. In 2020, WHO experts reported that 19.3 million people were diagnosed to have cancer. Ten million people died as a result of the disease [Mycobacterium tuberculosis) [Based on the current situation, research works on anticancer drug development throughout the world have significantly increased. For more than a decade, coordination chemistry has been at the forefront in solving the problems presented above ,3. Coordculosis) ,8,9. Comculosis) ,12,13,14culosis) ,16,17. Eculosis) . In addiculosis) ,20.N′-benzylidenepyrazine-2-carbohydrazonamide) as the organic ligand , atomic absorption spectrometry (AAS), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). In order to predict their biological properties, ADME analysis was performed. The presented in vitro investigations concentrated on the cell line L229 and U87 MG cells, with human skin fibroblast BJ  as a control normal cell line. This investigation also demonstrated the influence of the presence of transition metals on the antifungal and antibacterial properties of the ligand.The ligand was tested in silico for antitumor activity . The proActivity was predicted for the obtained ligand L . This co3O4-type metal oxides. Thermal decomposition data calculated or derived from decomposition patterns are presented in 3∙CH3OH and Ni(L)Cl2∙C2H5OH complexes, the first step was the loss of the methanol or ethanol molecule, respectively. In the case of the complexes that did not contain alcohol molecules (Mn(L)Cl2, Co(L)Cl2), complexation significantly increased their thermal stability.n-transition metals Mn(II), Fe(III), Co(II), and Ni(II). The spectrum of the uncoordinated ligand consisted of two bands at 3448 cm−1 and 3310 cm−1, which correspond to stretching vibrations of the NH2 group. In the case of the Mn(II) and Co(II) complexes, these two bands were moved because of the presence of an amino group, which did not take part in the process of complex formation. Coordination compounds of Fe(III) and Ni(II) had strong, broad peaks in the region of 3650–3200 cm−1, which were assigned to ν(O-H) stretching vibrations due to the presence of H2O molecules.−1 and 1474–1426 cm−1 were attributed to the ν(C=N) and ν(C=C) vibration modes. In all complexes, these bands were in the regions of 1670–1625 cm−1 and 1486–1450 cm−1, respectively. These modes shifted towards the higher wavenumber region after complex formation, which confirmed the involvement of the nitrogen atom in bonding with metal ions. Moving towards the lower wavelengths, we observed bands in the ranges 1394–1306 cm−1 and 970–690 cm−1 that corresponded to the β(CH) and γ(CH) modes, respectively. Their intensities were lower than those in the free ligand as a result of the coordination of the metal (II) ion with two nitrogen atoms. In the range 1220–1020 cm−1 appeared bands attributed to ν(C–N), belonging to alkylamines. In the case of coordinated ligands, these peaks were shifted to lower frequencies (1230–1040 cm−1). From the obtained spectra, it was clear that the ligand behaved as a bidentate ligand. The azomethine nitrogen atom and the nitrogen atom from the pyrazine ring were involved in metal(II) coordination.The strong absorption peaks observed in the pure ligand in the regions 1625–1600 cm50: 400 mg/kg) and the complexes into toxicity class 3 (predicted LD50: 200 mg/kg) for the Ni(II) complex, toxicity class 4 (predicted LD50: 400 mg/kg) for Co(II) complex, and toxicity class 4 (predicted LD50: 1000 mg/kg) for the Mn(II) complex and Fe(III) complexes.The ADME analysis confirmed the effectiveness of the tested compounds crossing the blood–brain barrier, except for the pure ligand . The ligWe screened the free ligand and its four complexes for activity against Gram-positive and Gram-negative bacteria, as well as against yeasts. Vancomycin (Van), ciprofloxacin (Cip), and nystatin (Nys) were used as the standard drugs .S. Typhimurium, E. coli, P. mirabilis, K pneumoniae, and P. aeruginosa), with MIC > 1000 mg/L, except for Mn(L)Cl2, which had mild bioactivity (MIC range 500–1000 mg/L). Moderate bioactivity (MIC range 125–250 mg/L) was exhibited against S. aureus S. epidermidis, M. luteus, E. faecalis, B. subtilis, and B. cereus by the free ligand and against M. luteus, E. faecalis, and B. subtilis by the Mn(L)Cl2 compound. The compound Fe(L)Cl3∙CH3OH had very strong selective, bacteriostatic activity against staphylococci , and the Co(L)Cl2 complex was less active but also revealed good bioactivity against Gram-positive cocci Cl2, which showed moderate activity against yeast (126–500 mg/L). The lowest value (MIC = 250 mg/L) was obtained for the Mn(II) complex against C. parapsilosis.The tested compounds showed no activity against reference Gram-negative bacteria (ve cocci . All com50 = 62.157 ± 3.58 µg/mL) and glioblastoma cells (IC50 ≈ 100 µg/mL for both cell lines) (50 > 100 µg/mL). A statistically significant decrease in cell viability was found for the compounds Co(L)Cl2 and Ni(L)Cl2∙C2H5OH starting at the concentration of 25 µg/mL (2 and Ni(L)Cl2∙C2H5OH revealed cytotoxicity against U87 MG cells   . All tes25 µg/mL . In all 25 µg/mL . The comctively) . The oth2 and Co(L)Cl2 Cl3∙CH3OH showed weak activity on all three tested cell lines.A significant decrease was also observed in the viability of LN229 cells after incubation with the compounds Mn(L)ClCo(L)Cl2 ; however−1) with an accuracy of recording of 1 cm−1 using KBr pellets. The thermolysis of the compounds in the air atmosphere was studied using the TG–DTG technique in the temperature range of 25–800 °C at a heating rate of 10 °C·min−1; TG and DTG curves were recorded on a Netzsch TG 209 apparatus under air atmosphere (v = 20 mL·min−1) using ceramic crucibles. Ceramic crucibles were also used as a reference material.All of the chemicals used for the synthesis were purchased from Sigma-Aldrich, Alfa Aesar, and POCH and used without further purification. The contents of Mn(II), Fe(III), Co(II), and Ni(II) in solid complexes were determined by an F-AAS spectrometer with a continuum source of light and using air/acetylene flame. Absorbance was measured at analytical spectral lines; the limit of quantification was 0.04 mg/L. Solid samples were decomposed using an Anton Paar Multiwave 3000 closed system instrument. Mineralization was carried out for 45 min at 240 °C under a pressure of 60 bar. The contents of carbon, hydrogen, and nitrogen were determined by a vario MICRO Elementar Analysensysteme GmbH. The FTIR spectra were recorded with an IRTracer-100 Schimadzu Spectrometer  of cyanopyrazine was dissolved in 30 mL of MeOH, and 0.5 mL (3.3 mmol) of DBU  was added. The mixture was refluxed for 1 h and cooled, and the carbimidate precipitate was filtered off. Thus, 13.5 g (82%) of the product was obtained. Carbimidate was recrystallized from methanol. Analytical data were in accordance with the literature ,30.First, 1.37 g (10 mmol) of the carbimidate was suspended in 5 mL of EtOH, and 1 mL of 32 mmol) 80% hydrazine hydrate was added. The mixture was refluxed for 15 min and then cooled. The carbohydrazonamide was isolated by filtration. Thus, 0.905 g (66%) of the product was obtained. Carbohydrazonamide was recrystallized from ethanol. Analytical data were in accordance with the literature ,32. mmol 80%−1; 1H NMR : δ 7.15–7.30 , 7.44–7.48 , 7.95–7.97 , 8.54 , 8.74–8.75 , 8.79 , 9.40  ppm [First, 0.685 g (5 mmol) of carbohydrazonamide was dissolved in 15 mL of MeOH while hot, and 0.510 mL (5 mmol) of benzaldehyde was added. The mixture was left at room temperature for 0.5 h and then cooled, and the resulting imine was filtered off. Thus, 0.996 g (89%) of the product was obtained. The product was recrystallized from methanol. M.p. 161–164 °C; FTIR: 3465, 3312, 1625, 1598, 1568, 1520, 1475, 1449, 1167, 1154, 1019, 948, 158, 691, 648, 526, 503, 488 cm Hz) ppm .2 (C12H11N5MnCl2)Mn(L)Cl , (yield 52%), anal. calculated (%): Mn, 15.65; C, 41.05; H, 3,16; N, 19.95. found (%): Mn, 15.58; C, 38.07; H, 2.93; N, 18.56. FTIR spectra : ν(NH) 3448, 3342; ν(C=N) 1628; ν(C=C) 1485; δ(NH) 1447; β(CH) 1373, 1308; ν(C–N) 1175; ν(N-N) 1036; γ(CH) 872, 689.3∙CH3OH (C13H15N5OFeCl3)Fe(L)Cl (419.53 g/mol), (yield 52%), anal. calculated (%): Fe, 13.31; C, 37.22; H, 3.61; N, 16.70. found (%): Fe, 11.12; C, 39.21; H, 3.53; N, 17.31. FTIR spectra : ν(NH) 3374, 3323; ν(C=N) 1669; ν(C=C) 1462; δ(NH) 1445; β(CH) 1401, 1308; ν(C–N) 1176; ν(N-N) 1044; γ(CH) 862, 695.2 (C12H11N5CoCl2)Co(L)Cl (355.11 g/mol), (yield 39%), anal. calculated (%): Co, 16.59; C, 40.58; H, 3.13; N, 19.73. found (%): Co, 16.91; C, 40.09; H, 3.13; N, 19.39. FTIR spectra : ν(NH) 3447, 3339; ν(C=N) 1630; ν(C=C) 1485; δ(NH) 1445; β(CH) 1396, 1306; ν(C–N) 1176; ν(N-N) 1043; γ(CH) 886, 692.2∙C2H5OH (C14H17N5ONiCl2)Ni(L)Cl (400.95 g/mol), (yield 31%), anal. calculated (%): Ni, 14.64; C, 41.94; H, 4.28; N, 17.47. found (%): Ni, 14.44; C, 41.13; H, 4.22; N, 17.00. FTIR spectra : ν(NH) 3421, 3296; ν(C=N) 1636; ν(C=C) 1485; δ(NH) 1445; β(CH) 1394, 1306; ν(C–N) 1173; ν(N-N) 1045; γ(CH) 858, 694.www.way2drug.com/Cell-line accessed on 23 April 2022, a freely available web service for in silico prediction of human cell line cytotoxicity for druglike compounds [http://www.way2drug.com/Cell-line) accessed on 23 April 2022 [The ligand was analyzed using the service at ompounds . The celril 2022 .http://www.swissadme.ch/) (accessed on 23 April 2022); BOILED-Egg [The ADME analysis was performed using the SwissADME service  , Gram-positive bacteria  and fungi .The complexes were screened for antibacterial and antifungal activities by the microdilution broth method using Mueller–Hinton broth for growth of bacteria or Mueller–Hinton broth with 2% glucose for growth of fungi . The min6 CFU/mL for bacteria and 5 × 104 CFU/mL for yeasts (CFU—colony-forming units). After incubation (35 °C for 24 h), the MICs were assessed visually as the lowest concentration of the extracts showing complete growth inhibition of the reference microbial strains. Appropriate DMSO control , a positive control (containing inoculum without the tested derivatives), and a negative control (containing the tested derivatives without inoculum) were included on each microplate.The complexes, dissolved in dimethyl sulfoxide (DMSO), were first diluted to the concentration (1000 mg/L) in an appropriate broth medium recommended for bacteria or yeasts. Then, using the same media, serial twofold dilutions were performed in order to obtain final concentrations of the tested derivatives ranging from 1.95 to 1000 mg/L. Sterile 96-well polystyrene microtitrate plates  were prepared by dispensing 200 µL of the appropriate dilution of the tested derivatives in broth medium per well. The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 0.5 McFarland standard, and 2 µL was added to the wells to obtain final densities of 1.5 × 10Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) was determined by subculturing 5 µL of the microbial culture from each well that showed growth inhibition, from the last positive culture, and from the growth control onto the recommended agar plates. The plates were incubated at 35 °C for 24 h, and the MBC/MFC was defined as the lowest concentration of the extracts without growth of microorganisms. Each experiment was repeated in triplicate. The highest MIC value was noted.5 cells/mL and incubated at 37 °C with 5% CO2 air. When 70–80% culture confluence was achieved, the compounds were added. The cells were incubated for 48 h with the tested compounds in concentrations