Objective: To prepare and validate the chemical structure of the cefotaxime dimer and cefotaxime trimer impurities available from pharmaceutical industry.Methods: A polymer stock solution of cefotaxime sodium was obtained using a concentrated solution degradation method. The cefotaxime dimer and trimer impurities were separated and purified by preparative reversed-high-performance liquid chromatograph (RP-HPLC), and the pure cefotaxime dimer and trimer were obtained by a freeze-drying method. The two impurities were characterized by infrared (IR) spectroscopy, ultraviolet (UV) spectroscopy, mass spectroscopy, and nuclear magnetic resonance . The groups corresponding to the characteristic absorption peaks in the UV and IR spectra were analyzed, and the 1H and 13C NMR signals were assigned.Results: The cefotaxime dimer was isolated and purified from the actual sample of industrial medicines, and chemical structure of the dimer is the same as in the dimers investigated earlier. The polymerization sites and stereoscopic configuration of trimer impurity was validated for the first time.Conclusion: This study is of great significance for the study of the structures and quality control of polymer impurities in cephalosporin drugs. Promoting the polymerization sites study and providing a technical basis for allergic study of polymer impurities. However, the raw materials and preparation process of β-lactam antibiotics may result in polymer impurities during production, transportation, storage, and even use. Polymer impurities, mainly including dimers, trimers, tetramer, and so on, are generated through covalent polymerization between β-lactam antibiotic drug molecules. β-lactam antibiotic molecules typically contain free polar groups such as amino and carboxyl groups, as well as structurally unstable quaternary lactam rings and easy-to-fall-off three-digit side chain groups. As a result, they are prone to polar groups attacking the amide bond of the quaternary lactam ring, resulting in fully polymerized polymer impurities, as well as attacking the methylene group of three-digit side chains, resulting in incompletely polymerized polymer impurities that fall off three-digit side chains, and a few polymer impurities with an unclear connection sites. These can be as the multivalent haptens, which are associated with allergic reactions and can seriously threaten people’s health . Therefohttps://pubmed.ncbi.nlm.nih.gov). ChP 2020  were obtained by a preparative chromatography and freeze-drying method. Finally, using infrared (IR) absorption spectroscopy, ultraviolet (UV) absorption spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR) methods, the chemical structures and connection sites of the dimer and trimer were studied by comparison with the spectra of cefotaxime sodium . The cheThe UV visible spectrophotometer was a SHIMADZU UV-2600 . The IR spectrometer was a PerkinElmer Spott40 0FT-IR/NIR . The NMR spectrometer was a Bruker Avance 500 . The high-resolution mass spectrometer was a Q Exactive Focus . The software was Xcalibur 4.0. The preparative RP-HPLC system 2545B consisted of a 2545 binary pump, a 2998 photodiode array (PDA) detector, an SFO column oven, and a 2767 autosampler and fraction collector . The software was MassLynx V4.1. The rotary evaporator was a Hei-VAP Advantage ML/HB/G3 . The freeze-dryer was an Alpha 2-4 LSC Basic .d6 (DMSO-d6) was purchased from Beijing Chemical Works . A milli-Q water purification system  was used to further purify the distilled water. Cefotaxime sodium, as a raw material, was provided by the National Institutes for Food and Drug Control of the People’s Republic of China. The purities of the cefotaxime dimer and trimer were >90.0%, as determined by HPLC, which were suitable for structural identification.HPLC-grade acetonitrile was purchased from Thermo Fisher Scientific . Glacial acetic acid (98.0%) was supplied by Sigma-Aldrich Co. Ltd , and dimethyl sulfoxide-−1, the column temperature was 30°C, the detection wavelength was 235 nm, and the injection volume was 20 µl.Mobile phase A was 0.05 mol/L phosphate buffer (pH 6.25) and methanol . Mobile phase B was 0.05 mol/L phosphate buffer (pH 6.25) and methanol . The gradient elution procedure was as follows: the proportion of phase B was maintained at 6.0% from 0 to 15.0 min, at 20% from 15.0 to 23.0 min, at 55% from 23.0 to 40.0 min, and at 100% from 40.0 to 54.0 min. Next, the proportion of phase B was maintained at 6% from 54.0 to 55.0 min and that of phase A was maintained at 6% from 55.0 to 65.0 min. The column was an Zorbax SB-C18  , the flow rate was 1.0 ml min−1, the column temperature was 30°C, the detection wavelength was 254 nm, the injection volume was 1,000 μl, the fraction collecting time ranges were 11.3–12.0 min (fractions containing trimer impurities) and 12.2–13.1 min (fractions containing dimer impurities). The sample solution was a polymer stock solution.Mobile phase A was 0.005 mol/L phosphate buffer . Mobile phase B was an acetonitrile. An isocratic elution was performed with A:B (v/v) = 95:5. The semi-preparative column was a TSK-Gel G2000SW (21.5-mm inner diameter (I.D.) × 30 cm, 13 μm), the flow rate was 5.0 ml min−1, the column temperature was 30°C, the detection wavelength was 235 nm, the injection volume was 1,000 μl, the fraction collecting time ranges were 33.4–35.3 min for the cefotaxime dimer and 38.0–39.9 min for the cefotaxime trimer, and the solutions were cefotaxime dimer and cefotaxime trimer stock solutions.Mobile phase A was water and acetic acid . Mobile phase B was acetonitrile. A gradient elution was performed as follows: the proportion of phase B was 10.0% at 0.0 min, 15% from 10.0 to 15.0 min, 18% from 15.0 to 30.0 min, 30% from 30.0 to 42.0 min, 40% from 42.0 to 45.0 min, 45% from 45.0 to 50.0 min, 10% from 50.0 to 51.0 min, and 10% from 51.0 to 55.0 min. The semi-preparative column was a CAPCELL PAK CA18 MGII , the flow rate was 5.0 ml minFirst, 2, 2, and 1.5 mg of cefotaxime, cefotaxime dimer, and cefotaxime trimer were accurately weighed, respectively. The measured samples were then transferred into 100-, 50-, and 25-ml volumetric flasks, respectively. And the samples were dissolved and diluted with distilled water. The solutions were then shaken well, and the UV spectra in the wavelength range of 200–350 nm were obtained.−1.Approximately 1.0 mg each of cefotaxime, cefotaxime dimer, and trimer were accurately weighed. Total reflectance infrared spectroscopy (ATR) was conducted in the wavenumber range of 600–4,000 cm−1. The scanning voltage (+) was 3000 V, the capillary temperature (+) was 350°C, the sheath gas (+) was 35 Lh−1, the auxiliary gas (+) was 10.00 Lh−1, the maximum spray current (+) was 100.00, the probe heater temperature (+) 350°C, the S-RF level was 50.00, and the ion source was heated electrospray ionization (HESI). A full-scan MS experiment (MS1) was conducted in the positive ion mode with a resolution of 70,000 and a scanning range of m/z = 200–2000. An MS/MS experiment (MS2) was conducted with a resolution of 17,500, a separation window of m/z = 3.0 (N) CE of 13 V, and a default charge state of 1.High-resolution mass spectroscopy (HR-MS) was performed by direct injection at a rate of 10 μl·mind6 to a concentration of approximately 20, 20 and 15 mg/ml respectively, and tetramethylsilane (TMS) was added as the internal standard peak (δH 0.00 and δC 0.00). The operating frequencies of the 1H and 13C NMR were 600.13 and 150.90 MHz, respectively, the experimental temperature was 24.5°C, and the spectral widths were 11,904.76 and 35,714.29 Hz, respectively. The operating frequency of the attached proton test (APT) spectrum was 150.90 MHz, and the spectral width was 35,714.29 Hz. Bruker standard pulse sequence was used for the 1H–1H correlated spectroscopy (COSY), 1H-13C heteronuclear single quantum (HSQC), and 1H-13C heteronuclear multiple bond correlation (HMBC) experiments. The spectral widths of F2 (1H) and F1 (1H) of 1H–1H COSY were both 7,812.50 Hz, the sampling data array was t2 × t1 = 2048 × 128, and the number of transients averaged was 4. The F2 (1H) and F1 (13C) spectral widths of HSQC and HMBC were 7,812.50 and 33,201.94 Hz, respectively, the sampling data array was t2 × t1 = 4,096 × 128, and the number of transients averaged was 8. The NMR spectra were processed using the Mestrenova 14.0.0 software.First, the cefotaxime sodium, its dimer and trimer were separately dissolved in DMSO-A concentrated solution degradation method:Approximately 2.0 g of cefotaxime raw material was transferred into a 100-ml volumetric flask, and distilled water was added to dissolve and dilute it to a concentration of approximately 20 mg/ml. The mixture was shaken well to produce a solution, after which it was allowed to stand at 25 ± 2°C for 7 days. It was then freeze-dried and stored for future use.By applying the method described in The UV spectra in Similar to the cefotaxime dimer, the cefotaxime trimer  exhibite−1 was assigned to carbonyl stretching vibrations of the β-lactam ring. The peak at 1728 cm−1 was assigned to the carbonyl stretching vibrations of the acetyl group. The peak at 1,669 cm−1 was assigned to C=N stretching vibrations of C=N-OCH3. The peak at 1,624 cm−1 was assigned to C=O stretching vibrations of COOH. The peak at 1,531 cm−1 was assigned to N-H bending vibrations and C-N stretching vibrations. The peaks at 1,384 and 1,351 cm−1 were assigned to CH3 stretching vibrations. The peaks at 1,232 and 1,037 cm−1 were assigned to the stretching vibrations of CO-O-R and C-O on the three side chains, respectively. The peak at 1,110 cm−1 was assigned to C-N stretching vibrations.The IR spectrum in −1 was assigned to the carbonyl stretching vibrations of the β-lactam ring. The peak at 1727 cm−1 was assigned to the carbonyl stretching vibrations of the acetyl group. The peak at 1,670 cm−1 was assigned to the C=N stretching vibrations of C=N-OCH3. The peak at 1,626 cm−1 was assigned to the C=O stretching vibrations of COOH. The peak at 1,531 cm−1 was assigned to the amide N-H bending vibrations and C-N stretching vibrations. The peak at 1,362 cm−1 was assigned to the bending vibrations of CH3. The two spectral bands at 1,232 and 1,036 cm−1 were assigned to the stretching vibrations of CO-O-R and C-O on the three side chains, respectively, and the peak at 1,109 cm−1 was assigned to C-N stretching vibrations.The dimer  had nineThe above IR spectrum analysis showed that the IR spectra of the cefotaxime dimer and trimer were basically the same as that of cefotaxime sodium , indicatm/z = 851.088 87 [M + H]+ and m/z = 426.048 28 [M+2H]2+ were detected under positive mode, which indicated that the relative molecular weight of the dimer was 850.09192 amu, and the annotated molecular formula was C30H30N10O12S4. The relative error between the experimental and theoretical molecular weight values was −0.8300 ppm. The results showed that the dimer had one C2H4O2 group fewer than two molecules of cefotaxime , which was determined to be the side-chain substituent (acetoxy) of cefotaxime. The secondary mass spectrum  of cefotaxime. The secondary mass spectrum  and two-dimensional (2D) NMR spectra , the pro1H NMR spectrum of