The nanohybrids were characterized using Fourier transform infrared (FT-IR) spectroscopy, ultra-violet visible near infrared (UV/VIS/NIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA) and transmission electron microscopy (TEM) measurements. The TGA measurements confirmed 20%, 60%, and 80% loading of pyrrole in AgFeO2 and hence the nanohybrids were designated as 20%-Ppy/AgFeO2, 60%-Ppy/AgFeO2, 80%-Ppy/AgFeO2 respectively. IR and X-ray photoelectron spectroscopy (XPS) studies confirmed polymerization of pyrrole and formation of the nanohybrids while XRD reflected high crystallinity of the nanohybrids. The photocatalytic activity of Ppy/AgFeO2 nanohybrids was investigated against 2,4,6-trichlorophenol  under sonophotocatalytic conditions using visible light irradiation. The nanohybrids were observed to completely degrade the organic pollutant within a short span of 40 min. The degradation kinetics fitted the pseudo-first order model. The fragments were analyzed using LCMS studies which revealed the formation of diols as degraded products. The nanohybrids revealed immense potential for rapid as well as eco-friendly destruction of organic pollutants in wastewater.With the aim to develop a visible light driven eco-friendly photocatalyst, the present work reports for the first time the synthesis of polypyrrole/AgFeO 2 nanohybrids for highly efficient visible light driven eco-friendly photocatalysis.Synthesis of polypyrrole dispersed AgFeO However, the photocatalytic performance of this semiconductor is largely limited by its high electron–hole recombination rate.20 With the aim to improve the photocatalytic performance of this semiconductor via sensitization with a conducting polymer, the present work reports the synthesis of Ppy dispersed AgFeO2 nanohybrids through sonochemical polymerization. The nanohybrids were characterized using FTIR, UV-visible, XRD and TEM measurements. Sonophotocatalytic activity of PPy/AgFeO2 nanohybrids was explored by degrading 2,4,6-trichlorophenol under visible-light irradiation. The results demonstrated that the synthesized nanohybrids hold immense potential to be used as an effective photocatalyst under visible light irradiation.AgFeON-methyl-2-pyrrolidone (NMP)  were used without further purification.Pyrrole , 2,4,6-trichlorophenol , ferric nitrate , silver nitrate , ammonium hydroxide , ferric chloride , methanol , ethanol , acetone  and Pyrrole (0.050 mol) was added to ethanol (25 ml) in a 250 ml round bottom flask and stirred for 15 min at 30 °C. Ferric chloride (0.040 mol) dissolved in distilled water (25 ml) was then added to the above reaction mixture drop wise for a period of 30 min with continuous stirring at 30 °C. The reaction mixture was then sonicated for a period of 6 h at the same temperature. The suspension was then left undisturbed for 24 h at −5 °C overnight in a deep freezer. The polymer obtained was filtered, washed thoroughly with acetone and double distilled water to remove unreacted pyrrole and excess ferric chloride. A black precipitate of polypyrrole was obtained which was dried at 70 °C for 24 h in vacuum oven.21 The solutions were further stirred for 24 h and the resulting ruby-brown precipitate was collected by filtration. The precipitate was then washed several times with deionized water and dried at 60–70 °C in vacuum oven for 24 h. Ruby red color powder was obtained which was sintered at 700 °C in a muffle furnace to obtain the desired silver ferrite.A mixture of silver nitrate (0.014 mol) and ferric nitrate (0.036 mol) were dissolved in deionized water (100 ml) and were stirred for 4 h at 27 °C on a magnetic stirrer. The hydroxide solution was then heated at 70 °C for 1 h. Ammonium hydroxide solution (50 ml) was slowly added to the above solution until a pH value of 12 was attained.2 were prepared by homogeneously dispersing the synthesized silver ferrite (0.005 mol) in ethanol (50 ml) in a 250 ml round bottom flask. Pyrrole monomer (0.005 mol) was then added to the above solution followed by the addition of FeCl3·6H2O (0.004 mol) with continuous stirring at 30 °C. The reaction mixture was then sonicated for 8 h and the obtained nanohybrid was filtered, washed thoroughly with acetone and distilled water in order to remove the unreacted pyrrole and excess of ferric