in the range of 1–100 µg/mL or with DMSO as a vehicle in control cultures (DMSO concentration in the cultures did not exceed <0.5%).The studies were performed on two brain glioblastoma cell lines, U87 MG and LN229, and human skin fibroblast BJ  as a control normal cell line. LN229 cells were cultured in Dulbecco’s Modified Eagle’s Medium , and BJ and U87 MG cells were cultured in Eagle’s Minimum Essential Medium (EMEM) . The culture media were supplemented with 10% fetal bovine serum . The culture conditions were in accordance with the guidelines of ATCC for particular cell line. The cells were seeded into plates at a concentration of 1.5 × 1050 values were determined using the AAT Bioquest IC50 calculator  (accessed on 23 April 2022).Cytotoxicity was evaluated with the MTT assay . After 48 h of incubation with the compounds or DMSO, they were treated with MTT solution for 4 h. Obtained purple formazan crystals were dissolved in DMSO, and the resulting solution absorbance was measured using a PowerWave™ microplate spectrophotometer  at a wavelength of 540 nm . Each exp values were less than 0.05.The results were statistically analyzed with Statistica v. 13 . The data were expressed as mean ± SD. Statistical comparison of values was performed by one-way analysis of variance (ANOVA) and post hoc multiple comparisons with Tukey’s honestly significant difference (HSD) test. All parameters were considered statistically significantly different if N′-benzylidenepyrazine-2-carbohydrazonamide ligand and its four coordination compounds: Mn(L)Cl2, Fe(L)Cl3∙CH3OH, Co(L)Cl2 and Ni(L)Cl2∙C2H5OH. The ligand and all four complexes were stable at room temperature and, when heated, decomposed gradually. Investigations of the spectra of the obtained compounds suggested the bidentate manner of binding the metal centers via the azomethine nitrogen atom and the nitrogen atom from the pyrazine ring. The ADME analysis performed for both the ligand alone and its coordination compounds suggested potent biological activity. The synthesis of the complex was aimed at increasing the likelihood of crossing the blood–brain barrier. In silico research confirmed the assumptions about the anticancer effect. The glioblastoma lines were selected because the tested compounds showed a crossing of the blood–brain barrier and a high probability of being active against these tumor cells. The complexes Co(L)Cl2 and Ni(L)Cl2∙C2H5OH showed significant activity against U87 MG tumor cells (IC50 = 7.69 ± 2.17 and 42.82 ± 4.27 µg/mL respectively). It is also important to highlight that in all cases, complexation decreased cytotoxicity against normal cells. The free ligand exhibited moderate bioactivity against the reference Gram-positive cocci  and Gram-positive, spore forming bacilli (B. subtilis and B. cereus). Even though the tested complexes did not exhibit significantly higher bioactivity, Fe(L)Cl3∙CH3OH showed very strong selective, bacteriostatic activity against staphylococci , and the Co(L)Cl2 complex was less active but had good bioactivity against Gram-positive cocci. Mn(L)Cl2 showed moderate activity against yeasts (126–500 mg/L). The improvement in the biological activity resulting from the complexation highlights the great possibilities of coordination chemistry in terms of possible applications in medicine.We present the method of synthesis of the


Streptomyces jiujiangensis NBERC-24992, which showed significant anti-PRV activity. Eight benzoheterocyclic secondary metabolites, including three new compounds  and five known compounds , were isolated from the broth of NBERC-24992. The structures of the new compounds were identified by using extensive spectroscopic data, including mass spectrometry (MS), nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD). Compound 1 was found to be a novel heterocyclic compound with a tricyclic skeleton from natural product. All compounds were tested for antiviral activity, and 4 (virantmycin) showed an excellent effect against PRV and was better than ribavirin and acyclovir. Our study revealed that chlorine atom and tetrahydroquinoline skeleton were important active moiety for antiviral activity. Virantmycin could be a suitable leading compound for an antiviral drug against PRV.Pseudorabies virus (PRV) is a pathogen that causes Aujeszky’s disease (AD) in animals, leading to huge economic losses to swine farms. In order to discover anti-PRV compounds, we studied the extracts of the strain  Pseudorabies virus (PRV), also named suid herpes virus type 1, is a pathogen that causes Aujeszky’s disease (AD) in animals . The symStreptomyces jiujiangensis NBERC-24992 showed significant anti-PRV activity, and eight benzoheterocyclic secondary metabolites were isolated from the EtOAc extracts. Herein, we report the isolation, structure elucidation, and anti-PRV activity of compounds from the actinomycetes NBERC-24992.At present, no anti-PRV drugs have been approved, and, thus, the discovery of highly active compounds is required. Actinomycetes are Gram-positive bacteria that belong to the unique group of prokaryotic microorganisms which form mycelia and spores, such as fungi . Actinom1–3, virantmycin D–G) and five known compounds   .1 was obtained as a white amorphous powder. The molecular formula is presented as C19H27NO4 by HRESIMS , δ 7.57 , and δ 6.50 ; one methoxyl signal δ 3.27 (s); and three methyl signals δ 1.19 (s), δ 1.03 (s), and δ 0.90 (s). The 13C NMR and DEPT spectra of 1 presents 19 carbon signals, including seven quaternary carbons , four methine carbons , four methylene carbons, and four methyl carbons. One carbonyl (δ 169.9) and six aromatic  signals are displayed in the low field of the 13C NMR spectrum, indicating the presence of one aromatic system and one carbonyl group for five degrees of unsaturation. Thus, 1 has two other ring structures, besides benzene ring.Compound 1H-1H COSY spectrum, the correlations from H-6 to H-2, from H-2 to H-3, from H-8 to H-9, and from H-11 to H-12 are observed  and C-4 (δ 143.4) 25D −15.0° ; UV (MeOH) λmax (log ε): 230 (4.02), 310 (4.41) nm; CD  λmax(Δε) 207nm (−3.55), 233 nm (−0.91), 285nm (+2.03), 318nm (−0.80); 1H NMR  and 13C NMR  data, see m/z 356.1838 [M + Na]+ .Virantmycin D (2): white amorphous powder; [α]25D −127.9° ; UV (MeOH) λmax (log ε): 236 (3.98), 317 (4.44) nm; CD  λmax(Δε) 213 nm (−2.09), 316 nm (−4.94); 1H NMR  and 13C NMR  data, see m/z 372.1787 [M + Na]+ .Virantmycin E (3): white amorphous powder; [α]25D −11.2° ; UV (MeOH) λmax (log ε): 230 (4.02), 318 (4.42) nm; CD  λmax(Δε) 210 nm (−1.60), 225 nm (−0.49), 249nm (+0.26), 288 nm (−1.10), 315 nm (+0.37); 1H NMR  and 13C NMR  data, see m/z 390.1441 [M + Na]+ .Virantmycin F  using the MMFF94S force field with an energy cutoff of 5 kcal/mol. Subsequently, geometry optimizations and frequency analyses were implemented at the B3LYP-D3(BJ)/6-31G* level in CPCM methanol using ORCA5.0.1 . All con50 (50% cell culture infectious dose). After 48 h post infection, the supernatant was discarded and the cells were washed twice. A MTT assay was used for cell viability calculation. The effective antiviral concentration (with PRV virus) and cytotoxicity concentration (without PRV virus) were expressed as EC50 and CC50, respectively. EC50 and CC50 were calculated by using SPSS 16.0 software, and each dilution had four replicates. The selectivity index (SI) was used as the result of EC50/CC50. Ribavirin and acyclovir were used as the positive controls.The compounds were dissolved in DMSO to 20 mg/mL for storage. The Vero cells with 80% confluence in 96-well plates were added with different concentrations of compounds  in a DMEM medium (2% FBS) with or without PRV virus at 100 TCID4 was added in 5 μg/mL and 2.5 μg/mL. After 24 h incubation, the cells were washed with PBS twice, fixed with cold absolute ethanol at 4 °C for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h. The cells were then incubated with 1:200 anti-pseudorabies virus antibody in PBS containing 1% BSA at 4 °C overnight. After washing three times with PBS, the cells were stained with 1:2000 goat anti-rabbit IgG H&L. To show the nucleus of the cells, DAPI was used to stain the cellular DNA. The fluorescence images were observed using an inverted fluorescence microscope . Cell morphology (CPE) was also recorded to show virus proliferation in the cells by using an inverted microscope.The monolayers of Vero cells cultured in 24-well plates were treated with PRV for 1.5 h, the supernatant was discarded, and then compound 50) for 1.5 h, the supernatant was discarded, and the cells were washed twice. Then, compound 4 in different concentrations was added. Cell suspension was collected after 48 h for DNA extraction and qPCR. Total DNA was extracted by using a TIANamp Genomic DNA kit according to the protocol. Amplification reactions were conducted using the PrimeScript RT Master Mix with a CFX96 Real-time PCR Detection System (BioRad). The gene-specific real-time primers were GTGATGACCCACAACGGGTCAGCTCCTTGATGACC (PRV gE) and GACAACAGCCTCAAGATCG (GAPDH). Then, qPCR was conducted at 95 °C for 3 min, for a total of 40 cycles at 95 °C for 5 s and at 60 °C for 30 s, and a final dissociation stage was conducted at 95 °C for 10 s and at 65 °C for 1 min. Each experiment was repeated at least twice, and the relative gene expression level was assessed using the ΔΔCt method. PRV gE gene was detected, and GAPDH served as an internal control to normalize the loading of the template.The Vero cells were infected with PRV  and five known compounds (4–8), were isolated from the broth of S. jiujiangensis NBERC-24992. Compound 1 was found to be a novel heterocyclic compound with a tricyclic skeleton from natural product. Compound 4 (virantmycin) showed an excellent effect against PRV and was better than the positive controls ribavirin and acyclovir. Our study revealed that chlorine atom and tetrahydroquinoline skeleton were important active moieties for antiviral activity. Virantmycin could be a suitable leading compound for an antiviral drug against PRV.Eight benzoheterocyclic secondary metabolites, including three new compounds (