the dimer impurity and 30 carbon signals in the 13C NMR spectrum of the dimer. According to analysis, the 17-NH2 of cefotaxime sodium was a primary amino group, while the 17-NH of the dimer was a secondary amino group, indicating that one amino proton of the latter was replaced. In the 1H-NMR spectrum, the 17-NH proton of the dimer was not a single peak but multiple peaks, which indicated that this proton was coupled with other protons. In the 1H–1H COSY spectrum, the 17-NH proton  of the dimer had a correlation peak with H18 . In the HMBC spectrum, the two protons of H18 [δ4.19  and δ4.30 ] were remotely correlated with C15 (δ169.0). These results indicated that the dimer was connected by σ bonds between the 17-NH and 18-CH2 groups.A total of 30 proton signals were detected in the 1H NMR spectrum of the trimer impurity. The13C NMR spectrum of the trimer should show 44 carbon signals, but only 31 carbon signals were actually detected, possibly because the chemical environment contribution to the C36–C51 carbon signals in the trimer was similar to the chemical environment contribution to the C3–C33 carbon signals. This resulted in partial overlap of the carbon signals in the carbon spectrum. This overlap included three groups of CH3 carbon signals at δ21.1, δ62.5, and δ62.4, which were assigned to 2′-CH3, 11-OCH3 (29-OCH3), and 47-OCH3, respectively; four groups of CH2 carbon signals at δ26.2, δ63.2, δ26.9, and δ45.6, which were assigned to C4, C3α, C20 (C38), and C18 (C36), respectively; and seven groups of CH carbon signals at δ58.0, δ57.9, δ59.1, δ59.1, δ109.7, δ109.9, and δ109.4, which were assigned to C6, C22 (C40), C7, C25 (C43), C13, C31, and C49, respectively, according to the HSQC spectrum and the chemical shift rule of the carbon spectrum. The remaining groups of C signals were assigned according to the HMBC spectrum and the carbon spectrum data of the dimer, as shown in 2 of cefotaxime sodium was a primary amino group, while the 17-NH in the dimer and the 35-NH in the trimer were secondary amino groups, indicating that two amino protons of the trimer were replaced. In the 1H–1H COSY spectrum, the 17-NH  and 35-NH  protons of the trimer had correlation peaks with H18  and H36 . In the HMBC spectrum, the two protons of H18 [δ4.30 ] and H36 [δ4.30 ] were remotely correlated with C15 (δ169.0) and C33 (δ169.0). Furthermore, the molecular formulas of unsaturated cefotaxime sodium, its dimer, and its trimer were Ω = 11, 21, and 31, respectively. These results indicated that the trimer was connected by the σ bonds between the 17-NH and 18-CH2 groups and between the 35-NH and 36-CH2 groups.A total of 41 proton signals were detected in the 3 and 29- OCH3 in the dimer were δ62.5 and δ62.4, respectively. Compared with cefotaxime sodium, the dimer slightly shifted to the low field only by 0.1–0.2 ppm. The above analysis showed that the configurations of the two double bonds at positions 11 and 29 in the dimer were all Z-types. The chemical shifts of protons H13, H31, and H49 in the trimer were δ6.83, δ6.83, and δ6.73, respectively, which were basically consistent with the chemical shifts of H13 and H31 in the dimer, and there was no evident shift from the high field to the low field. The carbon chemical shifts of 11-OCH3, 29-OCH3, and 47- OCH3 in the trimer were δ62.5, δ62.5, and δ62.4, respectively. Compared with the dimer, there was also no evident shift. Hence, the configurations of the three double bonds at positions 11, 29, and 47 in the trimer were all Z-types.In theory, there were four double-bonded cis/trans isomers in the dimer  and eight double-bonded cis/trans isomers in the trimer . In a previous study, it was found that when the double bond configuration of amino thiaoxime changed from Z to E, the chemical shift of the protons on the five-membered thiazole ring shifted from high field to low field by about 0.5–0.7 ppm, and the carbon chemical shift of the amino thiaoxime methoxyl group shifted from high field to low field by 0.3–0.9 ppm . It is kJ = 4.8 Hz, those of H22 vs. H25 were J = 4.7 and 4.8 Hz, respectively, and those of H40 vs. H43 were J = 4.7 and 4.8 Hz, respectively, all of which were similar to those of H6 vs. H7 and H22 vs. H25 in the dimer. These results indicated that the three β-lactam rings were stable; so, the absolute configurations of cefotaxime trimers C6, C22, and C40 in the trimer were the same as those of C6 and C22 in the dimer. Furthermore, the absolute configurations of C7, C25, and C43 were the same as those of C7 and C25 in the dimer. The absolute configurations of the six chiral centers were all R types.There were four chiral centers in the dimer, including C6, C7, C22, and C25. The four carbon atoms were all tertiary carbons; so, we could infer whether their absolute configurations changed by the proton coupling constant. The data in Therefore, the dimer impurity from the actual sample of industrial medicines was named -3-(acetoxymethyl)-7-[(Z)-2-)-7-(Z)-2-((2-aminothiazol-4-yl)-2-(methoxyimino) acetamido)-2-carboxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-3-yl)methyl)amino)thiazol-4-yl)-2-(methoxyimino)acetamido]-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, which is considered the same as the dimer investigated  and publ1H-NMR and 13C-NMR signals were assigned by 2D NMR techniques. The above data confirmed the chemical structure of the dimer from the actual sample of industrial medicines is the same as in the dimers investigated earlier. And the connection sites and the absolute configurations of trimer impurity for the first time. These results have great significance for the study of the polymerization sites and quality control of the polymer impurities in β-lactam antibiotics.HPSEC preparation and RP-HPLC were used to prepare a concentrated degraded polymer solution for two-step purification and separation in this study. Combined with the characteristics of short preparation times of the HPSEC preparation method and the high separation effect of the RP-HPLC preparation method, the efficiency of separation and purification could be significantly improved. The UV, IR, HR-MS, 1D NMR, and 2D NMR spectra of cefotaxime sodium, its dimer, and its trimer were determined. The characteristic absorption peaks in the UV and IR spectra were assigned. The molecular weights and molecular formulas of the dimer and trimer impurities were determined by HR-MS, and the characteristic structural fragments were deduced by secondary mass spectrometry. All the


Dynamic light scattering over 90 days was used to characterize CVNE. Cytotoxic activity and quantification of cytokines were evaluated in peripheral blood mononuclear cell (PBMC) culture supernatants. CVNE achieved a drug loading of 4.29 mg/mL, droplet size of 165.70 ± 0.46 nm, polydispersity index of 0.14 ± 0.03, zeta potential of −10.25 ± 0.52 mV, and good stability for 90 days. CVNE showed no cytotoxicity at concentrations up to 200 µM in PBMCs. CV diminished the production of IL-2 in the PBMC supernatant. However, CVNE reduced the levels of the pro-inflammatory cytokines IL-2, IL-17, and IFN-γ at 50 µM. In conclusion, a stable CVNE was produced, which improved the CV immunomodulatory activity in PBMCs.Carvacrol (CV) is an essential oil with numerous therapeutic properties, including immunomodulatory activity. However, this effect has not been studied in nanoemulsion systems. The objective of this study was to develop an innovative carvacrol-loaded nanoemulsion (CVNE) for immunomodulatory action. The developed CVNE comprised of 5%  Origanum  have been studied  assay.CVNE and BNE showed an average cell viability greater than 95% at all tested concentrations . CV showed an average cell viability greater than 99% at a concentration of 50 µM, and greater than 90% at concentrations of 100 and 200 µM. Based on these results, concentrations of 25 and 50 µM were selected for assays of immunomodulatory activity.Inflammation is a key response that involves the participation of a variety of chemical mediators and signaling pathways involved in tissue repair. The inflammatory process can be characterized by the activation of cascades of mediators that regulate important mechanisms of the inflammatory response, such as vascular permeability, recruitment of leukocytes in the blood, and cytokine production ,43. The PBMCs consist of a group of cells, including lymphocytes and monocytes/macrophages, which play an important role in the inflammatory response . Thus, tThe levels of immunosuppressive cytokines (IL-10), inflammatory mediators (IL-6), and proinflammatory cytokines  were quantified in PBMCs of healthy individuals stimulated with anti-CD3 and anti-CD28 antibodies .p = 0.007 and p = 0.023) the levels of IL-2  when compared to the stimulus condition with anti-CD3 and CD-28 (1051 [202.7–1331] pg/mL) (CV (25 and 50 µM) reduced (] pg/mL)  in the P51 202.7–31 pg/mL)p = 0.015) (165.4 [54.08–454] pg/mL) when compared to the anti-CD3 and anti-CD-28 condition (235.4 [83.67–728.7] pg/mL). At a concentration of 50 µM, the CVNE formulation reduced the levels of IL-2 (p = 0.015) (746.8 [126.4–1322] pg/mL), IFN-γ (p = 0.015) , and IL-17 (p = 0.031) (142 [49.50–325.4] pg/mL) (The CVNE formulation at 25 µM reduced IL-17 levels (] pg/mL)  when com69.44–11,9 pg/mL),.4 54.08–4 pg/mL) p = 0.039) in IL-6 expression. The BNE oil phase was composed of MCT. MCT can be mixed with other oils because it has no biological activity. In addition, two non-ionic surfactants, Span 80 and Tween 80, were used in the aqueous phase. These surfactants were chosen because of their known lack of biological toxicity [BNE was formulated without the bioactive compound to analyze whether the constituents of the formulation could be toxic or possibly interfere with the production of cytokines. It was observed that BNE did not show cytotoxic activity and did not interfere with cytokine production, except for an increase  at 50 µM in PBMC culture supernatant. In this way, we were able to obtain an immunomodulatory potential, and we suggest that new studies be carried out, such as in vivo tests, to better understand the performance of CVNE.