chloride. Similar procedure was adopted for the synthesis of nanohybrids of Ppy and AgFeO2 using AgFeO2 (0.005 mol), pyrrole (0.0025 mol) and (0.02 mol) respectively.Nanohybrids of Ppy and AgFeOW0) was measured using an analytical balance. The specimen was heated in an electric furnace at 1000 °C for 30 min to burn out the organic matrix and then re-weighed (W1). As the latter weight was only of the inorganic material, Ppy weight fraction (wt%) was determined according to the formula:The amount of Ppy present in the nanohybrid ferrite was determined gravimetrically and compared with the weight loss obtained from thermogravimetric analysis (TGA). Ppy content was determined by the standard ash method as it was used as filler. The weight of each nanohybrid  was added to TCP solution (200 ml) which was sonicated and exposed to visible light irradiation simultaneously for a period of 120 min. For the degradation analysis, aliquots (5 ml) of TCP solution were taken out at regular intervals  and were centrifuged at a high speed (5000 rpm) using a centrifuge machine (model REMI R8C). Supernatant solutions were taken out and collected for determination of concentration of degraded dye which was monitored using a UV-visible spectrophotometer model Shimadzu UV-1800 at λmax of 2,4,6-trichlorophenol (310 nm).Photocatalytic experiments were performed by decomposing 2,4,6-trichlorophenol  under visible light in a photochemical reactor , fitted with an ultrasonic bath . A 300 W Xe arc lamp equipped with an ultraviolet cutoff filter was used to provide visible light with d spacing was calculated using Bragg's equation. Transmission electron micrographs (TEM) were taken on Morgagni 268-D TEM, FEI, USA. The thermal stability of AgFeO2, Ppy and Ppy/AgFeO2 nanohybrid was investigated using thermal analyzer STA 6000, Perkin Elmer. The samples were heated from 30–800 °C at the rate of 10°C min−1 in N2 atmosphere at flow rate of 20 ml min−1. For the detection and identification of degradation products, liquid chromatography-mass spectroscopy (LC-MS) was conducted using a Waters XEVO G2-S TOF, USA mass spectrometer equipped with an electrospray ionization interface (ESI) source and operated in negative polarity mode fitted with BEH C18 (1.7 × 50 mm) containing 2.1 packed particles. ACN and mild Q waters containing 0.1% formic acid, pH 2.7, were used as eluents. The experiments were carried out in triplicate for evaluating the effect of nanohybrid catalyst dosage in the degradation of 2,4,6 trichlorophenol.FT-IR spectra of the nanohybrids and the pristine compounds were taken on FT-IR spectrophotometer model Shimadzu IR Affinity-1 in the form of KBr pellets. UV-visible spectra were recorded on Lambda 750 S UV/VIS/NIR Spectrometer  using 2-propanol as solvent. X-Ray diffraction patterns were recorded on Philips PW 3710 powder diffractometer (Ni filtered Cu-Kα radiations). Peak parameters were analyzed using Origin 8.1 software. The 2 revealed 1 wt% loss up to 840 °C showing its excellent thermal stability −1 corresponding to the presence of OH groups on the iron oxide surface.10 The peak at 1629 cm−1 was attributed bending vibrations of O–H for group bound to Fe atoms while peaks at 1062 cm−1 and 684 cm−1 corresponded to Fe–O vibration modes.21 Two strong peaks were observed at 550 cm−1 and 475 cm−1 that were assigned to the stretching vibrations of the Ag–O bonds and the Fe–O. bonds, respectively.21 The IR spectrum of Ppy, revealed N–H stretching vibration peak centered at 3415 cm−1, C–C asymmetric stretching at 1450 cm−1, and pyrrole ring stretching at 1538 cm−1 21,22 The peak at 1039 cm−1 was attributed to C–H in-plane deformation, the peak at 1164 cm−1 was correlated to N–C stretching vibration, while the peak at 1304 cm−1 was associated with C–H in-plane vibration. The peak at 1164 cm−1 was attributed to C–N stretching vibration, and the peak at 1175 cm−1 revealed the in-plane deformation vibration of NH+, which is formed on the polymer chains by protonation. The peaks at 1039 cm−1 and 901 cm−1 were observed due to the presence of C–H and N–H in-plane deformation vibrations and C–C out-of-plane ring deformation vibration respectively. The peaks correlated to ring deformation were noticed at 906 cm−1, 895 cm−1, 781 cm−1 and 674 cm−1. The IR spectrum of the nanohybrid 