Esophageal squamous cell carcinoma is a severe malignancy for its high mortality and poor prognosis. Mainstay chemotherapies cause serious side effects for their ways of inducing cell death. Oridonin is the main bioactive constituent from natural plants that has anticancer ability and weak side effects. The proteomics method is efficient to understand the anticancer mechanism. However, proteins identified by proteomics aimed at understanding oridonin’s anticancer mechanism is seldom overlapped by different groups. This study used proteomics based on two-dimensional electrophoresis sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) integrated with mass spectrometry and Gene Set Enrichment Analysis (GSEA) to understand the anticancer mechanism of oridonin on esophageal squamous cell carcinoma (ESCC). The results showed that oridonin induced ESCC cell death via apoptosis by decreasing the protein expression of LASP1 and PDLIM1. Esophageal cancer is the seventh most common cancer worldwide and ranks as the sixth leading cause of cancer-related death, with 604,100 3.1%) new cases and 544,076 (5.5%) estimated mortality as reported in 2021 /OD control] × 100%. Representatively, the third day’s IC50s of oridonin on TE-8 and TE-2 are 3.00 ± 0.46 µM and 6.86 ± 0.83 µM, while Gemcitabine, anti-carcinomas chemotherapy medication /OD control] × 100%.Seed cells on 96-well plates by 5000 cells/well and treat with Oridonin, with, as indicated, final concentrations for a different duration. Fix cells in situ with 40%  after washing ,48.5/dish density. After attachment, treat cells with the indicated concentrations of oridonin for 24 h and then harvest. Collect both floating and adherent cells and fix in 70% ethanol at 4 °C overnight [Seed cells on 60 mm Petri dishes at 6 × 10vernight ,49. Then5/dish on 60 mm Petri dishes. After attachment overnight, treat cells with indicated concentrations oridonin for 24 h or 48 h. Collect and wash both floating and trypsinized cells with cold PBS [Seed cells at a density of 6 × 10cold PBS ,51. Dete5/dish on 60 mm Petri dishes and allow attachment overnight. Then, treat with oridonin for 48 h at indicated concentrations. Detect PE Active caspase-3 by following the manufacturer’s introductions . Briefly, resuspend the cell pellets in Cytofix/Cytoperm solution at 1 × 106 cells/0.5 mL. Incubate cells on ice for 20 min. Centrifuge and wash cell pellets twice with Perm/Wash buffer at 0.5 mL buffer/1 × 106 cells. Resuspend cells in Perm/Wash buffer with caspase antibody and incubate for 30 min. At the end of incubation, analyze the PE active caspase-3 on a flow cytometer . Quantify the non-apoptotic (M1) and apoptotic (M2) populations.Seed cells at a density of 6 × 10Lyse the cells treated by oridonin at 40 µM for 24 h or untreated in 2-dimensional electrophoresis (2-DE) rehydration buffer  and vigorously vortex them for 90 min, then ultrasonicate . Remove Excise differentially expressed protein spots from Coomassie blue-stained gels and expose them to an In-Gel Tryptic Digestion Kit  as described by the manufacturer. Briefly, subject excised samples to 200 µL Destaining Solution and incubated at 37 °C for 30 min with constant shaking. Reduce samples with 50 mM TCEP  and allow to incubate for 10 min at 60 °C, followed by alkylation with 106 mM Iodoacetamide (IAA) for 1 h in the dark, at room temperature. Shrink the gel piece with OPTIMA grade acetonitrile for 15 min. Discard the acetonitrile, and allow the gel piece to dry completely. Digest the prepared proteins with 10 ng/µL Trypsin  into a 25 µL Digest Solution. Digest samples at 30 °C for 12 h with constant shaking. The remove the remaining supernatant (35 µL) and place at −20 °C until liquid chromatography-tandem mass spectrometry (LC-MS/MS) acquisition.m/z, and an AGC target at 1 × 106. Set the data-dependent MS2 parameters to 17,500 resolution, AGC target 1 × 105, nce 27. Set the charge exclusion ions to unassigned, +1, +6–8, >8.Load peptides onto a Nano-LC C18 analytical column  with 4% solvent B  and 96% solvent A  at a flow rate of 0.5 µL/min for 10 min. Separate peptides with a Dionex Ultimate 3000 RSLCnano  at 10.1 min with the flow rate decreased to 0.3 µL/min and increased to 22% solvent B over 129.9 min. Increase Solvent B to 45% over 50 min and to 95% over 10 min. Maintain a high organic plateau of 95% solvent B for 19 min before decreasing to 4% over 1 min. Re-equilibrated the column with 4% solvent B for 30 min before the next sample injection for a total of 250 min runtime. Ionize the eluted peptides with a µPAC Flex iON Connect  with a nanoESI emitter  attached to a Nanospray Flex ion Source . Analyze the ionized peptides with a Q-Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer  with MS1 resolution at 70,000, scan range from 350 to 1400 Database searching: All MS/MS samples were analyzed using Sequest . Sequest was set up to search Human_Database_043021.fasta , assuming the digestion enzyme was trypsin. The fragment ion mass tolerance of 0.020 Da and precursor ion tolerance of 10.0 PPM were set for searching. The carbamidomethyl of cysteine was set as a fixed specified modification. Met-loss of methionine, met-loss + Acetyl of methionine, oxidation of methionine, and acetyl of the N-terminus were specified in Sequest as variable modifications.Criteria for protein identification: MS/MS-based peptide and protein identifications were validated by Scaffold . Peptide identification thresholds were set at ≥95.0% probability of the Peptide algorithm . Proteinp-value of the analysis was set at 0.05.R software (version 4.1.2) was used to read and analyze genes encoding the proteins identified by proteomics for their Gene Ontology (GO) annotations. The Bioconductor package “clusterProfiler” (version 4.2.1) was used to perform GO annotations and probe ID was annotated to gene ID through “org.Hs.eg.db” database ,57. The 4, 150 mM NaCl, 1 mM EDTA) with fresh Protease and Phosphatase Inhibitor . Total protein lysates were quantified by the Bradford Protein Assay  and separated on SDS-PAGE, followed by transfer to nitrocellulose membranes . Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody , rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody , and mouse β-Actin antibody . Then, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody to rabbit  and HRP-conjugated IgG to mouse . Enhanced chemiluminescence substrate  was used for development. The images were captured by the iBright FL1000 imaging system .Cells were cultured and treated with 30 µM oridonin for 48 h. Cell lysates were obtained using lysis buffer , 15% Glycerol, 1% Triton X-100, 8 mM MgSO


Rat IVDD model was next established to estimate therapeutic effect of this PBNPs@OBG hydrogel for IVDD treatment in vivo. On the whole, combination of the smart multifunctional hydrogel and nanotechnology-mediated antioxidant therapy can serve as a fire-new general type of therapeutic strategy for IVDD and other oxidative stress-related diseases.Developing smart hydrogels with integrated and suitable properties to treat intervertebral disc degeneration (IVDD) by minimally invasive injection is of high desire in clinical application and still an ongoing challenge. In this work, an extraordinary injectable hydrogel PBNPs@OBG  with promising antibacterial, antioxidation, rapid gelation, and self-healing characteristics was designed via dual-dynamic-bond cross-linking among the oxidized hyaluronic acid (OHA), borax, and gelatin. The mechanical performance of the hydrogel was studied by dynamic mechanical analysis. Meanwhile, the swelling ratio and degradation level of the hydrogel was explored. Benefiting from its remarkable mechanical properties, sufficient tissue adhesiveness, and ideal shape-adaptability, the injectable PBNPs containing hydrogel was explored for IVDD therapy. Astoundingly, the as-fabricated hydrogel was able to alleviate HThe online version contains supplementary material available at 10.1186/s12951-022-01633-0. Low back pain, a chronic disease, increases with age and causes disability and high medical costs around the world , whose mIn view of heavy economic burden and finiteness of surgical intervention, developing new therapeutics for the treatment of IVDD is a substantial need. Stem cells implantation can relieve IVDD or regenerate degenerated disc , 9; howe2O2) has not been clarified. But, systemic application of PBNPs is improper for IVDD due to the avascular structure of disc 2· . T. T1H NMReviously . The FT-reaction , which wreaction , 55. Thereaction . FirstlyThe rhodamine B-dyed dual-dynamic-bond cross-linked hydrogel was injectable, which could be administered by a syringe Fig. A–B. The S. aureus and anti-E. coli activities by agar diffusion method. As we anticipated, both the OBG and PBNPs@OBG had decent antibacterial activities against gram-positive S. aureus and gram-negative E. coli thanks to the presence of borax. The diameter of bacterial inhibition halos around the OBG and PBNPs@OBG were 10.2 ± 1.4 and 8.7 ± 2.0 mm for S. aureus. The zone diameter in the OBG and PBNPs@OBG group reached 5.5 ± 0.4 and 5.5 ± 1.4 mm for E. coli after 12 h. The results of 36 h were similar to those of 12 h  in vitro firstly. When PBNPs was mixed with H2O2, bubbles appeared  was used as a probe to test the ROS generation. H2O2 has been extensively employed to induce an excessive ROS environment in intervertebral disc degeneration [2O2-induced NP cells [2O2-induced group, which manifested decreased MMP. JC-1 exists in the form of green fluorescent monomer chiefly. The other way around, cells treated with the PBNPs@OBG produced higher red fluorescence and lower green fluorescence (JC-1 monomers), indicating the restoration of MMP  to produce H2O and O2 [2O2 decomposes into H2O and O2. Geometrically optimized PBNPs observed from different angles were shown in Additional file 2O2 onto PBNPs with adsorption energy of 0.76 eV. The H2O2* was decomposed into two HO* with an activation energy of 1.15 eV. The decomposition is an exothermic reaction that releases 1.22 eV. One H of OH* is transferred to another HO* to form H2O*, which is then desorbed with an energy of 0.79 eV. Another H2O2 molecule participates the reaction that generates an adsorbed O* and a H2O. The two O* form O2* with an activation energy of 1.59 eV. Then O2* desorbs from PBNPs, giving the final products to be H2O and O2. Yellow color in the charge density difference (CDD) implies charge gain and blue color implies loss of electric charge. All these figures are shown in the same iso-surface level at 0.003 e/(Bohr^3). Charge gain is observed in the bonding area between O atom and PBNPs substrate for H2O2, indicating an interaction between H2O2 and PBNPs. The area between O–O bond in H2O2 shows the depletion of charge, as indicated by blue color. Before H2O2 adsorption, Fe atom in PBNPs initially shows a charge loss of 0.89 e− by Bader charge analysis. After H2O2 adsorbed to the Fe atom, the charge loss of Fe decreases to 0.86 e−, telling electrons are slightly transferred from H2O2 to Fe atom. The loss of electrons of H2O2 is consistent with CDD results that there is a deficiency of charge in O–O bond area, which can accelerate the breakage of O–O bond. The O–O bond is expected to break to form two OH due to the charge loss. The OH shows charge gain between O and PB, indicating strong interaction. From Bader charge analysis, the Fe atoms with OH adsorbed show an electron loss of 0.95 and 1.50 e−, respectively, much higher than the original state without OH adsorbed, which are 0.89 and 1.44 e−, respectively. The Bader analysis also shows the charge is transferred to OH, and consistent with CDD results. Within OH, there is a charge depletion between O–H bonds, which implies H can be broke facilely. One H2O will be formed when H in one of the OH transferred to another. With the loss of one H for OH, the electron gains of O changed from 1.00 to 0.50 e−. H contributes a lot to the accumulation of electrons in O atom clearly. Single O shows forceful interaction with PBNPs as illustrated by the large area of yellow color between single O and PBNPs. The electron loss of Fe from 0.89 to 1.04 e− before and after O adsorption demonstrates that electrons are transferred to O. The two O atoms will merge together to form O2 molecule eventually. The tangy reciprocity between O atom and PBNPs is also reflected by the high activation energy as discussed earlier. O2 molecule shows a less charge gain between O and PBNPs compared with single O atom, which implies an easier desorption process. Bader charge manifests that the electron loss Fe for O2 adsorbed structure is similar to that without O2, further confirming the pregnable interaction between O2 and PBNPs, and an easier desorption of O2 molecule   staining was used to investigate fibrous tissue, margins, and NP morphology. From 4 to 8 weeks, in the Acupuncture group, the NP cells were replaced by disorganized hypocellular fibrocartilaginous tissue, and at the meantime the AF structure was wrecked along with atrophied NP volume. 