®) were purchased from Sigma-Aldrich . Polysorbate 80 (Tween 80®) was provided by Vetec Química Fina LTDA . MCTs (Mygliol 812®) were acquired from Sasol . Ethanol was sourced from Merck , and dimethyl sulfoxide (DMSO) was purchased from NEON . The anti-CD3 and anti-CD28 antibodies were purchased from Ebiosciences , Ficoll Paque Plus was purchased from GE Healthcare Biosciences , and the media were purchased from Gibco . The human ELISA kits were provided by BD Biosciences , eBioscience , and Invitrogen (USA for IL-1β).CV oil  and sorbitan monostearate 80 (Span 80® + 0.08 g Span 80®) were added to the oily phase. Finally, distilled water (9.3 g) was added. In the BNE, CV was replaced with MCT. The CVNE was prepared by ultrasonication, and the components were subjected to four homogenization cycles in a sonication apparatus  for 1 min at 300 W using an ice bath, followed by an ultrasonic bath for 1 min [To prepare the NECV, initially, all components of the oily phase were weighed (0.05 g CV + 0.45 g MCT). After that, the surfactants (0.12 g Tween 80n = 7) y = 0.01646x − 0.02384; R² = 0.9992). The CVNE was diluted in methanol (1:2000) and filtered using a 0.2 μm membrane filter  [The CV content in the nanoemulsions was determined by ultraviolet–visible spectrophotometry . Initially, the maximum absorption wavelength of CV was determined to be 276 nm in methanol by scanning UV-Vis spectroscopy ranging from 200 to 900 nm. The concentration of CV in the nanoemulsion was calculated using a calibration curve (( Brazil) . All meav/v). All measurements were performed in triplicate, and the results are presented as a mean with standard deviation.Particle size, PDI and zeta potential were determined using a Zetasizer instrument  using the dynamic light scattering technique at 25 °C. Measurements of mean droplet (Z-average) diameter of the intensity size distribution were carried out at a fixed scattering angle of 173°. The zeta potential was evaluated from the electrophoretic mobility under an electric field using the same equipment. Prior to measurement, the nanoemulsions were diluted with deionized water . The emulsion was then subjected to centrifugal acceleration at 3000 rpm for 30 min at 25 ± 2 °C .In thermal stress test, the nanoemulsion was gradually heated from 40 up to 80 °C, rising the temperature in intervals of 5 °C, keeping each temperature for 30 min . After cStability studies were carried out using CVNE and BNE over a period of 90 days. CVNE and BNE were kept in 10mL glass vials sealed with plastic caps and kept in a refrigerator at 4 ± 2 °C. The sampling time points were 1, 7, 20, 30, 60, and 90 days. The formulations were evaluated for changes in particle size, PDI, zeta potential, pH, and macroscopic aspect.The pH of the CVNE and BNE was measured using a multipurpose autotitrator  previously calibrated with buffer solutions of pH 4.0 and 7.0.For the macroscopic aspect, color, appearance of the nanoemulsion, and phase separation were assessed.6 cells/well) in RPMI-1640 (Gibco) medium supplemented with L-glutamine, 10% fetal bovine serum (FBS) (Gibco), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 200 U/mL penicillin/streptomycin (Gibco). Subsequently, the cells were stimulated with anti-CD3 and anti-CD28 antibodies and treated with CV, CVNE, or BNE at concentrations of 25 µM and 50 µM of CV. Eight healthy individuals were included in this study. Exclusion criteria for controls were regular use of medications, chronic inflammatory disease, or alcohol or cigarette consumption in the last 15 days. The protocol was approved by the ethics committee of Universidade Federal de Pernambuco (CEP/CCS/UFPE/CAAE Nº 63517517.1.0000.5208).PBMCs were isolated from the blood of healthy individuals after collection in heparinized tubes. The blood was centrifuged in Ficoll Paque Plus and PBMCs were separated. After separation, cells were counted and plated in 24-well plates  was added to each well of the plate, followed by the addition of 130 µL of 20% SDS after 3 h. Cell viability was assessed by the ability of cells to reduce MTT to formazan blue. The absorbance was measured 24 h later at a wavelength of 570 nm using a microplate reader . Four independent experiments were conducted.The cytokines IL-2, IL-6, IL-10, IL-17, IFN-γ, TNF-α, and IL-1β were quantified in the supernatant of PBMC cultures. Cytokine levels were quantified using human ELISA kits  for IFN-γ, TNF-α, IL-6, and IL-10, eBioscience, San Diego, CA, USA for IL-2 and IL-17, and Invitrogen, Carlsbad, CA, USA for IL-1β. The lower detection limits for IL-1β, IFN- γ, TNF-α, IL-2, IL-17, IL-6, and IL-10 were 2.0 pg/mL, 4.7 pg/mL, 7.8 pg/mL, 2.0 pg/mL, 4.0 pg/mL, 4.7 pg/mL, and 7.8 pg/mL, respectively. All tests were performed according to the manufacturer’s recommendations.p < 0.05.GraphPad Prism 6.0 software  was used for data analysis. In the stability study, one-way analysis of variance (ANOVA) was applied. In the in vitro study, the differences between conditions were calculated using Wilcoxon’s signed classification test. The results of continuous variables are expressed as mean ± standard error of the mean. The differences were considered significant when


Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C.E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named TanHcw were studied. TanHcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of TanHcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na+, K+ Mn2+ and Triton-X100, Tween80 increased the enzyme activity of TanHcw, whereas Zn2+, Mg2+, Hg2+, EMSO, EDTA and β-mercaptoethanol inhibited enzyme activity. Km, kcat and kcat /Km of TanHcw were 0.30 mM, 37.84 s−1, 130.67 mM−1 s−1 for EGCG, 0.33 mM, 34.59 s−1, 105.01 mM−1 s−1 for ECG and 0.82 mM, 14.64 s−1, 18.17 mM−1 s−1 for MG, respectively.The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in Hcw from H. camelliae has been identified and characterized. The biological properties of TanHcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase TanHcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.A novel tannase TanThe online version contains supplementary material available at 10.1186/s12934-021-01685-1. Tannase, also known as tannin acyl-hydrolase EC 3.1.1.20], catalyzes the hydrolysis of ester bonds in gallotanins, epigallocatechin gallate, epicatechin gallate, and gallic acid esters. The hydrolysis of tannic acids by tannase releases gallic acid, some galloyl esters, and glucose .1.20, ca. Rivas eherbaspirillum camelliae WT00C and WT00F were isolated from the tea plant (Camellia sinensis L) and classified as a novel species in the Herbaspirillum genus  version 1.11a (SHIMADZU Corporation), and kinetic parameters were determined by Lineweaver–Burk plot [2) value was equal to or large than 0.997. All data were also analyzed using the statistic software SPSS based on the non-linear regression method INVERSE, and analysis of variance gave P values of less than 0.005 in each case.Kinetic parameters of Tanurk plot  and checAdditional file 1: Fig. S1. Alignment of amino acid sequences of microbial tannases and 4 putative tannases in H. camelliae WT00C. * pfam07519: a conserved domain of tannases predicted by NCBI database ; Acinetobacter: A. bayli (Q8RLZ8); Xanthomonas: X. campestris (Q8P8Y5); Agrobacterium: A. fabrurn (Q8UK62); Bradyrhtzobium: B. japonicum (Q89C36); Pseudomonas: P. syringae (Q88IB4); Aspergillus: A. niger (EHA25030); Tan1-4: putative enzymes of Herbaspirillum camelliae. Fig. S2. Kinetic parameter determination of TanHcw at the optimal temperatures and pH. (a) Lineweaver-Burk plot for substrates EGCG at pH7.0 and 40 ºC; (b) Lineweaver-Burk plot for substrates ECG at pH7.0 and 40 ºC; (c) Lineweaver-Burk plot for substrates MG at pH6.0 and 30 ºC. Table S1. Kinetic parameters of TanHcw compared with other tannases.


The decision limit (CCα) values were in the range from 0.067 to 2103.84 μg/kg, while the range for detection capability (CCβ) was from 0.083 to 2482.13 μg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 μg/kg, and 0.081 to 328.13 μg/kg, respectively. The recovery of analytes ranged from 61.28% to 116.20%. The intra-day coefficient of variation (CV) was from 0.97 to 25.93% and the inter-day CV was 2.30–34.04%. The method has been used for the determination of 49 residues/contaminants in bovine meat. Application of the method in routine analysis in bovine samples, revealed in limited samples the presences of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively.A multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, β-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the RThe online version contains supplementary material available at 10.1186/s13065-021-00788-5. Important source of nutrition for many people around the world is meat. The safety of meat is seriously challenged by all kinds of low molecular weight organic contaminants, such as residues of veterinary drugs, agro-chemical residues, mycotoxins, food additives and environmental contaminants, which arouse considerable attention from people over the world , 14. LiqAmoxicillin (99.6%), ampicillin (99.8%), benzylpenicillin (99.3%), cloxacillin (98.7%), oxacillin (98.4%), clenbuterol HCl (99.1%), isoxsuprine HCl (100%), salbutamol (99.4%), zilpaterol HCl (96.0%), ractopamine HCl (95.5%), terbutaline hemisulfate salt (100.0%), taleranol (99.5%), 19 nortestosterone (99.8%), clostebol (99.1%), boldenone (99.1%), methyltestosterone (99.5%), testosterone (100.0%), carbofuran (99.9%), carbaryl (99.9%), parathion (99.7%), malathion (99.2%), diazinon (98.3%), dimethoate (99.8%), atrazine (99.5%), cypermethrin (98.4%), permethrin (98.1%), deltamethrin (99.9%), coumaphos (99.7%), dicholphos (99.8%), chlorpyrifos (99.8%), fenvalerate (99.4%) were purchased Sigma-Aldrich . Brombuterol (98.0%), mabuterol HCl (98.0%), cimbuterol (98.0%), clenpenterol HCl (98.0%) were obtained from Witega . Zeranol (99.9%), stanozolol (99.8%), ceftioflur (98.0%), cephalexin (96.6%), oxytetracycline (96.5%), enrofloxacin (99.74%), ciprofloxacin (98.0%), sulfadimidine (99.6%), sulfamethoxazole (99.7%), sulfadiazine (99.8%), sulfachloropiridazine (99.1%) and sulfadimethoxine (99.7%) were obtained from Dr. Ehrenstorfer GmbH ; ochratoxin A (≥ 98.0%) and zearalenon (99.0%) were obtained from Trylogy Analytical Laboratory, Inc. .Clenbuterol-d6 HCl (98.0%), brombuterol-d9 HCl (98.0%), mabuterol-d9 HCl (98.0%), clenpenterol-d5 HCl (98.0%), cimbuterol-d9 (98.0%), were obtained from Witega ; isoxsuprine-d5 hemifumarate (≥ 98.0%) and ractopamine-d6 HCl (≥ 98.0%) were obtained from the European Reference Laboratory (EURL) at RIKILT, The Netherlands, salbutamol -d9 (≥ 98.0%) was obtained from Dr. Ehrenstorfer GmbH ; zilpaterol–d7 (≥ 98.0%) and β-zearalenol-d4 (≥ 98.0%) were obtained from Toronto Research Chemicals Inc. , while terbutaline-d9 acetate hemihydrate (99.3%), flunixin-d3 (100.0%) and penicillin G-d7 N-ethylpiperidinium (98.1%) salt were obtained from Sigma-Aldrich .Individual stock standard solutions and internal standards were prepared in methanol (MeOH) at concentration from 0.528 to 3.610 mg/mL, while the concentration of ochratoxin A was 50 μg/mL and for zearalenone was 100 μg/mL after reconstruction in methanol. After preparation of individual stock standard solutions and internal standards, the standards were divided in groups in accordance with the Maximul Recommended Residue Levels (MRL) and Minimum Required Performance Limits (MRPL) values according to the acceptance criteria of European Commision , EuropeaThe initial mixed working standards with concentration at 10 µg/mL were prepared in MeOH and kept at − 18 °C. The groups of standards were: clenbuterol, brombuterol, mabuterol in group 1; cimbuterol, clenpenterol, isoxsuprine, ractopamine in group 2; salbutamol, terbutaline, zilpaterol in group 3; testosterone, methyltestosterone, boldenone, zeranol, 19 nortestosterone, stanozolol, clostebol, taleranol in group 4; amoxicillin, ampicillin, benzylpenicillin, cloxacillin, carbaryl, parathion, dimethoate, atrazine, permethrin, dicholphos, zearalenon, ochratoxin a in group 5; enrofloxacin, ciprofloxacin, oxytetracycline, sulfadimidine, sulfamethoxazole, sulfadimethoxine, sulfadiazine, sulfachloropiridazine in group 6; carbofuran, chlorpyrifos in group 