8 weeks after operation, the AF structure was destructed more seriously compared with 4 weeks. In the early stage (4 W), the PBNPs@OBG group showed more intact composition than the OBG and PBNPs group, merely manifested as mildly reduction of NP cells. NP in the OBG and PBNPs group collapsed gradually and the borders were blurred and indistinct. After 8 weeks, regions of the NP were replaced by the annulus fibrosus in large part, along with worsened NP status. Alternatively, the PBNPs@OBG group displayed a much more integrated morphological arrangement Fig. A. SafranThe cytotoxicity was determined by CCK-8 assay on human NP cells using the exudate of the OBG and PBNPs@OBG hydrogel  for disc replacement [It is still a challenge to search appropriate delivery strategies to alleviate IVDD. Gan et altibility , while txidation , howeverlacement . NeverthIn summary, we prepared a novel dual-dynamic-bond cross-linked injectable self-healing hydrogel with antibacterial and antioxidant properties as well as excellent mechanical characters for IVDD repair. The smart PBNPs@OBG hydrogel could be injected into the degenerated intervertebral disc via minimal invasive method and provide appropriate mechanical support. By taking advantages of the sustained release of PBNPs from the PBNPs@OBG hydrogel, long time retention of PBNPs in disc and effective antioxidant therapy could be achieved. Profit from versatile integration of hydrogels, as-fabricated PBNPs@OBG hydrogel could protect NP cells against ROS overproduction, restore the disc height, attenuate the decrease of water content, and reverse the IVDD disordered microenvironment. We believe that the smart multifunctional hydrogel could serve as a promising candidate for IVDD treatment.Additional file 1: Figure S1. Abridged general view of PBNPs formation. Figure S2. Dynamic light scattering (DLS) of the PBNPs. Figure S3. Thermogravimetry-differential scanning calorimetry (TG-DSC) analyses of the PBNPs. Figure S4. Adsorption isotherms of the PBNPs, demonstrating its mesoporous structure. Figure S5. Photographs of OBG hydrogel formation. Figure S6. SEM images of the OBG and PBNPs@OBG. Scale bar = 100 µm. Figure S7. EDS mapping images of the PBNPs@OBG. Scale bar = 100 µm. Figure S8. In vitro degradation percentages of the PBNPs@OBG in vitro, in PBS with different pH values of 7.4 and 6.5 at 37 °C. Figure S9. Photograph of the OBG and PBNPs@OBG in compression tests. Figure S10.A Weight lifting ability for the glass adhered by the PBNPs@OBG hydrogel. B Adhesiveness strengths of the OBG and PBNPs@OBG . Figure S11. H2O2 decomposed into H2O and O2 in the presence of PBNPs within 10 min. Figure S12. JC-1 staining quantified as the ratios of Red/Green fluorescence intensities. . Figure S13.A–B Effect of the PBNPs@OBG on NPs apoptosis after H2O2 treatment by annexin V-FITC and propidium iodide staining. . Figure S14. Geometrically optimized PBNPs observed from different angles. Figure S15. Charge density difference of different species adsorbed on the PBNPs substrates. The yellow color represents charge accumulation, while green color is the charge loses. Figure S16. Immunofluorescence staining of aggrecan, collagen II, MMP3, and MMP13 in NP tissues at 8 weeks after injection. Scale bar = 50 µm. Figure S17.A Living/dead staining images of NP cells in vitro. Scale bar = 200 µm. B CCK-8 assay of NP cells after being treated with different materials. Figure S18. Blood routine examination results of WBC at 4 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S19. Blood routine examination results of WBC at 8 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S20. Blood routine examination results of RBC at 4 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S21. Blood routine examination results of RBC at 8 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S22. Blood routine examination results of platelets at 4 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S23. Blood routine examination results of platelets at 8 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S24. Standard blood biochemical examination at 4 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S25. Standard blood biochemical examination at 8 weeks. 1. Control; 2. PBNPs@OBG; 3. OBG; 4. OBG; 5. Acupuncture . Figure S26. H&E staining of heart, liver, spleen, lung, and kidney at 4 weeks after operation. Scale bar = 100 µm. Figure S27. Histological screening at 8 weeks after operation. Scale bar = 100 µm. Table S1. Primer used for Qpcr.Additional file 2. Video of OBG hydrogel formation.


While, FTAs significantly decreased the expression levels of IL-1β, IL-6, MMP-1, MMP-3, PGE2, TGF-β, and VEGF. FTAs also enhanced the progress of apoptosis and arrested the cell cycle at G0/G1 phase to prevent excessive cell proliferation. In addition, FTAs inhibited the hyperactivity of NF-κB and JAK/STAT signaling pathways, and regulated the cascade reaction of mitochondrial apoptosis signaling pathway. The results suggested that FTAs exerted anti-inflammatory effects by inhibiting NF-κB and JAK/STAT signaling pathways, promoted apoptosis by stimulating mitochondrial apoptosis signaling pathway, and inhibited cell proliferation by modulating cell cycle progression.Aconiti Lateralis Radix Praeparata (“Fuzi” in Chinese) is one of the traditional herbs widely used to intervene rheumatoid arthritis (RA), while Fuzi total alkaloids (FTAs) are the main bioactive components. However, the treatment targets and specific mechanisms of FTAs against RA have not been fully elucidated. The purpose of the present study was to confirm the anti-rheumatism effects of FTAs and reveal its potential molecular mechanisms. In TNF- Rheumatoid arthritis (RA) is an autoimmune disease characterized by a systemic immune inflammatory response, which can accompany synovial tissue hyperplasia, joint swelling, bone destruction, chronic pain, and even loss of functions. The patient’s living situation is severely deteriorated with the progress of RA . The etiAconitum carmichaelii Debx. Aconiti Lateralis Radix Praeparata (“Fuzi” in Chinese) was first recorded in “Sheng Nong’s herbal classic,” and has a long history of clinical application in China. In the theoretical system of TCM, Fuzi is often combined with other herbs to form a common multi-drug combination pattern, which appears in numerous TCM medical preparations, such as “Shaoyao-Gancao-Fuzi Decoction,” “Guizhi-Shaoyao-Zhimu Decoction,” and recommended for the treatment of RA . DMEM/high glucose, PBS, PMSF, RIPA lysis buffer, phosphatase and protease inhibitor, SDS-PAGE gel kit, goat anti-rabbit IgG-HRP, histone H3, TBS buffer, Cell membrane-breaking kit, DAPI, antifade mounting medium, JAK2, Bax, IκBα were obtained from Servicebio Co., Ltd. . Trypsin 0.25% (1X) solution, penicillin-streptomycin were obtained from HyClone Co., Ltd. . FBS was obtained from Merck/Sigma Co., Ltd. . Commercially available ELISA kits for IL-1β, IL-6, MMP-1, MMP-3, PGE2, VEGF, TGF-β, and Annexin V-FITC and PI dual-staining apoptosis kit were obtained from Elabscience Biotechnology Co., Ltd. . BCA protein quantitative kit, protein loading buffer, ECL chemiluminescence kit (enhanced), CCK-8 kit were obtained from Biosharp Co., Ltd. . Tween 20, BSA, glycine were obtained from Bioforxx Co., Ltd. (Germany). Bcl-2, caspase-3, p-p65, β-actin were obtained from Affinity bioscience Co., Ltd. . IKKβ, p-IKKβ were obtained from Abcam Co., Ltd. . p-IκBα, STAT3 were obtained from Biosynthesis Biotechnology Inc. . P65 was obtained from Proteintech Group, Inc. . Cell cycle staining kit and cell membrane-breaking kit were obtained from Multisciences (Lianke) Biotech Co., Ltd. . Cy3-labeled goat anti-rabbit IgG was obtained from Beyotime Biotechnology Co., Ltd. . TPCA-1 was obtained from Selleck Chemicals Co., Ltd. .MH7A cells were obtained from Jennio Biotech Co., Ltd. . TNF-The raw Fuzi from Good Agricultural Practice (GAP) planting base was provided by Zhongba Fuzi Co., Ltd. . 3 kg of raw Fuzi was soaked in 0.1 mol/L hydrochloric acid  for 3 h and heated for extraction twice, 2 h each time. Then, we concentrated the extract to about 1 g/ml, adjusted the PH to 3, and passed it through styrene strong acid cation exchange resin at a flow rate of 5–6 BV/h. After adsorption, the extract was washed with distilled water to remove impurities. Immediately, a mixture of 70% ethanol containing 9% ammonia was eluted at a flow rate of 2-3 BV/h until there was no color reaction between the effluent and bismuth potassium iodide. Finally, the eluent was collected and concentrated to obtain about 7.0 g of FTAs.The quantitative analysis of FTAs was accomplished in UPLC-MS system with Waters ACQUITY BEH C18 column . The mobile phase of chromatographic separation was consisted of solvent A (acetonitrile) and solvent B . The gradient elution conditions was set as follows: 80%–0% B at 0–5 min, 0% B at 5–7 min, 0%–80% B at 7–8 min, 80% B at 8–10 min. The flow rate was set at 0.3 ml/min and the column temperature was maintained at 35°C. After equilibration, 0.2 μl sample was injected for detection.Aconitine, hypaconitine and mesaconitine were selected as the reference compounds for contents determination of FTAs, and they were 4.71 μg/mg, 59.11 μg/mg and 35.22 μg/mg in concentration, respectively. The chemical fingerprints of FTAs were displayed in 2 and 37°C, with saturated humidity. MH7A cells were passaged approximately every 3 days, and the 3-9 generations were used for all experiments. FTAs were dissolved in 0.1% DMSO and prepared to the corresponding concentrations with culture medium. The Ethics Committee of Sichuan Provincial People’s Hospital reviewed the experimental plan of this study.MH7A cells are highly consistent with the pathologic characteristics of parental fibroblast-like synoviocytes (FLSs), and they are a kind of mature immortalized FLS line widely used in RA studies. MH7A cells were sub-cultured in DMEM/high glucose containing 15% FBS and 1% penicillin-streptomycin. The environment was kept constant at 5% CO3/well for 24 h, followed by intervention with the different concentrations of FTAs . After this, 10 μl CCK-8 reagent was supplemented into all the culture wells and the cells were incubated for another 3 h, at 37°C. Finally, at 450 nm wavelength, the optical density (OD) value of the microtiter plate was detected by the microplate reader. (drug[OD]-blank[OD])/(control[OD]-blank[OD]) × 100% was used to calculate the viability of MH7A cells.Due to the high toxicity of FTAs, it is necessary to determine the effects of FTAs on MH7A cells viability via CCK-8 assay. MH7A cells were incubated in 96-well plate with the density of approximately 5×10α and treated with low, medium and high concentrations of FTAs  for 24 h. After incubation, the supernatant samples of MH7A cells were collected and the contents of IL-1β, IL-6, MMP-1, MMP-3, PGE2, TGF-β, and VEGF were detected by ELISA kits. In addition, MH7A cells were incubated with 0.5 μM TPCA-1 for 24 h, and then cultured with 20 ng/ml TNF-α and 1 μg/ml FTAs for another 24 h. Finally, the contents of IL-1β and IL-6 were detected by ELISA kits.MH7A cells were incubated in 96-well plate, then exposed to 20 ng/ml TNF-α , p-IκBα , IKKβ , p-IKKβ , p65 , p-p65 , Bcl-2 , Bax , caspase-3 , JAK2 , STAT3  and β-actin , and incubated overnight at 4°C. Following this, the incubation was continued with HPR-conjugated antibody  at room temperature for another 1 h. Lastly, the protein signals were displayed after being processed by ECL kit, and the gray levels of the bands were read and recorded with ImageJ software.The total proteins of MH7A cells were obtained after treatment with RIPA lysis buffer supplemented with a mixture of phosphatase and protease inhibitors. After centrifugation, the supernatants were collected and transferred to BCA protein quantitative kit for protein concentration detection. Before transferring to PVDF membranes, the equal amount of protein samples were fractionated by 10% SDS-PAGE. Subsequently, the TBS-T buffer containing 5% skimmed milk was selected to block the PVDF membranes for 1.5 h. Immediately, the membranes were coated with the diluted primary antibodies of IκBFor immunofluorescence assay, MH7A cells were placed on the slides, treated with BPS-Triton X-100 (0.1%) mixed reagent for 20 min, and blocked with serum-supplemented medium at room temperature for 35 min. Immediately, the primary antibodies were diluted to the corresponding concentrations with PBS, and the cells were treated with p-p65, Bax overnight at 4°C. Then, the Cy3-labeled goat anti-rabbit IgG was added and treated for another 50 min. Subsequently, the cells were washed twice with PBS and further incubated with DAPI fluorescent dye for 10 min. Finally, the Nikon fluorescence microscope system was selected to photograph the above slides, and the fluorescence intensity was recorded with Image-Pro Plus v.6.0  processing program.Before the experiments, the MH7A cells were washed and centrifuged to discard the supernatants. For apoptosis assay, 500 μl diluted 1×Annexin V binding buffer was selected to re-suspend the MH7A cells. Immediately, 3 μl of Annexin-V-FITC and PI staining reagents were supplemented to the cell suspensions, respectively. After being mixed, the cells were incubated at room temperature for 15 min, under light-avoiding condition. Then, the apoptosis rate of MH7A cells was detected by Bechman Coulter flow cytometer. For cell cycle assay, the MH7A cells were treated with 1 ml DNA staining reagent and 10 μl permeabilization reagent. After being mixed, the cells were incubated at room temperature for 30 min, under light-avoiding condition. Then, the cell cycle of MH7A cells was detected by Bechman Coulter flow cytometer. The numbers of gap 1- (G1-), synthesis- (S-), and gap 2/mitotic- (G2/M-) phase cells were quantified using Modfit cell cycle analysis software.t-test, and p < 0.05 was regarded to represent statistically significant.All the experimental results were the average of three repeated experiments, and recorded as mean ± SD. SPSS 25.0  software was applied to perform statistical analysis of the raw data. The difference analysis among the multiple groups was carried out by One-Way ANOVA and Student’s Due to the high toxicity of FTAs, medication safety must be ensured first. The MH7A cells were intervened by different concentrations of FTAs for 12, 24, and 48 h, and the cytotoxicity of FTAs was detected by CCK-8 method. As demonstrated in α-induced inflammatory mediators were analyzed by ELISA assay. As demonstrated in α significantly boosted the levels of IL-1β, IL-6, MMP-1, MMP-3, PGE2, TGF-β, and VEGF. While the various concentrations of FTAs could significantly reverse the TNF-α-induced augmented expression of the above inflammatory mediators. In conclusion, the results suggested that FTAs could significantly inhibit inflammatory responses in MH7A cells induced by TNF-α.The imbalance of cytokines expression is closely associated with the autoimmune inflammation of RA. Therefore, some biological agents play an anti-RA role by targeting the relevant cytokines. However, whether FTAs can regulate the expression of cytokines needs to be further explored, and in the present study, the effects of FTAs on TNF-α stimulation significantly upregulated the contents of phosphorylated p65, IKKβ and IκBα, while FTAs could abolish the abnormal phosphorylation of p65 , IKKβ  and IκBα (except for 1 μg/ml). Furthermore, TNF-α stimulation also significantly increased the protein signal intensity of JAK2 and STAT3, while high concentration of FTAs significantly decreased the protein signal intensity of JAK2 and STAT3  and caspase-3, while Bcl-2  was decreased on the contrary. As demonstrated in In order to further explore the potential targets involved in the promoting effect of FTAs on apoptosis, western blot and immunofluorescence were employed to analyze the levels of apoptosis-associated proteins in mitochondrial apoptosis signaling pathway. As demonstrated in α single treatment groups, different concentrations of FTAs could significantly increase the number of cells arrested at G0/G1 phase (except for 1 μg/ml), while decrease the number of cells arrested at S (except for 1 μg/ml) and G2/M phases, respectively. The above results indicated that FTAs could exert anti-RA effects by arresting the G1/S transition, and thus inhibited the hyperproliferation of MH7A cells.In addition to focusing on the effects of FTAs on apoptosis, flow cytometry was also used to investigate whether FTAs interfered with the cell cycle of MH7A cells. As demonstrated in in vitro experiments to further investigate the pharmacological mechanisms of Fuzi.Chinese herbal medicine has the pharmacological characteristics of “multiple components, multiple targets, multiple links and multiple effects,” and has irreplaceable capability in the treatment of various difficult and miscellaneous diseases . Under tβ, TNF-α, IL-6, IL-17, and IL-23 are closely related to the bone metabolism, inflammation and immune processes of RA. For example, IL-1β induces inflammation by activating monocytes and macrophages. It can also induce the fibroblasts proliferation, osteoclasts and chondrocytes activation, resulting in synovial hyperplasia, bone absorption and cartilage damage (α and IL-6 are involved in the induction of osteoclastogenesis. Due to abnormal expression of the above cytokines in RA patients, osteoclasts are dysfunctional in regulating bone mineral density and bone absorption, ultimately leading to bone erosion (β exerts a complex immunomodulatory effect in the progression of RA disease, and numerous evidences indicate that TGF-β is capable of inducing cell proliferation, differentiation, migration, apoptosis and even angiogenesis, bone and cartilage repair and destruction. However, the relationship between it and the pathophysiological process of RA needs further study (β, IL-6, MMP-1, MMP-3, PGE2, TGF-β, and VEGF in TNF-α-induced MH7A cells, suggesting that FTAs exert anti-inflammatory functions by regulating the synthesis and release of these cytokines.Cytokines are important mediators in regulating immune response, immune pathology and cell differentiation. In the pathophysiological process of RA, the imbalance of cytokines may cause immune cells to infiltrate synovium and joint tissues . The acte damage . TNF-α a erosion . After b erosion . The abn erosion . While P erosion . In addi erosion . TGF-β eer study . The aboin vitro cultured synovial fibroblasts. Studies in animal models of arthritis also suggest that NF-κB exhibits a strong driving force in the pathophysiological process of RA (β, IL-6, TNF-α, and MMPs, can directly or indirectly induce related receptor proteins to activate NF-κB (The activation of NF-κB transcription family members is one of the key links to induce chronic inflammatory changes in RA. The NF-κB signaling pathway is activated in synovial tissues of RA patients and ss of RA . Clinicass of RA . Some inte NF-κB . During te NF-κB . In addite NF-κB .α, and IL-17, which are closely associated with RA. Among them, TNF-α and IL-17 synergistically increase the synthesis and release of MMPs, thereby activating chondrocytes and FLSs (β and IL-6 were still observed, but the efficacy was significantly reduced. Similar to other Chinese herbal medicines, FTAs have “multi-components” and “multi-targets” characteristics and are involved in regulating many signaling pathways, but according to the above results, it is certain that NF-κB and JAK/STAT are the important regulatory pathways of FTAs that exert anti-RA effects.Similarly, JAK/STAT signaling pathway has a non-negligible ability in chronic inflammation, which is also a potential therapeutic target for RA, and biological agents such as JAK inhibitors have been put into clinical application . Differeand FLSs . The aboα-induced MH7A cells, and the inhibitory effects enhanced with higher dose. Meanwhile, FTAs promoted apoptosis of MH7A cells by acting on mitochondrial apoptosis signaling pathway, and guided cell cycle arrest at G0/G1 phase to prevent excessive cell proliferation. The results suggested that FTAs could exert anti-RA effects by inducing cell apoptosis and suppressing cell proliferation.In healthy humans, a delicately regulated process of apoptosis is essential for coordinating the growth and death of tissue cells within the body. However, in RA patients, FLSs have apoptotic resistance, leading to synovial hyperplasia accompanied by the release of related cytokines . The proTo sum up, these experimental results confirmed that FTAs could treat RA by reversing the imbalance of pro-inflammatory cytokines, preventing excessive cell proliferation and promoting apoptosis. While, NF-κB, JAK/STAT and mitochondrial apoptosis signaling pathways were the main regulatory targets . This st


The toxic equivalent concentrations in birds from Qilianyu Island were significantly and positively correlated with PCBs concentrations, indicating that high concentrations of non- and mono-ortho-PCB congeners may induce adverse effects on bird species.Six biota species were collected from Qilianyu Island, South China Sea to determine the bioaccumulation and biomagnification of polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane and its metabolites (DDTs). Concentrations of ΣPCBs and ΣDDTs in biota from Qilianyu Island ranged from 6.88 to 519.1 ng/g lipid weight (lw) and 7.0 to 19,413 ng/g lw, respectively. Significant differences for PCBs and DDTs concentrations were found among the six biota species from Qilianyu Island. The levels of PCBs and DDTs in intermediate egret were significantly higher than the other five biota species, which can be attributed to their different feeding and living habits. Significantly negative relationships between concentrations of PCBs and DDTs and δ13C values in the six biota species confirmed that dietary source is an important factor to determine the levels of PCBs and DDTs in biota species. ΣPCBs, ΣDDTs, PCB 28/31, PCB 52, and  Polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) are two typical types of chlorinated persistent organic pollutants (POPs) and have been of great concern for several decades due to their persistence, toxicity, bioaccumulation and long-range transport. PCBs were mainly used as dielectric and hydraulic fluids in industrial products such as transformers, capacitors, and electric motors from the 1930s to 1970s . It is eThe South China Sea (SCS) is one of the richest areas of marine biodiversity in the world, surrounded by several developing countries that are considered as potential sources of POPs ,9. POPs In this study, six biota species, including two birds and four fishes, were collected from Qilianyu Island, SCS and analyzed for PCBs and