7; malathion, diazinon, coumaphos in group 8. Working standards for oxacillin, cephalexin, deltamethrin and fenvalerate were prepared at 10 µg/mL and ceftioflur and cypermethrin at 100 µg/mL.Mixed internal standard working solutions including brombuterol-d9 HCl, cimbuterol-d9, clenbuterol-d6 HCl, clenpenterol-d5 HCl, isoxsuprine-d5 hemifumarate, mabuterol-d9 HCl, ractopamine-d6 HCl, salbutamol -d9, terbutaline-d9 acetate hemihydrate, zilpaterol-d7) at 100 ng/mL and  at 10 μg/mL were prepared and kept at − 18 °C.LC–MS/MS grade MeCN, water and MeOH, HPLC grade ethylacetate (EtOAc), dichloromethane, ammonium hydroxide, n-hexane, acetic acid, ammonium acetate were obtainedfrom Carlo Erba Reagent S.A.S ; LC–MS/MS grade formic acid was from Merck  from Waters .A grand total of 100 random samples of fresh bovine meat were collected from local markets of North Macedonia. The collected samples were transported directly to the laboratory at 4 °C and then subjected to the following examination.All collected samples were homogenized. After homogenization, all samples were mixed and prepared one sample which was used for validation purposes. This process was intended to acquire appropriate-representative control sample. The control sample was checked with the analytical method to avoid pre-existing contamination with the analytes of the method’s scope. In the next step, 10 g of meat sample was fortified with the analytes and the internal standards and let to stand for 20 min. Then, 20 mL of extraction mixture  was added and shaken vigorously for 1 min on a vortex mixer. The mixture was then shaken for 30 min with an automated shaker and centrifuged at 8000 rpm for 10 min, at 0 °C. The extraction step was repeated and the combination of the supernatants was transferred to a 50 mL tube and kept at − 80 °C, for 20 min. The solution was filtered through filter paper  and evaporated to nearly dryness under stream of nitrogen in a water bath at 35 °C. The extract was dissolved in 10 mL of MeOH:water  and the solution shaken for 1 min on a vortex mixer followed by cleanup by solid phase extraction using Oasis HLB cartridges. The cartridge was activated and conditioned by passing through 5 mL of MeOH and 5 mL water before supernatant was loaded and cartridges washed with 5 mL of water, and then vacuum-dried for 10 min. The residues were eluted into a test tube using 4 mL MeOH:MeCN:ammonium hydroxide  followed by 4 mL MeOH:dichloromethane . The samples were evaporated to dryness under stream of nitrogen at 35 °C and the residue reconstituted with 1 mL of MeCN:water . Defatting was attained by adding 3 mL of n-hexane of the reconstituted residues in 1 mL on MeCN:water. The solution was shaken vigorously for 1 min on a vortex mixer. After that, the lower layer was centrifuged at 10,000 rpm for 3 min and the extract was filtered through a 0.45 µm membrane filter into autosampler vials prior to LC–MS/MS analysis.The analysis was performed with a Waters  quadrupole LC–MS/MS equipped with a binary pump, vacuum degasser, thermostated autosampler and thermostated column manager. MassLynx software  version 4.1 was used for instrument control, data acquisition and calculation of results. Chromatographic separation was carried out using an Kinetex C18 column .A gradient mobile phase was used where mobile phase A consisted of 0.1% formic acid solution in water containing 5 mM ammonium acetate and mobile phase B consisted of 0.1% formic acid solution in MeCN. The elution program was as follows: 0–1 min, 95–80% A; 1–4 min, 80–60% A; 4–8 min, 60–95% A; 8–12 min, 95% A while the flow rate was 0.2 mL/min. The column temperature was 40 °C and injection volume was 10 µL.The MS/MS acquisition was carried out using electrospray ionization positive and negative mode (ESI + and ESI −). The main MS conditions were optimized and finally set as follows: capillary voltage of 3.0 kV; source temperature of 150 °C; desolvation temperature of 400 °C; cone gas at 100 L/h; and desolvation gas at 300 L/h. Three multiple reaction monitoring (MRM) transitions of banned analytes were chosen, while for analytes with permitted limits, 2 MRM transitions were chosen. Analysis of internal standards involved one MRM transition. The optimized MRM conditions for each analyte are given in Table The analytical method was validated according to the European Commission decision 2002/657 and validation approach of the SANTE/12682/2019 guidelines [Multi-class and multi-residue LC–MS/MS analytical method, was developed for simultaneous analysis in the single run in (electrospray ionisation) ESI + and ESI −. For both ionization modes, the best sensitivity for all drugs was determined to provide the highest signals for quantification and confirmation. In positive ion mode were detected 93.55% of compounds, while in negative ionization mode were identified 6.45% of targeted compounds. The performance criteria for the analytical methods for detection of residues are prescribed in Commission Decision 2002/657/EC, therefore, this document is crucial for consultation from the laboratories for analyses of residues. According to this document, for the confirmation of banned substances (Group A) a minimum of four identification points are required, while for the confirmation of permitted substances (Group B) the minimum number of identification points is set to three. To achive maximum response for all compounds the individual standard solution with concentration from 1 μg/mL, was injected directly in the MS/MS detector. The MS/MS optimization parameters are summarized Table In order to achieve high sensitivity, good ionization, and sufficient separation with minimum interference from matrix, mobile phase and gradient are very important parameters in LC method. Different mobile phases were tested to provide the best chromatographic results, consisting of MeOH with 0.1% formic acid or MeCN with 0.1% formic acid, MeOH and MeCN (1:1) with 0.1% formic acid and water acified with formic acid  and 5 mmol/L ammonium acetate. According to Ren et al. , Xie et In this study, the best sensitivity with better chromatographic separation and peak shape was attained using water with 0.1% formic acid and 5 mmol/L ammonium acetate as aqueous phase and MeCN with 0.1% formic acid as organic phase. Also, the peak shape and tailing were improved with addition of 5 mM ammonium acetate. Due to the differences in physiochemical characteristics of target compounds, a gradient program described in section LC–MS/MS analysis was applied in order to elute 49 compounds within 12 min.The optimization of extraction methods for multi-class veterinary drugs and contaminants is challenging in part because of differences in structures and physicochemical properties; potential interfering matrices and fats/lipid and proteins in foods such as meat. Thus, the sample preparation protocol that includes simultaneous extraction of all components is an important part of a multi-residue and multi-class analysis.Better recoveries were obtained for most of the compounds when a mixture of MeCN and EtOAc acidified with 1% acetic acid was used. The effect of sample amount (5 g and 10 g) and extraction steps on recovery was investigated  for each compound are given in Table 2 values of most compounds was ≥ 0.990, but in 13 compounds R2 was below 0.99. The results correspond with the results obtained from Kaufman et al. 2008 and can probably be explained by the enzymatic/chemical instability or the extreme polarity of the compounds. The coefficient of corelation revealed good linearity in the concentration range for each compound.The linearity of the analytical method was determined at five differenct concentration levels for each compound. Therefore, matrix-matched calibration curve and the internal standard were utilized in the method for quantification to reduce the matrix effect. Although the method covered various different classes of veterinary pharmaceuticals, pesticides and mycotoxines the use of internal standards was only feasible for β-agonists, some antibiotics: amoxicilin, ampicilin, benzylpenicilin, oxacilin, cloxacilin, ciprofloxacin, enrofloxacinand for one mycotoxin– zearalenon . The linear range and coefficient of corelation  and the detection capability (CCβ) were calculated according to the 2002/657/EC requirements. The LODs were calculated from the lower concentrations of analyte i.e., the lower standards which were used for calibration curve (n = 6), and as mean value, plus 3.3 times of the calculated standard deviation and for LOQ this was plus 10 times of the calculated standard deviation. The CCα values were in the range of 0.067–2103.84 μg/kg, while the range for CCβ was from 0.083 to 2482.13 μg/kg. The LOD values were in the range of 0.059–291.36 μg/kg, while the range for LOQ was from 0.081 to 328.13 μg/kg. The gained results showed good sensitivity because the values of the LOD, LOQ, CCα and CCβ were in accordance with the requirements of 2002/657/EC. The results are show in Table Recovery and intra-day precision were evaluated by preparing spiked samples at three different concentration levels using six replicates for each concentration level in one day, while inter-day precision were evaluated by analysis of spiked samples at the same concentration levels on three consecutive days, also in six replicates for each concentration level. The spiked concentration levels and results for recovery and precion are shown in Table Statistical evaluation revealed that average recoveries were in the range of 61.28 (amoxicillin at 50 μg/kg) to 120%  and CV for the intra-day precision were in the range of 0.97 (for permethrin at 75 μg/kg) to 25.93% (isoxsuprin at 0.2 μg/kg). For inter-day precision the CV was from 2.27% (for cypermetrine at 3000 μg/kg) to 34.04% (for ciprofloxacin at 50 μg/kg). For all investigated compounds, the average recoveries and CV for precision was acceptable and in agreement with the criteria of Commission Decision 2002/657/EC and SANTE/12682/2019. To further demonstrate the suitability of the method the laboratory will be participate in proficiency tests in the near future.A total of 87 import and local samples from bovine meat were analysed, in order to test the applicability of the established method for the routine analysis. After the sample analysis the trace residues of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively, were confirmed in three samples. The chromatograms from positive samples are given in Additional file A multi-class and multi-residue analytical methods including use of stable isotopes were developed for analysis of residues of veterinary drugs and other contaminants in bovine meat. The veterinary drugs included antimicrobials , anabolic hormones, lactones, β-agonists and from pesticides and mycotoxins . Thirteen (13) stable isotopes were used as internal standards to cover wide range of analytes including veterinary drug residues, pesticides and mycotoxins. The developed method showed good performance characteristics of the method comply with EU recommendations.Additional file 1. Recoveries from investigation of sample amount and extraction steps.Additional file 2. Chromatograms of positive samples from bovine meat.


Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.A recently-validated and underexplored drug target in  Mycobacterium tuberculosis remains high, with 2019 seeing approximately 10 million people infected and 1.4 million deaths worldwide . H. HPseudoUnless otherwise stated, chemicals, media and reagents used in this study were supplied by Sigma-Aldrich , Thermo Fisher Scientific , Duchefa Biochemie  or New England Biolabs . Sanguinarine chloride for kinetic screening was supplied by Sapphire Biosciences .bpsA was described previously [pcpS, which expresses an untagged version of the broad-spectrum PPTase PcpS to covert apo-BpsA to holo-BpsA and not co-purify during Ni-NTA chromatography, was as previously described [pptT was constructed by amplifying pptT from M. tuberculosis H37Ra genomic DNA using the primers CCCCCATATGGACGGTAGGCACGCTG and CCCCCTCGAGTCATAGCACGATCGCGGT (restriction sites underlined). The amplified gene was ligated into pET28a(+) between the NdeI and XhoI restriction sites.Construction of the BpsA expression plasmid pCDFDUET1::eviously . Construescribed . pET28a(E. coli BL21(DE3) ΔentD [600 of 0.1 overnight and incubated at 37 °C, 200 rpm until an A600 of between 0.6 and 0.8 was obtained. The cultures were then chilled on ice for approximately 30 min. Protein expression was induced with the addition of IPTG to a final concentration of 0.5 mM. The cultures were then incubated for 24 h at 18 °C, 200 rpm before harvesting by centrifugation . The cell pellets were then stored at −80 °C until needed.All protein expression used 3) ΔentD  as an exv/v glycerol to enhance protein stability. The eluted PPTases were then desalted using a HiTrap desalting column  with a desalting buffer of 50 mM Tris-Cl (pH 7.8) and 12.5% glycerol (v/v). The buffer composition was then adjusted to a final concentration of 40% (v/v) glycerol, and aliquots were stored at −80 °C until needed. Apo and holo-BpsA were purified as previously described [PPTases were purified using standard Tris-Cl Ni-NTA chromatography buffers with the following modification: the bind, wash and elute buffers were supplemented with 25% 2 and 5 mM ATP was added to individual wells of a standard 96 well plate. Next, 10 µL holo-BpsA , 10 mM MgClescribed ) was add2, 5 mM ATP, 5 mM L-glutamine, 10 µM CoA and 0.6 µM apo-BpsA was added to each well. The plate was then shaken at 1000 rpm for 10 s and incubated at room temperature for 1 h. The indigoidine was then resolubilised by the addition of 200 µL DMSO and incubated at 37 °C with shaking at 200 rpm for 0.5 h. A Perkin Elmer Enspire plate reader was then used to record the absorbance of each well at 590 nm.To determine the optimal concentration of PptT for the detection of inhibitors, 10 µL of PPTase mix comprising 50 mM Tris-Cl (pH 8.0) and PptT at a range of concentrations (0–0.5 µM) was added to individual wells of a standard 96 well plate. To initiate indigoidine synthesis, 40 µL of a reaction mix comprising 50 mM Tris-Cl (pH 8.0), 10 mM MgClZ-factor of the screen, 80 reactions were established in columns 2–11 of a standard 96 well-plate. Each was prepared from 30 µL of reaction mix comprising 50 mM Tris-Cl (pH 8.0), 0.01% (v/v) Triton-X, 10 mM MgCl2, 5 mM ATP, 5 mM L-glutamine and 10 µM CoA. Next, 10 µL PPTase mix containing either 0.4 µM PptT and 50 mM Tris-Cl (pH 8.0) (or else unadulterated 50 mM Tris-Cl (pH 8.0) as a negative control) was added to each well. To initiate indigoidine synthesis, 10 µL of 0.6 µM apo-BpsA in 50 mM Tris-Cl (pH 8.0) was added to each well. The plate was then shaken at 1000 rpm for 10 s and incubated at room temperature for 1 h. The indigoidine was then resolubilised by the addition of 200 µL DMSO and incubated at 37 °C with shaking at 200 rpm for 0.5 h. A Perkin Elmer Enspire plate reader was then used to record the absorbance readings at 590 nm. To calculate the Z-factor, the following equation was used , with σ1280 collection was first diluted to a working stock of 500 mM per compound. To screen the collection, 88 reactions at a time were established in columns 1–11 of a standard 96 well-plate. Each was prepared from 30 µL of reaction mix comprising 50 mM Tris-Cl (pH 8.0), 0.01% (v/v) Triton-X, 10 mM MgCl2, 5 mM ATP, 5 mM L-glutamine and 10 µM CoA. Next, a CyBio liquid handler was used to pin 2 µL of compound into columns 2–11 and 2 µL of DMSO into column 1. To screen for PptT inhibitors, 10 µL of PPTase mix comprising a freshly-prepared stock of 0.4 µM PptT and 50 mM Tris-Cl (pH 8.0) was added to wells A1–C1 and columns 2–11 (see apo-BpsA in 50 mM Tris-Cl (pH 8.0) was added to each well. To screen for holo-BpsA inhibitors, an equivalent screening format was established only using 0.6 µM holo-BpsA and with no PPTase mix added. Each plate was then shaken at 1000 rpm for 10 s and incubated at room temperature for 1 h. Indigoidine was then resolubilised by the addition of 200 µL DMSO and incubation at 37 °C with shaking at 200 rpm for 0.5 h. A Perkin Elmer Enspire plate reader was then used to record the absorbance readings at 590 nm. The Python package Pandas [The Sigma-Aldrich LOPAC2–11 see . To inite Pandas .50 values of inhibitors, a two-fold serial dilution between 40 µM and 0.625 µM of each compound was established in 30 µL reaction mixes comprising 50 mM Tris-Cl (pH 8.0), triton-X 0.01% (v/v), 10 mM MgCl2, 5 mM ATP, 5 mM L-glutamine and 10 µM CoA in replicate rows of a 96 well plate. To screen for PptT inhibitors, 10 µL of PPTase mix comprising a freshly-thawed stock of 0.4 µM PptT in 50 mM Tris-Cl (pH 8.0) was added. To initiate indigoidine synthesis, 10 µL of 0.6 µM apo-BpsA in 50 mM Tris-Cl (pH 8.0) was added to each well. The plate was then shaken at 1000 rpm for 10 s and incubated at room temperature for 1 h. Indigoidine was resolubilised by the addition of 200 µL DMSO and incubation at 37 °C with shaking at 200 rpm for 0.5 h. A Perkin Elmer Enspire plate reader was then used to record the absorbance readings at 590 nm. The percentage inhibition at each compound concentration was then compared to a DMSO only control, and GraphPad Prism 9.0 was used to fit a four parameter dose–response curve.To calculate the ECv/v) across individual rows of a standard 96 well plate. Next, 35 µL of a PPTase mix comprising a freshly-thawed stock of 0.4 µM PptT, 50 mM Tris-Cl (pH 8.0) and Triton-X 0.01% (v/v) was added to each well. To initiate indigoidine synthesis, a 35 µL reaction mix comprising 0.01% (v/v) Triton-X, 50 mM Tris-Cl (pH 8.0), 10 mM MgCl2, 5 mM ATP, 5 mM L-glutamine, 10 µM CoA and 0.6 µM apo-BpsA was added to each well. The plate was then shaken at 1000 rpm for 10 s, and absorbance readings at 590 nm were read every 20 s. To determine the EC50 values, the maximum indigoidine synthesis velocities were determined as described previously [To calculate the inhibition of PptT by 6-nitroso-1,2-benzopyrone and sanguinarine chloride, a serial dilution between 200 µM and 0.2 µM was established for each compound in 30 µL 50 mM Tris-Cl (pH 8.0) and Triton-X 0.01%  without exceeding the well capacity of a standard 96-well plate  to initiate indigoidine synthesis, where possible. Following incubation for 1 h at room temperature, each 50 µL reaction was stopped by the addition of 200 µL of DMSO to solubilise the indigoidine, after which the A590 was measured  and 0.01% Triton X-100 (to minimise nonspecific inhibitory activity from compounds that induce protein aggregation ) was alimeasured C. The ca1280) to identify inhibitors of PptT  of each compound added to a PptT/0 values . In one  of PptT . Other tsubtilis .1280 collection against the SARS-CoV-2 protease using a FRET-based reporter and noticed that 8 of our 17 candidate PptT inhibitors  also gave hits as strong protease inhibitors [holo-BpsA at a higher concentration of 40 µM -eicosadienoic acid, disulfiram and sanguinarine chloride.In other work, we recently screened the LOPAChibitors . This suof 40 µM . In thisAspergillus fumigatus in a PPTase dependant manner [B. subtilis [M. tuberculosis PptT [50 value of 4.4 ± 1.2 µM for 6-nitroso-1,2-benzopyrone (50 value of 8.3 µM we calculated from our endpoint assay. However, the kinetic assay data for sanguinarine chloride was more variable (We next sought to benchmark our DMSO-resolubilisation end-point assay against the kinetic assay we had previously described . For thit manner , and thesubtilis  and M. tsis PptT . We usedzopyrone , which ivariable , and GraM. tuberculosis. We validated the assay by screening the LOPAC1280 collection from Sigma-Aldrich. To the best of our knowledge, this is the first reported direct chemical library screen for inhibitors of PptT. From this screen we identified 18 inhibitors, but further investigation indicated that ten of these were non-specific compounds that also inhibited holo-BpsA when added at higher concentrations. Of the remaining eight, two were novel PPTase inhibitors that may be specific for PptT, while six had previously been identified as inhibitors of the B. subtilis canonical Type II PPTase Sfp, providing evidence by association for the efficacy of our screen. Finally, we compared our optimised end-point assay against a kinetic BpsA assay we had employed earlier [Here, we report on the development, optimisation and validation of a simple high-throughput assay to rapidly identify inhibitors of the promising antibiotic target PptT from  earlier  and concTwo other assays have been developed to evaluate possible inhibitors of PptT. The first uses a scintillation proximity assay that is substantially more complex than our simple colourimetric BpsA assay, requiring radiolabelled CoA and a scintillation counting machine, and validation against PPTase inhibitors has not yet been reported . The secB. subtilis Sfp [M. tuberculosis HR7Rv against approximately 90,000 small molecules, with PptT subsequently identified as the drug target [M. tuberculosis offer substantial promise in enabling selection of a suitable target upfront [PPTases have been discussed in the literature as promising drug targets for almost two decades . Despiteilis Sfp , but hasg target . While wg target . Moreoveg target . Convers upfront  and faci upfront . Our ass upfront . This pr


GelMA had a concentration-dependent increase in stiffness, but cells decreased this stiffness over time, across concentrations. Viscoelastic changes in terms of stress relaxation increased progressively in 5% GelMA, while mathematical Maxwell analysis showed that the relative importance (iR) of the fastest Maxwell elements increased proportionally. The 10% GelMA only showed differences at 7 d. In contrast, ASCs in 15% GelMA caused slower stress relaxation, increasing the iR of the slowest Maxwell element. We conclude that GelMA concentration influenced the stiffness and number of Maxwell elements. ASCs changed the percentage stress relaxation and iR of Maxwell elements transforming hydrogel viscoelasticity into a more fluid environment over time. Overall, 5% GelMA induced the most favourable ASC response.The extracellular matrix provides mechanical cues to cells within it, not just in terms of stiffness (elasticity) but also time-dependent responses to deformation (viscoelasticity). In this work, we determined the viscoelastic transformation of gelatine methacryloyl (GelMA) hydrogels caused by adipose tissue-derived stromal cells (ASCs) through mathematical modelling. GelMA-ASCs combination is of interest to model stem cell-driven repair and to understand cell-biomaterial interactions in 3D environments. Immortalised human ASCs were embedded in 5%, 10%, and 15% ( In tissues, cells reside within the extracellular matrix (ECM). The ECM consists of fibrous proteins such as collagens, that provide mechanical strength and architecture; enmeshed in a water-retaining gel of negatively charged polysaccharides, such as glycosaminoglycans (GAGs) ,2. The EWithin a 3D environment, MSCs will experience biochemical and biomechanical stimuli. Overall, the ECM regulates cellular behaviour via activation of intracellular signalling pathways ,8,9. In In 3D culture, cells will initially attach to their surrounding ECM and, to put it simply, will “push and pull” on their adjacent environment. This cell-induced strain will be initially resisted by the matrix (elastic response), but this resistance will decay over time (viscous response) . For exaWhile the examples amassed to date offer invaluable knowledge about the influence of viscoelasticity on cellular processes, the current literature derives mostly from non-(bio)degradable hydrogels and at short time scales ,22,23. T2 side chains with methacrylic anhydride (MA) [In this study, we employed adipose tissue derived stromal cells (ASCs) loaded into gelatine methacryloyl (GelMA) hydrogels. ASCs are multipotent and can differentiate into adipogenic, chondrogenic, and osteogenic phenotypes, among others . In compide (MA) . This pride (MA) . These cide (MA) . Combinaide (MA) ,38,39.In the literature, data that describe the impact of stromal cells over matrix time-dependent mechanics is scarce. Therefore, in our study, we evaluated the changes in GelMA stiffness (elastic modulus) and viscoelasticity (stress relaxation) caused by embedded ASCs cultured over a period of 14 d. The stress relaxation data were analysed using a generalized Maxwell model. ASC morphology and viability was also assessed. Subsequently, we mathematically modelled the viscoelastic changes caused by ASCs-matrix interaction in GelMA hydrogels in vitro.1H-NMR spectra derived from gelatine and GeIMA batches used in these experiments are shown in Examples of the w/v GelMA were 20.00, 10.00, and 6.70, respectively. After reaching equilibrium swelling at 1 d, the calculated swelling ratio were 14.19 ± 1.28, 6.41 ± 0.43, and 4.54 ± 0.46 for 5%, 10%, and 15% GelMA, respectively. Analyses showed that cell-free 5%, 10%, and 15% GelMA hydrogels differed after 1 d with 5% GelMA having the highest water content compared to 10% GelMA and 15% GelMA. Compared to 15% GelMA, 10% GelMA hydrogels also contained more water (The mass swelling ratio of GelMA hydrogels based on Equation (2) is shown in re water . −1 was 6.94 ± 1.89 kPa; 72.08 ± 9.50 kPa; and 214.50 ± 24.53 kPa, respectively. Stiffness of cell-free GelMA did not change during the 14 d of study. The elastic modulus at 0 d of cell-loaded GelMA hydrogels did not differ from cell-free hydrogels at the same timepoint (The elastic modulus (stiffness) at 0 d of cell-free (control) 5%, 10%, and 15% GelMA hydrogels at a strain rate of 0.2 simepoint a. In cell-loaded 5% GelMA, the elastic modulus at 0 d (6.39 ± 0.59 kPa), steadily decreased at 7 d (4.96 ± 0.75 kPa) and 14 d (4.86 ± 0.99 kPa). The decrease in stiffness was also detected between cell-loaded and cell-free hydrogels at 1 d, 7 d, and 14 d a. The elastic modulus of cell-loaded 10% GelMA at 1 d (70.83 ± 15.32 kPa) decreased after 7 d (64.41 ± 8.66 kPa) and 14 d (68.81 ± 13.43 kPa) a. DifferIn cell-loaded 15% GelMA hydrogels, their elastic modulus decreased at 14 d (189.90 ± 13.67 kPa) compared to 0 d (217.20 ± 20.74 kPa), 1 d (216.20 ± 21.01 kPa), and 7 d (216.40 ± 22.19 kPa) a. DifferThe percentage of stress relaxation of cell-free 5%, 10%, and 15% GelMA hydrogels in 100 s at 0 d was 7.2 ± 1.48%, 7.34 ± 1.12%, and 6.95 ± 0.64%, respectively. This initial percentage of stress relaxation did not change significantly after 14 d in culture conditions in cell-free hydrogels b. FurtheCell-loaded 5% GelMA hydrogels showed a gradual and significant increase in stress relaxation percentage. Compared to 0 d (8.19 ± 1.12%), stress relaxation percentage increased at 7 d (10.49 ± 2.02%) and 14 d (11.03 ± 2.20%). Differences were also found between cell-loaded hydrogels between 1 d (8.80 ± 1.02%) and 14 d. Higher stress relaxation percentage among cell-free and cell-loaded hydrogels at 7 d and 14 d was also found b. Unlike cell-loaded 5% GelMA, cell-loaded 10% GelMA hydrogels showed higher percentage of stress relaxation only at 7 d (7.77 ± 0.84%) compared to 7 d cell-free hydrogels (6.65 ± 0.61%) b. In contrast to previous GelMA concentrations, in 15% GelMA cells decreased the percentage of stress relaxation, from 0 d (8.00 ± 1.37%) to 7 d (6.28 ± 1.34) and 0 d to 14 d  and 7 d and 1 d to 14 d b. The avτ) values  of certain Maxwell elements behaved similarly across GelMA concentrations irrespective of cells. For example, the iR of the first and second elements in 10% and 15% GelMA remained below 3%  of individual Maxwell elements over time.In this study, we found that ASCs modify not only the stiffness but the viscoelasticity of GelMA hydrogels. We showed that cells transformed hydrogel viscoelasticity into a more fluid-like environment. We established that cell-induced viscoelastic changes can be accurately quantified using a generalised Maxwell model of viscoelasticity. Through this model we found that GelMA concentration influenced the elastic modulus (stiffness)and number of Maxwell elements, whereas cells influenced percentage stress relaxation and relative importance  [GelMA hydrogels showed a concentration-dependent rise in stiffness. The elastic modulus of our hydrogels, at a strain rate of 0.2 sDoF 56%) ,32,40. SDoF 56%) . Since tDoF 56%) . Cells did not interfere with GelMA photopolymerisation and crosslink formation, nor was cell viability affected by exposure to UV/VIS-light radiation. Compared to more concentrated hydrogels, 5% GelMA had greater cell viability, marked spreading, and elongated morphology over 14 d. Lower cell viability has also been reported in other hydrogels used for 3D cell culture with increasing polymer concentrations . In our Viscous dissipation of mechanical stress (stress relaxation) depends on the water content of a hydrogel, i.e., its swelling ratio. Our data showed that the GelMA swelling ratio was inversely proportional to the polymer concentration, which corroborates previous literature ,40,44,45iR at the cost of a decrease in the fourth element, i.e., longest element. For 10% GelMA, only the fourth element changed after 14 d, but no statistical changes were reflected in other elements, likely due to small changes in their iR. In contrast to previous concentrations, in 15% GelMA, ASCs slowed down the stress relaxation. Maxwell analysis indicated that the iR of the fourth element increased, with changes reflected on the third element. These findings suggest that matrix mechanics can lead to diverse viscoelastic outcomes in ASC loaded hydrogels. Cell-induced alterations in the architecture and composition of the ECM may explain the contrasting results in viscoelasticity among GelMA concentrations. For example, deposition of hygroscopic ECM molecules such as GAGs has been associated with increased viscous dissipation [Maxwell analysis of the viscoelastic properties of GelMA, indicated that the number of Maxwell elements increased alongside stiffness, an observation also made in other hydrogel systems ,46. In osipation . Additiosipation . Tightlysipation , the ovesipation ,53. Furtsipation ,54,55. A® > 600MW, Leverkusen, Germany) was dissolved in 1x Dulbecco´s phosphate-buffered saline  and 0.6 mL of MAA  added per gram of gelatine at 60 °C under gentle stirring. After three hours, the solution was diluted in an equal volume of 1× DPBS and centrifuged at 2000× g for 5 min to separate any unreacted MAA. The supernatant was collected, and its pH adjusted to 7.4 with 2 M sodium hydroxide (NaOH). The pre-GelMA stock solution was dialysed for a week in a 14 kDa cut-off cellulose membrane (Sigma-Aldrich) against demi-water at 50 °C with twice daily changes of demi-water. The remaining solution was lyophilised in a Free Zone® 2.5 Plus freeze dryer  at −80 °C for five days. The lyophilized GelMA was stored in the dark at −20 °C until further use.Medical grade gelatine type A, 99.8 kDa MW, 262 g Bloom , was assessed through 1H-Nuclear Magnetic Resonance (1H-NMR) as described previously [2O at 40 °C. The samples were placed inside 5 mm diameter, 100 MHz Wilmad® NMR tubes  and analysed with an AvancedTM Neo 600 MHz spectrometer . Each sample underwent 16 cycles with a 5 s delay between cycles. The resulting 1H-NMR-spectra were analysed using MestReNova® v14.0 . The chemical shift scale was adjusted using the D2O peak and normalised using phenylalanine’s aromatic rings. The DoF were calculated by dividing the area of the lysine peak before (b) and after (a) functionalization as mentioned in Equation (1).The efficiency of substitution of NHeviously . For thiw/v) concentrations at 60 °C and mixed with lithium phenyl-2,4,6-trimethylbenzoylphosphinate , a photoinitiator, at 0.5% (w/v) concentration. Once dissolved, the GelMA working solution was sterilised with a 0.2 µm polyethersulfone (PES) membrane filter  and stored at −20 °C. Lyophilised GelMA was dissolved in 1× DPBS at 5%, 10%, and 15%  containing 10% Fetal Bovine Serum , 1% Penicillin–Streptomycin  and 2 mM L-glutamine  and cultured at 37 °C, 5% CO2. Upon reaching 80–90% confluence in tissue culture plates, cells were detached using 0.5% Trypsin EDTA (Gibco) and centrifuged at 2000× g for 5 min. The cell pellet was resuspended in fresh culture medium and the number of live cells calculated using both a Z2TM Coulter Counter® particle count and size analyser  and by trypan blue staining. Aliquots containing 7 × 105 cells were centrifuged again, the supernatant removed, and the cell pellet resuspended in 350 µL of either 5%, 10%, or 15% GelMA working solution thawed at 37.5 °C. For hydrogel moulding, polycaprolactone  containers of 2.4 mm (h) × 14 mm (d) were 3D printed under sterile conditions with a Biobots I bioprinter (Allevi Inc.) at 130 °C, 90 PSI. Hydrogels were cast in the PCL moulds and photopolymerised using the bioprinter’s UV lamp (405 nm) at 7 mW/cm2 for 5 min. Cell-free GelMA hydrogels were used as controls. After photopolymerisation, all hydrogels were carefully removed from the moulds and incubated at cell culture conditions  until subsequent analysis.Immortalised human adipose tissue-derived stromal cells (iADSC13) between passages 19 to 23, negative for ®, Eugene, OR, USA) and 2 µM propidium iodide  dissolved in culture medium in the absence of FBS. Hydrogels were gently shaken at room temperature for 10 min to facilitate diffusion of the working solution and then incubated at 37 °C, 5% CO2 for an additional 20 min. The hydrogels were washed again with DPBS and imaged with an EVOS® FL digital inverted microscope  using the following light cubes: GPF (λex 470/22 nm/λem 525/50 nm), and Texas Red (λex 585/29 nm/λem 628/32 nm) to visualise calcein AM and PI, respectively, at 10x magnification. Micrographs were analysed using using ImageJ Ver 1.52p  [Cell viability, spreading, and morphological examination of ASCs were assessed using a Live/Dead fluorescent staining at 0 d, 1 d, 7 d, and 14 d. Cell-loaded hydrogels were washed three times with DPBS and mixed with the Live/Dead working solution composed of 5 µM calcein-AM (Life Technologiesry 2021) . All imaWs) on a balance  before being placed inside a RapidVap® vacuum evaporation system (Labconco Corporation). Hydrogels were dried at 45 °C using negative pressure at 25% vortex speed for 1 d. The remaining weight (Wd) was measured, and the swelling ratio calculated according to Equation (2).The swelling ratio tells us the fluid to solid ratio in the hydrogel, i.e., weight of DPBS present in the hydrogel divided by the dry weight of GelMA. Acellular GelMA hydrogels were incubated in 1× DPBS for 1 d to reach swelling equilibrium. Afterwards, hydrogels were briefly blotted against paper to remove excess liquid drops and weighed . At first the hydrogel was compressed to 80% of its original thickness  at a strain rate ) was divided by the constant strain of 0.2 to obtain the value of relaxing modulus E(t). A generalised Maxwell model shown in Equation (3) was then fitted to the experimental data to get the values of iE and iτ for individual Maxwell elements. The iτ was the relaxation time constant for each individual Maxwell element and was the ratio of iη (dashpot) and iE (spring) for that element. The number of Maxwell elements necessary to fit the experimental data was determined by both looking at the fit visually and with the help of the plot which shows a decrease in Chi2 value with the addition of every extra Maxwell element. The number of Maxwell elements were chosen for which no further decrease in Chi2 was observed.Changes in the mechanical stability and viscoelasticity of cell-loaded and cell-free (control) GelMA hydrogels were monitored at 0 d, 1 d, 7 d, and 14 d, by performing a stress relaxation test in compression on the Low-Load Compression Tester (LLCT) ,59,60. DiR) of each Maxwell element in terms of percentage within the relaxation process was expressed as the proportion of its spring constant, iE, to the sum of all spring constants (Equation (4)) [The relative importance (ion (4)) ,59,60. or median values with quartiles. p values below 0.05 were considered statistically significant.All statistical analyses were performed using GraphPad Prism v9.1.0 . All data were searched for outliers using the robust regression and outlier removal (ROUT) test and analysed for normality using Shapiro–Wilk and D’Agostino & Pearson tests ,62,63. BiR of individual Maxwell elements over time. Whether changes in the iR of each Maxwell element are due to matrix degradation, ECM deposition, or cellular processes that induce permanent deformation of the hydrogel structure, remains an intriguing question.We conclude that ASCs transformed GelMA viscoelasticity into a more fluid environment and altered hydrogel mechanical stability. Out of the concentrations studied here, 5% GelMA provided the most conducive biomechanical environment for ASC cells to spread and remain viable. Their cellular response in turn, caused the most viscoelastic remodelling towards viscoelastic fluid, observed as changes in elastic modulus, stress relaxation, and


Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.Emergence of bovine mastitis caused by The online version contains supplementary material available at 10.1186/s13567-021-01014-9. Prototheca spp. are unicellular achlorophyllous algae, 3–30 µm in diameter, that lack a specific glucosamine cell wall or chloroplasts; specific species include P. bovis, P. ciferrii, P. cerasi, P. pringsheimii, P. blaschkeae, P. wickerhamii, P. xanthoriae, P. cookie, P. xanthoriae, P. cutis, P. miyajii, P. tumulicola, P. moriformis, P. stagnora, and P. ulmea [Prototheca spp. is characterized by an abrupt decrease in both milk production and quality, an increased somatic cell count, and frequently culling, with substantial economic losses [Prototheca spp., P. bovis was the causative pathogen of bovine mastitis, whereas P. ciferrii occasionally causes granulomatous lesions in experimentally infected bovine udders and protothecosis in humans [Prototheca spp. mastitis has been reported in many countries, including Canada, Poland, Italy, Brazil, and Japan [Mastitis is common in dairy cattle worldwide, causing serious reductions in milk yield and quality and large financial losses , 2. InfeP. ulmea . Bovine c losses . Among Pn humans , 8. Protnd Japan –11.Escherichia coli and Staphylococcus aureus, causing an aggravated inflammatory response and damage in bMECs [An inflammatory response, a typical feature of bovine mastitis, is characterized by release of inflammatory cytokines such as IL-1β, TNF-α and IL-18. Numerous signal molecules or pathways are involved in regulation of an inflammatory response, including reactive oxygen species, inflammasome and NF-κB pathway , 13. Thein bMECs , 20. Actin bMECs . Furtherin bMECs , 23.Prototheca spp. mastitis, there are interstitial infiltrates of macrophages, plasma cells and lymphocytes into the mammary gland, and an antiserum against bovine keratin had weak positive expression in damaged mammary tissue [P. bovis or P. ciferrii increased expression of cytokine mRNA in bMECs [P. bovis or P. ciferrii are not well characterized. Therefore, mitochondrial damage, inflammatory cytokines including TNF-α, IL-1β and IL-18, and protein expression in the NF-κB/NLRP3 pathway that regulate inflammation were measured to characterize and compare the pathogenesis of inflammatory responses in bMECs induced by infection with P. bovis versus P. ciferrii.Mitochondrial reactive oxygen species (mtROS) also activate the NLRP3 inflammasome, promoting inflammation and enhancing immune responses , 25. Accy tissue . We repoin bMECs ; howeverCell Counting Kit-8 (CCK-8), NADP + /NADPH assay kit, Bicinchoninic acid (BCA) protein assay kit, radioimmunoprecipitation assay (RIPA) lysis buffer, Mito-Tracker Green staining solution and Hoechst 33342 live cell staining solution were purchased from Beyotime . ELISA assay kit was purchased from mlbio . 4’, 6-Diamidine-2’-phenylindole dihydrochloride (DAPI), coverslips and Triton X-100, penicillin, streptomycin and bovine serum albumin (BSA) was purchased from Solarbio . Enhanced chemiluminescence (ECL) kits were obtained from Thermo Fisher Scientific Pierce . Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s medium (DMEM) were purchased from Hyclone . Rotenone was purchased from MCE . Mito-SOX red mitochondrial superoxide indicator was purchased from Yeasen . Trizol Reagent, cDNA synthesis superMix and Two-step RT-PCR superMix were purchased from TransGen Biotech . Primary antibodies, including NLRP3, ASC, Caspase-1, IL-1β and α-Tubulin, were purchased from Proteintech , and NF-κB p65, Phospho-NF-κB p65, IκBα and Phospho-IκBα were purchased from Cell Signaling Technology . Peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Proteintech .Prototheca bovis was isolated in 2016 from 105 clinical mastitis milk samples collected on 6 large (> 500 cows) Chinese dairy farms, whereas the 58 P. ciferrii isolates were recovered in the same year from environmental samples from 3 large dairy farms, located in suburbs of Beijing, Tianjin and Shandong [Prototheca spp. were characterized as P. bovis and P. ciferrii by several methods. Firstly, based on cellular fatty acid pattern, P. bovis had more eicosadienoic acid (C20:2) compared to P. ciferrii [Prototheca (450 bp) fragment internal amplification control Proto18-4f (GACATGGCGAGGATTGACAGA) and Proto18-4r (AGCACACCCAATCGGTAGGA) primers (2.5 µL each primer), DNA template (1 µL), ddH2O (4 µL), and 2 × EasyTaq PCR supermix (10 µL) was amplified under specific conditions . Amplified fragments were sent for sequencing . Then, P. bovis and P. ciferrii were characterized by genotype-specific primers [P. bovis and P. ciferrii genotypes were further confirmed by restriction fragment length polymorphism analysis targeting the cytb gene fragment [P. bovis and P. ciferrii genotypes in the isolates recovered from clinical mastitis milk and environmental samples. These strains in within a species (P. bovis and P. ciferrii), strains had the same genotype, colony morphology and similar biochemical characteristics. We randomly selected 3 strains of each species for the following experiments, which were performed independently in triplicate. The 3 strains of P. bovis and 3 strains of P. ciferrii were isolated from clinical mastitis milk and environmental samples of 3 large farms located near suburbs of Beijing, Tianjin and Shandong, respectively [P. ciferrii grew more slowly than P. bovis on SDA and their colonies had differences in morphological characteristics; P. ciferrii produced small colonies with smooth surface and folded edges compared to P. bovis, whereas the latter had more eicosadienoic acid (C20:2) compared to the former [P. bovis and P. ciferrii isolates were multiplied by streaking on sabouraud dextrose agar (SDA) and incubated at 37 °C for 48 h. Then, a single colony was placed in sabouraud dextrose broth (SDB) and incubated for 72 h. Thereafter, organisms were diluted in DMEM to achieve required concentrations.ciferrii . Secondl2 at 37 °C, and cells from passages 2–8 were used for experiments. Before infection, cells were put in 6-well plates (1 × 106 cells per well) and cultured overnight. Next, cells were infected with P. bovis or P. ciferrii at a 5:1 multiplicity of infection  and incubated in 5% CO2 at 37 °C for 12 h. Then, culture supernatants were collected and frozen (−80 °C) to subsequently determine cytokine concentrations, whereas cells were collected to extract and characterize proteins. Each experiment was conducted in triplicate.The MAC-T line of bMECs  was used for cell culture. bMECs were placed in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 U/mL) and grown in cell culture plates. Cells were incubated in 5% COThe bMECs were fixed as described  and tran3 cells/well, allowed to adhere overnight, and then treated for 12 h with various concentrations of rotenone  used to enhance mitochondrial reactive oxygen species production . Then, bMECs were washed 3 times with PBS and 10 µL CCK-8 solution added to each well. After incubation for 1.5 h at 37 °C with 5% CO2, OD values were read at 570 nm.Cell viability was measured with a Cell Counting Kit-8 (CCK-8). The bMECs were seeded into 96-well plates at a density of 5 × 10P. bovis or P. ciferrii at a 5:1 MOI. After 12 h, bMECs were washed 3 times with PBS and 2 mL of warm (37 °C) Mito-Tracker Green staining solution was added. After incubation for 30 min at 37 °C, Mito-Tracker Green staining solution was removed and 2 mL fresh cell culture solution (37 °C) was added. Then, 10 μL Hoechst 33342 live cell staining solution was added to each well. After incubating for 10 min at 37 °C, the dye-containing culture medium was aspirated, cells were washed 3 times with culture medium and observed with laser scanning confocal microscopy .The bMECs were cultured in 6-well plates overnight and then infected with P. bovis or P. ciferrii at a 5:1 MOI. After 12 h, Mito-SOX red mitochondrial superoxide indicator was used to label mitochondrial reactive oxygen species. To induce accumulation of mtROS (positive control), bMECs were treated with 2.5 μM rotenone for 12 h. Next, cells were incubated with Mito-SOX (5 μM) in the dark for 10 min at 37 °C and then washed 3 times with PBS. Nuclei were stained with 300 nM 4. 6-diamimo-2-phenyl indole (DAPI) for 5 min at 37 °C and washed with PBS. Slides were covered with glass cover slips and intracellular mtROS assessed with laser scanning confocal microscopy (Olympus-FV3000).To detect intracellular mtROS production, bMECs were seeded into 6-well plates with cell climbing films and infected with P. bovis or P. ciferrii at a 5:1 MOI for 12 h. The NADPH content in cells was determined with a commercial NADP + /NADPH Assay Kit, according to the manufacturer’s protocol. Briefly, 200 μL NADP + /NADPH extract was added into each hole of the 6-well plate, gently blown to promote cell lysis, and supernatant collected for subsequent experiments. Then, 50 μL supernatant and 200 μL of G6PDH working solution were added into each 96-well plate. After incubation for 10 min at 37 °C, 10 μL chromogenic solution was added into each well and after incubation for 20 min at 37 °C, absorbance was measured at 450 nm.The bMECs were cultured into 6-well plates overnight and then infected with P. bovis or 3 P. ciferrii isolates at a 5:1 MOI for 12 h, and 10 µM of 2--2-oxoethyl) triphenyl-phosphonium chloride, monohydrate (mito-TEMPO), a mitochondria-targeted superoxide dismutase mimetic with superoxide and alkyl radical scavenging properties, was used to scavenge superoxide. Cytokines in supernatants of bMECs culture medium were quantified by ELISA kits, according to the manufacturer’s instructions. Cell culture supernatants were collected and concentrations of TNF-α, IL-1β and IL-18 in supernatants were measured, based on OD values at 450 nm.The bMECs were infected with the 3 g for 15 min at 4 °C and supernatant collected. Total mRNA of bMECs was extracted with mRNA extraction kit according to manufacturer’s instructions. Relative expression levels of TNF-α, IL-1β and IL-18 mRNA were determined using the StepOnePlus Real-Time PCR systems. Data were analyzed according to the 2−∆∆Ct method and results were expressed as relative mRNA levels [The bMECs were treated as described above for ELISA, washed 3 times with PBS, and cells collected for total RNA extraction. Trizol Reagent was pre-chilled on ice and 1 mL added to cell