DDTs. The main objective of this study was to investigate the bioaccumulation and biomagnification of PCBs and DDTs in biota from Qilianyu Island. The ecological risks of PCBs and DDTs to biota species were also evaluated.Ardea intermedia, n = 6), red-footed booby , peacock grouper , goldspot seabream , Japan surgeonfish , and striated surgeonfish , were collected from Qilianyu Islands, SCS. Intermediate egret is a migratory bird, and the other five species are native. Fishes were collected by fishing nets. Birds were found dead or stranded on the beaches of Islands. All biota samples were immediately transported to the laboratory and stored at −20 °C until chemical analysis.A total of six biota species, including intermediate egret (v/v = 1:1) for 45 min. The extraction solutions were concentrated and converted to hexane. A total of 1 mL of the extract was used for the gravimetric determination of lipid content. Then, 9 mL of the extract was used for PCBs and DDTs analysis, added into concentrated sulfuric acid to remove lipid, and extracted with hexane. The extraction solutions were cleaned up on a multilayer column filled with 1 cm anhydrous sodium sulfate, 8 cm acidified silica, and 8 cm neutral silica from top to bottom. The extract was eluted with 30 mL hexane/dichloromethane (v/v = 1:1), and the eluate was concentrated to near dryness under a gentle nitrogen flow and reconstituted in 100 μL of hexane. Internal standards  were spiked before instrumental analysis.Muscles were excised from each biota sample and then freeze-dried and ground. Approximately 1 g of muscle samples were spiked with surrogate standards  and then treated by microwave-assisted extraction with 30 mL acetone/hexane , and its metabolites  were analyzed by an Agilent 7890 gas chromatograph coupled with an Agilent 5975B mass spectrometer using electron impact in the selective ion-monitoring mode and separated by a DB-5MS  capillary column. The heating program was as follows: the initial oven temperature was set at 120 °C, then increased to 180 °C at a rate of 5 °C/min, further increased to 240 °C at a rate of 1 °C/min, and finally raised to 290 °C at a rate of 6 °C/min (held for 17 min). Then, 1 μL of sample was injected in the pulsed splitless mode. The monitored and quantitative ions of PCBs and DDTs were given elsewhere [Thirty-four PCB congeners , DDT  and triplicate spiked matrices were passed through the whole analytical procedure to ensure method QC. PCB 28 was detected in the procedural blanks with mean concentrations of 2.88 ng/mL and subtracted from the samples. The average recoveries of PCB congeners in the spiked blanks and matrices ranged from 106.5% to 129.4% and 109.6% to 124.1%, respectively. The relative standard deviations were less than 6%. The mean recoveries of surrogate standard in all samples were 70.1 ± 2.7% for PCB 30, 74.3 ± 2.8% for PCB 65, and 74.7 ± 2.2% for PCB 204, respectively. Method detection limits (MDLs) were defined as 3 times the signal-to-noise levels for the undetected compounds in blanks. MDLs were set as three times the standard deviation of the target value in blanks. Based on the average lipid weight (lw) of samples, the MDLs ranged from 0.15 to 1.10 ng/g lw for PCBs and 0.19 to 6.76 ng/g lw for DDTs, respectively.13C and ẟ15N analysis were freeze-dried and ground into powder. A roughly 0.4 mg sample was placed into a tin vessel and analyzed for stable carbon and nitrogen isotopes by a Flash 2000 series elemental analyzer coupled to a Delta V isotope ratio mass spectrometer . The analytical precision was less than 0.2‰. The abundance of stable isotope values is calculated by the following formula:13C or δ15N, and R is the corresponding ratio of 13C/12C or 15N/14N.The samples for ẟp < 0.05. Concentrations were not normally distributed and log10 transformed before statistical analysis. Independent-sample t-test was used to compare the differences of δ13C and δ15N values between birds and fishes. One-way analysis of variance (ANOVA) was used to analyze the differences in concentrations of PCBs and DDTs among the six biota species. Linear regression analysis was used to investigate the correlations between concentrations and δ13C values, concentrations and δ15N values, PCBs, and TEQ concentrations in biota.Concentrations were expressed as a lipid weight (lw) basis. Statistical analysis was performed with SPSS 22 . The level of significance was set as 13C and δ15N values are often used to infer the carbon source and trophic level in biota [13C and δ15N values were observed in biota from Qilianyu Island (p < 0.001). The δ13C values increased the order of intermediate egret (−23.3 ± 0.41‰) < red-footed booby (−18.7 ± 0.13‰) < Japan surgeonfish (−14.5 ± 0.29‰) < peacock grouper (−14.2 ± 0.39‰) < striated surgeonfish (−13.1 ± 0.07‰) < goldspot seabream (−12.5 ± 0.24‰)  > goldspot seabream (8.92 ± 0.13‰) > intermediate egret (8.29 ± 0.31‰) > red-footed booby (8.24 ± 0.11‰) > striated surgeonfish (6.36 ± 0.15‰) > Japan surgeonfish (5.26 ± 0.06‰). The δ15N values of two herbivorous fishes were significantly lower than those of two carnivorous fishes and two bird species, suggesting that the two fishes had a lower trophic level.δ± 0.24‰) . The δ13F = 47.3, p < 0.001). The levels of PCBs in intermediate egret were significantly greater than the other five biota species (p < 0.001), which can be explained by their different feeding and living habits. Intermediate egret is a migratory bird, and the other five biota species often live in the surrounding of Qilianyu Island. In addition, significantly negative relationships between PCBs concentrations and δ13C values were found among the six biota species  > red-footed booby (90.5 ± 11.0 ng/g lw) > goldspot seabream (85.1 ± 12.6 ng/g lw) > peacock grouper (54.7 ± 10.1 ng/g lw) > striated surgeonfish (32.4 ± 9.0 ng/g lw) > Japan surgeonfish (30.6 ± 7.8 ng/g lw) a. Signif species a, furtheo,p′-DDE, o,p′-DDD, p,p′-DDMU, p,p′-DDE, and p,p′-DDT were detected in the intermediate egret. o,p′-DDE and p,p′-DDE were detected in the red-footed booby. For four fish species, only p,p′-DDE were detected. The concentrations of DDTs in biota from Qilianyu Island ranged from 7.0 to 19,413 ng/g lw. The highest mean concentrations of DDTs were found in the intermediate egret (5595 ± 2847 ng/g lw), followed by red-footed booby (82.3 ± 29.3 ng/g lw), goldspot seabream (31.6 ± 5.5 ng/g lw), peacock grouper (30.3 ± 6.8 ng/g lw), striated surgeonfish (23.3 ± 9.0 ng/g lw), and Japan surgeonfish (16.0 ± 3.2 ng/g lw) (p < 0.05), which were similar to PCBs distribution pattern. Significantly negative relationships between DDTs concentrations and δ13C values were seen among the six biota species (ng/g lw) b. The co species b, indicameta-para positions on the phenyl rings, the four PCB congeners were slowly cleared in bird species [Penta-, hexa-, and hepta-PCBs were the main homologue profiles in the intermediate egret and red-footed booby, contributing more than 79% to the total PCBs. PCB 153, 180, 118, and 138 were the main PCB congeners in bird species . Similarp,p′-DDE was the most abundant compound in six biota species from Qilianyu Island and contributed more than 95% to the total DDTs  also indicated that DDTs in biota from Qilianyu Island were derived from historical use rather than recent input.tal DDTs . The rat15N values were used to investigate the biomagnification of these contaminants. Concentrations of ΣPCBs and PCB 28/31 were positively and significantly correlated with δ15N values in all six biota species  for wildlife risk assessment suggested by the World Health Organization (WHO) were used to evaluate the toxic equivalent (TEQ) concentrations of these PCBs [ortho-PCB congeners (PCB 77 and 126) and mono-ortho-PCB congeners  were detected in bird samples. The concentrations of TEQs in intermediate egret and red-footed booby ranged from 270–2586 pg/g lw and 0.07–0.11 pg/g lw, respectively. The levels of TEQs in the intermediate egret were much higher than red-footed booby (p < 0.001). The TEQ concentrations in birds from Qilianyu Island were significantly and positively correlated with PCBs concentrations (ortho-PCB congeners may induce adverse effects on bird species.Non- and mono-ese PCBs . Becausetrations . A signiIt has been reported that bird populations had declined due to bioaccumulation of DDTs . The con13C values. The δ15N values of two herbivorous fishes were significantly lower than those of two carnivorous fishes and two bird species, suggesting that the two fishes had lower trophic level. Significant differences for PCBs and DDTs concentrations were found among the six biota species from Qilianyu Island. The levels of PCBs and DDTs in intermediate egret were significantly higher than the other five biota species, which can be attributed to their different feeding and living habits. Dietary source is an important factor to determine the concentrations of PCBs and DDTs in biota species. ΣPCBs, ΣDDTs, PCB 28/31, PCB 52, and p,p′-DDE were biomagnified in the biota species from Qilianyu Island, and native species are suitable for investigating the biomagnification of these contaminants. The high concentrations of non- and mono-ortho-PCB congeners may induce adverse effects on bird species, and the high concentrations of DDTs may cause reproductive effect on intermediate egret.This study investigated the bioaccumulation and biomagnification of PCBs and DDTs in six biota species from Qilianyu Island, SCS. Biota with a more marine feeding strategy had higher δ


During fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes.gadE), a periplasmic chaperone (hdeB), and reactive oxygen species (ROS) scavengers (sodB and katE). Directed evolution was used to construct an acid-responsive asr promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5–6), first by cell growth using the laboratory Escherichia coli strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing E. coli strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8.In this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system (E. coli upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.Our results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of The online version contains supplementary material available at 10.1186/s12934-022-01795-4. Microbial production of chemicals and fuels is a growing industry, and has been playing an increasingly significant role in the global economy, e.g., it accounts for more than 2% of the national gross domestic product (GDP) in the USA . MicroorOver the years, several genome-wide evolution engineering strategies have been widely used to enhance the stress resistance for microbial cells . Among tSOD1, the glutamate-cysteine ligase gene GSH1, the glutathione-disulfide reductase gene GLR1 of the antioxidant system, the glucose-6-phosphate dehydrogenase gene ZWF1 of the glycolysis pathway, and the acetyl-CoA synthase gene ACS1, which is involved in the acetyl-CoA synthesis, leading to an improved ethanol titer by 49.5% at the shake flask scale [Escherichia coli DH10B, the DNA binding protein gene hu, which is involved in DNA protection, the RNA binding protein gene rbp, which is involved in RNA protection, and the ATP-dependent serine protease gene clpP, which is involved in misfolded protein degradation, were overexpressed, leading to an increase in survival rate of over 600-fold for the best strain upon acid shock at pH 1.9 [Synthetic biology offers a useful approach to design