samples for 5 min to lyse cells. Mixed liquid was centrifuged at 12 000 × A levels . Primer The bMECs were treated as described above for ELISA, washed 3 times with PBS and then fixed in 4% paraformaldehyde for 30 min and subsequently permeabilized in 0.25% Triton X-100. Cells were incubated with 3% bovine serum for 30 min at room temperature and then incubated overnight at 4 °C with the following primary antibodies: NLRP3, NF-κB p65, Phospho-NF-κB p65, ASC, and IL-1β. Next, samples were washed with PBS and incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) for 1 h at room temperature. Then, samples were washed with PBS and stained with DAPI for 20 min. After washing with PBS, slides were covered with glass cover slips and observed under a laser scanning confocal microscope (Olympus-FV3000).g, 4 °C) for 15 min. Total protein concentration in the supernatant was determined with a BCA protein assay kit. Protein samples were denatured in boiling water for 10 min and then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. These membranes were blocked with 5% nonfat dry milk for 2 h at room temperature, then incubated overnight at 4 °C with the following primary antibodies: α-Tubulin, NLRP3, NF-κB p65, Phospho-NF-κB p65, IκBα, Phospho-IκBα, ASC, Caspase-1, and IL-1β. For α-Tubulin, membranes were incubated with mouse anti-α-Tubulin antibody, whereas for all other proteins, they were incubated with secondary antibody against rabbit IgG for 1 h at room temperature. After washing with Tris-buffered saline, the membrane was developed using ECL reagents and visualized with a chemiluminescence system. Results were normalized to α-Tubulin, and band density was analyzed with Image J .The bMECs were treated as described above for ELISA and then lysed on ice and the cell lysate suspension collected and centrifuged  of 3 independent experiments .After visually confirming that the data were normally distributed, independent Student’s P. ciferrii, mitochondria had relatively minor damage, including slight vacuolization , mitochondria had reticulated morphologies and mitochondrial cristae were clearly visible with TEM , with the highest NADPH in the P. bovis infection group , with more profound increases in bMECs infected with P. bovis (P < 0.05)    Figure A. In conP. bovis increased the green fluorescence intensity of NF-κB p65 and p-NF-κB p65 in bMECs  compared to the control ; there were fewer profound increases induced by P. ciferrii, but p-IκBα (P > 0.05) was higher than the control    compared to the control      and IL-1β protein in P. bovis and P. ciferrii infected bMECs were downregulated (P < 0.05)  were isolated from different samples, the pathogenicity of strains in each species to bMECs was similar, as both P. bovis and P. ciferrii induced mitochondrial damage and mtROS accumulation, with the former causing more profound damage.Pathogenic infections can cause mitochondrial damage, including swelling and vacuolation, increase ROS, decrease membrane potential, and increase oxidative stress, both in vitro and in vivo –33. Mitooduction  and mito in vivo , 37. In P. bovis or P. ciferrii stimulated inflammatory responses, characterized by release of inflammatory cytokines which activate immune effector cells to eliminate invading pathogens. There were significant increases in production of TNF-α, IL-18, and IL-1β at 12 h after bMECs were infected with P. bovis or P. ciferrii, indicating a marked inflammatory response. However, responses to P. bovis were more severe than P. ciferrii, consistent with its greater pathogenicity. However, scavenging mtROS with mito-TEMPO significantly decreased cytokine production. Therefore, we inferred that infection with P. bovis or P. ciferrii induced inflammatory responses in bMECs that were mitigated by suppression of mtROS.Increased mitochondrial ROS promoted inflammatory responses in peritoneal mesothelial cells, macrophages and T cells , 38. InfP. bovis or P. ciferrii activated the NF-κB pathway, upregulating expression of IκBα and NF-κB p65 proteins. Furthermore, expression levels of phosphorylated IκBα and NF-κB p65 proteins were upregulated after P. bovis or P. ciferrii infection, with greater upregulation of protein expression in the NF-κB pathway induced by P. bovis, indicating higher pathogenicity. Activation of the NF-κB pathway promoted inflammation, including a massive increase in cytokine production. In addition, mtROS activation of the IKK complex and subsequent signaling through the NF-κB pathway led to secretion of proinflammatory cytokines by inducing the intermolecular disulfide linkage of nuclear factor IκBα essential modulator [P. bovis or P. ciferrii, mito-TEMPO decreased expression of various proteins in the NF-κB pathway, including IκBα, NF-κB p65, p-IκBα and p-NF-κB p65. Thus, infection of bMECs with Prototheca spp., especially P. bovis, caused overexpression of proteins in the NF-κB pathway and enhanced inflammatory responses through generation of mtROS.Inflammatory responses have many regulatory mechanisms, including the NF-κB and NLRP3 inflammasome pathways , 40. In odulator , whereasodulator . In the P. bovis or P. ciferrii infection in bMECs promoted activation of NLRP3 inflammasomes to varying degrees, modulating upregulation of expression of NLRP3, ASC, and Caspase1 proteins, and promoting cleavage of Caspase1. We inferred that infection with either P. bovis or P. ciferrii contributed to the assembly of ASC, Pro Caspase1, and NLRP3 during inflammasome formation. Although P. bovis or P. ciferrii infections in bMECs promoted NLRP3 inflammasome activation, P. bovis induced larger increases in proteins of the NLRP3 inflammasome, indicating greater pathogenicity. Furthermore, expression levels of proteins of the downstream genes Pro IL-1β and IL-1β were upregulated in cells infected with P. bovis or P. ciferrii, although P. bovis caused more pronounced increases. Therefore, P. bovis induced a greater inflammatory response than P. ciferrii vai the NLRP3 inflammasom pathway.Activation of inflammasomes is critical in inflammatory responses, with key roles in regulating inflammation caused by pathogenic bacteria , 47. TheP. bovis or P. ciferrii infections induced mtROS in bMECs. However, treatment with mito-TEMPO downregulated expression levels of NLRP3, ASC and Caspase1 proteins in bMECs infected with P. bovis or P. ciferrii, whereas expression of Pro IL-1β and IL-1β proteins was also downregulated. Therefore, we inferred that mtROS has an important role in activation of NLRP3 inflammasomes and enhances production of IL-β, resulting in an inflammatory response in bMECs. Mito-TEMPO suppressed expression of proteins in NF-κB and NLRP3 inflammasomes pathways and reduced inflammatory responses in P. bovis- and P. ciferrii-infected bMECs  of P. ciferrii and P. bovis infections of bMECs. The coefficient of variation (cv) is the standard deviation divided by the mean. We calculated the cv values among biological replicates within P. bovis and P. ciferrii, respectively. These cv values reflect the variation among different biological replicates and the cv values were in the range of 0.001–0.098, therefore we consider the variation among biological replicates was acceptable.Additional file 2. The coefficient of variation values (Western blot) of P. ciferrii and P. bovis infections of bMECs. The coefficient of variation (cv) is the standard deviation divided by the mean. We calculated the cv values among biological replicates within P. bovis and P. ciferrii, respectively. These cv values reflect the variation among different biological replicates and the cv values were in the range of 0.006–0.298, therefore we consider the variation among biological replicates was acceptable.


Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung infections in cystic fibrosis patients. In this study, we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin, colistin and ivacaftor in rat plasma, human epithelial cell lysate, cell culture medium, and drug transport media. An aliquot of 200 μL drug-containing rat plasma or cell culture medium was treated with 600 μL of extraction solution (acetonitrile containing 0.1% formic acid and 0.2% trifluoroacetic acid (TFA)). The addition of 0.2% TFA helped to break the drug-protein bonds. Moreover, the addition of 0.1% formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility. After vortexing and centrifuging, the sample components were analyzed by HPLC-MS/MS. The multiple reaction monitoring mode was used to detect the following transitions: 585.5–101.1 (colistin A), 578.5–101.1 (colistin B), 393.2–337.2 (ivacaftor), 332.2–314.2 (ciprofloxacin), 602.3–101.1 ) and 595.4–101.1 (polymyxin B2 as IS). The running time of a single sample was only 6 min, making this a time-efficient method. Linear correlations were found for colistin A at 0.029–5.82 μg/mL, colistin B at 0.016–3.14 μg/mL, ivacaftor at 0.05–10.0 μg/mL, and ciprofloxacin at 0.043–8.58 μg/mL. Accuracy, precision, and stability of the method were within the acceptable range. This method would be highly useful for research on cytotoxicity, animal pharmacokinetics, and in vitro drug delivery.  •The method is highly useful for analysis on cytotoxicity, pharmacokinetics, and in vitro drug delivery for cystic fibrosis.•The method is the first validated method for analysis of colistin, ivacaftor and ciprofloxacin simultaneously.•The method could be employed in analysis of plasma, epithelial cell, cell culture medium, and drug transport media.•Trifluoroformic acid was used as a drug-protein bond cleavage, and formic acid was added to increase the response of drugs.•The method could detect the three model drugs within 6 min. In the later stages of the disease, some patients are infected with more resistant and difficult-to-treat isolates 2+. The dominant product ions for colistin A were m/z 241.0 [6-methyloctanoicacid-L-Dab-γ-NH2+H]+ and m/z 101.1 [L-Dab-γ-NH2]+ [m/z 578.5 [M+2H]2+. The dominant product ions for colistin B were m/z 227.2 [6-methylheptanoicacid-L-Dab-γ-NH2+H]+ and m/z 101.1 [L-Dab-γ-NH2]+ [m/z 602.3 [M+2H]2+ and polymyxin B2 ion m/z 595.4 [M+2H]2+ were measured.The precursor ion of colistin A was the doubly charged ion -γ-NH2]+ . The pre-γ-NH2]+ . These i-γ-NH2]+ . Polymyxm/z 393.2 [MH]+ and the dominant daughter ion was m/z 337.2 [MH–C4H9]+, which was the result after a tertbutyl cleavage. The ion m/z 288.2 originated from the breaking of the amide bond. The ions selected for ciprofloxacin were m/z 332.2, m/z 314.2 and m/z 288.2, which correspond to [MH]+, [MH–H2O]+ and [MH–CO2]+, respectively [For ivacaftor, the precursor ion was ectively . The pot3.43.4.1In the blank plasma and HBSS, no obvious interference was observed at the retention time of the analyte and the IS, which indicates that the method has satisfactory selectivity.3.4.2We optimized the ratio of needle washing solution as 50% water: 25% acetonitrile: 25% isopropanol. Both water-soluble colistin and ciprofloxacin or water-insoluble ivacaftor could be cleaned. After injection of the highest concentration of the analyte, no significant carryover was observed.3.4.3As shown in 3.4.4The linear equations are listed in 3.4.5For inter-day and intra-day experiments, the precision was within the acceptable range (CV ≤ 15%) . The acc3.4.6For the stability experiment, the samples were stored at 4 °C for one week and one month respectively, and compared with the freshly prepared samples. The accuracy of each drug was within ±15%, which is considered to be relatively stable under such conditions.4The new HPLC-MS/MS method established here could be used for rapid and accurate analysis of colistin, ivacaftor and ciprofloxacin in various media simultaneously. The total retention time of less than 6.0 min is efficient for analyzing large quantities of samples. The sample pretreatment procedure could be applied to rat plasma, cells lysate, cell culture medium, and transport medium. This method would be useful in cystic fibrosis related pharmacokinetic, in vitro cytotoxicity and drug transport studies.The authors declare that there are no conflicts of interest.