synthetic acid-tolerance modules or other stress-tolerance modules based on stress resistance mechanisms and stress-tolerance genes mining, in a systematic, predictable, and transferrable fashion , 22. To sk scale . In anott pH 1.9 . It is nt pH 1.9 , 25.E. coli uses a range of physiological, metabolic, and proton-consuming AR mechanisms to survive acid stress [E. coli.The acid resistance (AR) of microorganisms is a complex polygenic trait, e.g. d stress . In our d stress , or the d stress , we founE. coli under mild acidic condition, by fine-tuning the expression of the genes involved in the proton-consuming acid resistance system (gadE), a periplasmic chaperone (hdeB) and ROS scavengers (sodB and katE) which participate in the aforementioned three stress resistance systems. The transcriptional regulator GadE is the key activator of the proton consumption system AR2 [sodB and katE enhanced the titer of 5-aminolevulinic acid by 117% in a 5-L fermenter [In this study, we designed a set of synthetic acid-tolerance modules to improve the acid resistance of stem AR2 –31. Peristem AR2 , 33. Supstem AR2 , 35. A permenter .gadE, hdeB, sodB and katE into synthetic acid-tolerance modules. The different modules variants were cloned into the laboratory E. coli strain MG1655, and were then screened and analyzed for cell growth at pH 5.0. Subsequently, 33 synthetic acid-tolerance modules were evaluated in an industrial lysine-producing E. coli strain, MG1655 SCEcL3, using multiple 10-mL micro-bioreactors and 1.3-L parallel bioreactors, resulting in a best strain with improved l-lysine yield at pH 6.0 compared with the parent strain at pH 6.8. This work provides an example of semi-rational design of industrial E. coli strains for improving the production robustness at low fermentation pH.With the goal of achieving a just-enough and just-in-time gene expression, we used directed evolution to construct an acid-responsive promoter pool with a gradient of strengths, from which four promoter variants with different strengths were assembled with the four genes asr) promoter [asr sequence using degenerated primers . About 6000 clones were randomly picked for a first round of screening. It is noteworthy that many of these variants lost the activity or acid-responsiveness [asr promoter variants with strengths ranging from 22 to 134% of the wild type (at pH 5.0), and the ratio above 1.8 were obtained  then a medium-throughput screening guided by lysine productivity performed using an industrial strain, an industrial medium and the small-volume bioreactor Micro-Matrix which can handle 24 strains at once; (3) finally the industrial strain variants were analyzed in a laboratory-scale parallel-bioreactor system, which can handle 8 strains at once. We surmise that although cell growth is not necessarily correlated with high production, it is a prerequisite for high production [Stepwise screening is an efficient strategy for engineering stress tolerant strain , 45. Ouroduction , 47. ThuE. coli under mild acidic condition (pH 5.0), we individually overexpressed the four genes gadE, hdeB, sodB, katE, and the katE-sodB cascade, under the control of three different asr promoter variants, P24E8, P77C4 and P50G1, using plasmid pACYC184 backbone , respectively, indicating that gadE alone conferred little acid tolerance to E. coli. The overexpression of hdeB enhanced the acid resistance, as the final OD600 of P24E8-hdeB, P77C4-hdeB and P50G1-hdeB at pH 5.0 reached 99%, 106% and 134% of that of the wild type strain, respectively. The overexpression of katE and katE-sodB improved the cell growth at pH 5.0, as the final OD600 of P24E8-katE, P77C4-katE and P50G1-katE reached 105%, 133% and 110% of that of the wild type strain, respectively, and the final OD600 of P24E8-katE-sodB, P77C4-katE-sodB and P50G1-katE-sodB were 106%, 121% and 112% of that of the wild type strain, respectively. This indicated that hdeB and katE-sodB can enhance acid resistance of E. coli in mild acidic conditions under the control of the selected asr promoter variants. On the other hand, the overexpression of sodB alone did not enhance the final OD600 of the strains with any of the promoters, and the lag phase was prolonged. These results are consistent with the fact that the enzyme SodB produces H2O2 which might actually damage the intracellular environment [600 of the strain harboring P77C4-katE-sodB was lower than that of the strain harboring P77C4-katE, given that sodB and katE forms a cascade relationship [E. coli MG1655, we thus included this cascade in the following analyses, but used separate promoters in order to individually fine tune the two genes   Fig. A. The reasr promoters in these 244 strains are shown in Additional file 600 ratios and the maximum specific growth rate ratios, compared with the wild type strain. The standard deviations of the final OD600 ratio and maximum specific growth rate ratio of each strain were mostly below 5%, with an average of 2.3% for the final OD600 ratio and 1.5% for the maximum specific growth rate ratio. In terms of growth, 24 strains showed a final OD600 higher than 120% of that of the wild type strain. The maximum specific growth rates were 55–103% of that for the wild type strain, indicating that the overexpression of the four genes also brought burden to E. coli. The growth curves of the top five recombinant strains and the wild type at pH 5.0 are shown in Fig. 600 of the five best strains MG 1124, MG 3124, MG 1314, MG 1344 and MG 1321 were 0.71, 0.69, 0.68, 0.67 and 0.67, which were 151%, 147%, 145%, 143% and 143% of the OD600 of the wild type strain , respectively , respectively.The four-gene synthetic acid-tolerance module library had 256 possible distinct variants. We randomly picked 832 clones in order to achieve approximately a 95% coverage of the library, and we characterized them by cell growth assay using an automated turbidimeter (Bioscreen C) . Among tasr promoter variants were set as the ordinal levels, and the final OD600 ratios were set as the responses. The result showed that hdeB and sodB were significantly correlated to the final OD600 (p < 0.05), but the p-value for katE (0.056) was slightly above the statistical threshold. Surprisingly, the contribution of gadE to growth was not significant, with a p-value of 0.862.To understand the contribution of each gene in the four-gene modules for the acid tolerant phenotype, a multi-factorial analysis of variance (ANOVA) was performed  , but with the same four asr promoter variants used for the four-gene modules . Among these strains, MG 0414 and MG 0314 grew better than MG 1124 . The maximum specific growth rates of MG 0414, MG 0314, MG 0323, MG 0333 and MG 0324 were 0.77 h−1, 1.11 h−1, 0.59 h−1, 0.69 h−1, and 0.72 h−1, which were 78%, 112%, 60%, 70%, and 73% of that of MG 1124 . The final OD600 of MG 0414 and MG 0314 were higher than their corresponding four-gene counterparts harboring also gadE. These results indicated that for the lab strain MG1655 used in this step, the hdeB-sodB-katE modules were sufficient to confer acid tolerance to the cells at pH 5.0, in terms of final OD600, or the further overexpression of gadE might actually bring more burden to the strain.We then constructed a new recombination strain library co-expressing just the three genes that showed contribution to the phenotype in the ANOVA test , and the maximum specific growth rates were 0.80 h−1 and 0.77 h−1, which were 93% and 90% of that of the wild type strain . Thus, the two modified strains maintained advantages, albeit of reduced extents, over the wild type in terms of cell growth, whereas the maximum specific growth rates were comparable to that of the wild type strain.The strains MG 1124, MG 0414, along with the wild type strain were then tested at the shake flask scale. The final OD600, respectively) and the wild type at pH 5.0 by a DCFH-DA-based fluorescent assay [In order to shed some light over the acid tolerance mechanism, we determined the intracellular ROS levels in strains MG 1124, MG 0414  were 0.5 μM/min and 0.4 μM/min, respectively, essentially equivalent to that of the wild type strain (0.5 μM/min). At the stationary phase (16 h) the rates of ammonia release in MG 1124 and MG 0414 were 6.7 μM/min and 10.2 μM/min, which were 48% and 73% of that at wild type strain (13.9 μM/min). These results indicated that in our design the expression of ybaS was not involved in the mechanism of enhanced acid tolerance.In addition, we examined the releases of GABA and ammonia in the strains during the growth at the shake flask scale , then the four best strains were further evaluated in 1.3-L parallel bioreactors (T&J-Minibox). The medium used was an industrial medium provided by China Oil & Foodstuffs Corporation (COFCO)  see “”. The in600 was 2.1 at pH 6.0, corresponding to 58% of that at pH 6.8 . For strains harboring acid-tolerance modules, the lysine titers of the four best strains SC 2121, SC 3124, SC 1344 and SC 3331 were 5.6 g/L, 5.4 g/L, 4.8 g/L and 4.7 g/L, which were 121%, 111%, 103% and 101% of that for the parent strain at pH 6.0 , respectively. It is worth to note that the titers of SC 2121 and SC 3124 at pH 6 were comparable or higher than that of the parent strain at pH 6.8 , indicating that the synthetic acid-tolerance modules could enhance the robustness of lysine production at pH 6.0. In addition, the lysine yields of the same four best strains were 110%, 113%, 90% and 111% of that of the parent strain at pH 6.0 , or 86%, 89%, 70% and 87% of that at pH 6.8 , respectively. The final OD600 of the four strains were all higher than that of the parent strain at pH 6.0 , or 92%, 72%, 89% and 77% of that for the parent strain at pH 6.8 , respectively.The parent strain SCEcL3 was fermented at pH 6.0 or pH 6.8 for 48 h, while the other strains carrying the synthetic modules were fermented at pH 6.0 for 48 h. As shown in Fig. 600 of the four strains were 23.4, 22.3, 22.9 and 19.5, which were 142%, 135%, 139% and 118% of that of the parent strain at pH 6.0 , or 148%, 135%, 139% and 118% of that for the parent strain at pH 6.8 , respectively , and with the lysine titer and yield comparable to those of the parent strain at pH 6.8. Moreover, we also evaluated SC 3124 at pH 6.8, and compared to the parent strain, only the difference in biomass was significant , suggesting that the synthetic acid-tolerance module improved the performance of the best strain by mostly enhancing the growth robustness.We then further evaluated the same four strains in 1.3-L parallel bioreactors. Compared to the parent strain, the lysine titers of SC 2121, SC 3124, SC 1344 and SC3331 were 68.0 g/L, 69.3 g/L, 62.0 g/L and 61.0 g/L, which were 113%, 115%, 103% and 101% of that of the parent strain at pH 6.0 , or 116%, 118%, 106% and 104% of that of the parent strain at pH 6.8 , respectively . As GadE is responsible for regulating the AR2 system, we attempted to replace it with the two AR2 component genes coding for GadB (variant GadB(dHT) which has enhanced activity at pH 4–6 [gadE could up-regulate the lysine-dependent acid resistance system (AR4) [Along this line, it is interesting to note that at the cell growth assay level, we found that t pH 4–6 ), and Ybem (AR4) . This reSaccharomyces cerevisiae strains [sodA, sodB from E. coli and an endogenous cAMP phosphodiesterase PDE2. It appeared that PDE2 alone activated a number of stress tolerance pathways, and thus the effects of other factors like the heat shock proteins, ubiquitins and autophagy proteins were likely masked under the overexpression of PDE2 [PDE2 like regulators with an appropriate activation range could also be tested for E. coli. The best strain finally was subjected to adaptive evolution by atmospheric and room temperature plasmas (ARTP), yielding a final strain with an enhanced ethanol titer of 6.9% in a large scale 200-L fermenter.Recently, a combinatorial strategy was used for engineering multiple-stress-tolerant ethanol-producing  strains . The stu of PDE2 –61. Thisasr promoter library with an enlarged operation range both in terms of absolute promoter strength as well as of pH response ratio at different pH (pH 5.0 vs pH 7.0). This promoter is regulated by RstBA and PhoRB systems. The RstA box is located at − 77 to − 55 of the promoter region, and the PhoB is located at − 39 to − 22, thus overlaps the -35 element , ROS scavenging enzymes (sodB and katE), and a transcription factor for the AR system (gadE). The functional optimization of these modules can be realized by the use of acid-responsive promoters of different strengths, and screened with a stepwise protocol, from a growth-guided high-throughput screening to a production performance-guided fermentation evaluation.This study demonstrates a useful strategy to improve growth robustness and productivity for industrial The strains and plasmids used in this study are listed in Additional file E. coli MG1655 strains were cultured in lysogeny broth (LB) medium at 37 °C [E. coli MG1655 strains were tested in LB supplemented with 20 g/L glucose (LBG medium) for the cell growth assay. For fermentation of E. coli MG1655 SCEcL3 strains, the seed medium consisted of sucrose (3 g/L), yeast extract (5 g/L), tryptone (7 g/L), ammonium sulfate (5 g/L), potassium dihydrogen phosphate (5 g/L), magnesium sulfate (0.5 g/L), ferrous sulphate (0.012 g/L), manganous sulfate (0.012 g/L), sodium glutamate (5 g/L), l-threonine (0.3 g/L), l-methionine (0.3 g/L) and pyruvic acid (0.3 g/L), and the fermentation medium consisted of glucose (30 g/L), phosphoric acid (0.6 g/L), magnesium sulfate (2 g/L), ammonium sulfate (10 g/L), corn steep liquor (0.325 g/L), potassium chloride (0.5 g/L), betaine (2.2 g/L), ferrous sulphate (0.032 g/L), manganous sulfate (0.032 g/L), l-threonine (0.25 g/L), cupric sulfate (6.8 mg/L), zinc sulfate (7.65 mg/L), and thiamine (5.6 mg/L). For the strains harboring plasmids with chloramphenicol or ampicillin resistance, 34 μg/mL chloramphenicol or 50 μg/mL ampicillin was added to the medium.For cloning, all at 37 °C . All E. asr promoter library was constructed by the Gibson assembly [E. coli XL1-Blue. Transformed cells were cultivated on LB agar plates overnight at 37 °C. The library size was estimated to be 105. Plasmids were then extracted and transformed by electroporation into E. coli MG1655 for screening.The synthetic assembly , based oasr promoter variants, the cells were grown overnight (about 16 h) in LBG medium (pH 7.0) at 37 °C. Then the cultures were diluted to initial OD600 0.05 in 200 μL LBG medium (pH 7.0) or LBG medium (pH 5.0) which was acidified by HCl in 96-well black flat clear bottom polystyrene microplates. The cultures were grown at 37 °C for 16 h, and OD600 and fluorescence of mCherry  were determined by a Tecan infinite 200 pro microplate reader . The clones with a fold of the fluorescence/OD600 ratio between pH 5.0 and pH 7.0, i.e. pH response ratio, above 1.8 were selected to perform the second screening to verify the performance. Each experiment was performed in three biological replicates. Sequences of the mutated region at asr promoter variants in this study are available in Additional file For the screening of the gadE, hdeB, sodB and katE individually, the plasmids  containing individual genes with three Pasr variants  and the rrnB terminator were constructed using the Gibson assembly based on the pAm plasmid series containing different Pasr variants  overexpressing the katE-sodB cassette were constructed based on plasmids pAk1, pAk3 and pAk4 containing katE in a similar fashion  and the rrnB terminator were PCR-amplificated by a pair of primers BsaI-f1-f2-F/BsaI-f1-f2-R with BsaI restriction sites from plasmids pAg1, pAg2, pAg3 and pAg4, respectively. Then the fragments were inserted into the pMD19T vector using the pMD™19T Vector Cloning Kit, to generate plasmids pMg1, pMg2, pMg3 and pMg4. For the gadE-hdeB-sodB-katE acid-tolerance modules, all the pMD19T-derived plasmids  and the destination plasmid pAcc1 (constructed in house based on plasmid pACYC184) were assembled using the Golden Gate assembly. For the hdeB-sodB-katE acid-tolerance modules, all the pMD19T-derived plasmids except for pMg1, pMg2, pMg3 and pMg4  and the destination plasmid pAcc2 (constructed in house based on plasmid pACYC184) were assembled. Then the plasmids were transformed by electroporation into E. coli MG1655, and the transformed cells were cultivated on LB agar plates overnight at 37 °C.The assembly . First, E. coli MG1655 and strains harboring acid-tolerance modules were grown overnight (about 16 h) in LBG medium (pH 7.0) at 37 °C, and then the cultures were diluted to initial OD600 0.05 in 300 μL LBG medium (pH 5.0) which was acidified by HCl. Then the cultures were incubated at 37 °C in 100-well Honeycomb microplates monitor by automated turbidimeter  for online monitoring of OD600 for 24 h. Each experiment was performed in three biological replicates.The cell growth assays on were performed using a modification of the protocol as described in our pervious study . Wild tyμ is the population mean, αi, βj, γk and δl are main effects of four factors due to the ith, jth, kth and lth level of the factors, ε is the random error, and the other parameters accounting for the interactions among the factors. In this study, α, β, γ and δ denoted the genes gadE, hdeB, sodB and katE as the four factors, and the levels of the factors were the four asr promoter variants of different strengths, P24E8, P10G6, P77C4 and P50G1.The Analysis of Variance (ANOVA) was performed using Design Expert software version 10 for Windows (Stat-Ease Inc.). The ANOVA F-statistics test and the general linear regression model were used . BrieflyStatistical significance analysis was performed using the two-tailed Student’s t-test with a confidence interval of 95%, and p-value ) below 0.05 indicates significance.E. coli MG1655 SCEcL3 was provided by Nutrition and Health Research Institute , a unit of COFCO. E. coli MG1655 SCEcL3 and SCEcL3 harboring synthetic acid-tolerance modules were grown overnight at 37 °C in seed medium. The overnight cultures were diluted (1.5:10) into 24-well Micro-Matrix plates containing 3.6 mL fermentation medium  ammonia) in Micro-Matrix . The fermentation was performed at 37 °C, 300 rpm for 48 h. The dissolved oxygen (DO), pH and temperature were monitored online. The initial pH of the fermentation medium for all the strains was pH 6.8, and after the pH gradually decreased during fermentation, the wild type strains were controlled at pH 6.8 or pH 6.0, while other strains were controlled at pH 6.0, using 5% (w/v) ammonia. DO and temperature were controlled at 40–60%, and 37.0 ± 0.1 °C, respectively, during the 48 h fermentation period. After 48 h fermentation, samples were taken for measuring optical density, residual glucose and lysine titer. OD600 was measured by ultraviolet spectrophotometer and the titers of glucose and lysine were measured by SBA-40D biosensor analyzer  [The lysine producing parent strain , China) .600 reaching 2.0–3.0, and 75 mL seed inoculum was then inoculated into 425 mL fermentation medium in the 1.3-L parallel bioreactors. The DO, pH and temperature were monitored online. The pH of the cultures was controlled at 6.8 or 6.0 by the addition of 5% (w/v) ammonia. DO, temperature, and glucose were controlled at 40–60%, 37.0 ± 0.1 °C, and 0.4–1.0% (w/v), respectively, during the 48 h fermentation period. DO was monitored online and was controlled by adjusting the agitation rate from 200 to 1000 rpm with an aeration rate of 0.5 vvm. Samples were taken periodically for measuring optical density, residual glucose and lysine. Each experiment was performed in three biological replicates.Fermentation test was performed in T&J-Minibox 1.3-L × 8 parallel bioreactors . Briefly, cells were activated in seed medium and inoculated at 1% (v/v) into 500 mL flasks containing 100 mL seed medium at 37 °C, 250 rpm until the OD600/mL with 1 mL citrate buffer . After incubation at 37 °C for 1 h, the cultures were harvested by centrifugation and the supernatant was used for subsequent analysis. Briefly, in a 100 μL reaction mixture, 10 μL of supernatant was mixed with 0.5 mM NADP+, 0.5 mM dithiothreitol, 0.5 mM α-ketoglutarate and 10 μg GABase . The reaction mixture was incubated at 37 °C for 1 h, and then measured at 340 nm using a Tecan infinite 200 pro microplate reader . The concentration of GABA was calculated using a standard curve obtained with a set of GABA standard solutions of different concentrations.The assays of GABA and ammonia release were followed as previously described with minor modifications . For the600/mL with 20 mL citrate buffer . The concentration of ammonium ions was continuously monitor using a SevenCompac™ S220 electrochemical analytical meter  with an ammonia ion selective electrode .For the ammonia release determination assay, cultures grown in LBG medium were harvested in exponential phase (3 h) or in stationary phase (16 h). The pellet was washed twice with citrate buffer  and resuspended to 1 ODE. coli MG1655, MG 1124 and MG 0414 were grown overnight (16 h) in LBG medium (pH 7.0) at 37 °C, and then the cultures were diluted to initial OD600 0.05 in 250 mL LBG medium (pH 5.0) which was acidified by HCl in shake flasks. Then the cultures were incubated at 37 °C, 250 rpm. The cultures of different time points  were harvested and washed twice with 50 mM PBS (pH 7.4).The 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) based assay for measurement of the intracellular ROS level was followed as previously described . The samAdditional file 1: Figure S1. Schematic diagram of the construction of the synthetic asr promoter library. Figure S2. Distribution of 177 unique asr promoter variants in terms of fluoresce/OD600 at pH 5.0 and 7.0. Figure S3. Statistics of nucleotide frequency in N9 sequences of two groups of asr promoter variants with the pH response ratios above 1.8. Figure S4. Intracellular ROS level in strains MG 1124, MG 0414 and E. coli MG1655 (WT) cultured for 24 h in LBG at pH 5.0. Figure S5. Relative strength of the synthetic asr promoter library entries at different pH.Additional file 2: Table S1. Summary of the relative strengths of the asr promoter library entries at pH 5.0. Table S2. Multi-factorial ANOVA analysis of the recombinant strains in gadE-hdeB-sodB-katE library. Table S3. The release of GABA and ammonia in the exponential phase and in the stationary phase. Table S4. Strains and plasmids used in this study. Table S5. Primers used in this study. Table S6. Sequence of DNA fragments or genes used in this study. Table S7. Sequences of the mutated region at asr promoter variants in this study with relative strength at pH 5.0 and pH response ratio.Additional file 3. Growth characteristics  of the strains containing the gadE-hdeB-sodB-katE acid-tolerance modules.Additional file 4. Growth characteristics  of the strains containing the hdeB-sodB-katE acid-tolerance modules.