20%-Ppy/AgFeO2, 2. A broad NH stretching peak was noticed around 3424 cm−1 due to interaction between the –NH group of Ppy with Fe2+ ions of the AgFeO2 suggesting that each AgFeO2 particle was completely coated with Ppy. The C–C asymmetric stretching peak was noticed at 1401 cm−1, while pyrrole ring stretching peaks were noticed at 1558 cm−1, 1216 cm−1 and 1048 cm−1 respectively. Strong absorption peaks were noticed at 537 cm−1 and 460 cm−1 which were assigned to the stretching vibrations of the Fe–O bonds in tetrahedral positions and the Ag–O bonds, respectively.19,20 However, upon increasing the loading of Ppy upto 60%, shifting of N–H vibration peak to 3434 cm−1 was noticed while the characteristic peaks attributed to AgFeO2 were observed at 537 cm−1, and 460 cm−1. The nanohybrid 80%-Ppy/AgFeO2 exhibited a pronounced shift in the N–H stretching vibration peak which was observed at 3454 cm−1 indicating strong interaction of the Fe–O and Ag–O bond of AgFeO2 with Ppy. The presence of the peaks associated with polypyrrole as well as AgFeO2 confirmed polymerization of the former as well as formation of the nanohybrid.21The IR spectrum of AgFeO PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bonds and other absorption peak at about 450 nm associated with polaronic transitions.22 The UV spectrum of AgFeO2, Eg) was calculated to be 1.73 eV as reported by other authors (shown in inset).23 The UV-visible spectra of the Ppy/AgFeO2 nanohybrids revealed an increase in the intensity of absorption band corresponding to AgFeO2 with the increase in the loading of Ppy. The intensity of this peak was noticed to be highest for 80%-Ppy/AgFeO2 suggesting intense interaction of the two components with the increase in the loading of Ppy, while it was observed to be lowest for 20%-Ppy/AgFeO2.. The band gap was calculated to be 1.65 eV, 1.68 eV and 1.7 eV for 80%-Ppy/AgFeO2, 60%-Ppy/AgFeO2 and 20%-Ppy/AgFeO2 respectively, confirming synergistic interaction between Ppy and AgFeO2 that improved the optical properties of the nanohybrid.The UV-vis spectrum of Ppy, 2, exhibited a fused tubular morphology with particles ranging between 90–100 nm 2, 2 morphology revealing the formation of tubular structures of dense as well as hollow particles ranging between 50–60 nm. The TEM of 60%-Ppy/AgFeO2 2, 2 was found to be predominant while at higher loadings, the morphology revealed aggregation and formation of clusters of Ppy as well as AgFeO2 particles.The TEM of Ppy θ = 25.28°, confirming the amorphous nature of the polymer.24 The XRD profile of AgFeO2, θ = 32.36°, 38.26°, 44.30°, 64.57°, 77.40°assigned to scattering from d(101), d(104), d(009), d(0012), d(114), planes, respectively. The (104) reflections were intense suggesting the preferred orientation of the grains along the c-axis. All diffraction peaks were correlated to the presence of α-AgFeO2 which is predominantly a 3R structure (JCPDS: 75-2147) though a small amount of 2H structure was also present (JCPDS: 25-0765).18,19 The peaks associated with metallic Ag, Ag2O, and FeOOH were not found which confirmed the formation of delafossite type structure. The XRD pattern, of Ppy/AgFeO2 nanohybrids, θ = 27.76° corresponding to d(006) plane. This peak appeared to be quite pronounced in the nanohybrids, showing the dominance of 2H type polystructure of AgFeO2. The intensity of the peak observed at 2θ = 32.36° corresponding to d(101) plane also appeared to be quite prominent in case of the nanohybrids besides the other peaks.The XRD of profile of pure Ppy, d(006) plane and the increase in the intensity of the peak corresponding to d(101) plane were attributed to the changes in the lattice symmetry of the AgFeO2, as the 3R polytype has rhombohedral symmetry, while the 2H polytype consists of stacking alternate layers with hexagonal symmetry. The 2H delafossite possesses a layered structure with one layer composed of close-packed Ag+ ions and the other as edge-shared Fe3+O6 octahedra containing the iron cations inside. The Ag+ ions are coordinated by two oxygen ions with the linear O–Ag–O bonds while, each of the Fe3+ cation is coordinated with six oxygen ions. It can therefore be concluded that the dispersion of Ppy in AgFeO2 was found to change the symmetry of the later, while the overall morphology of the nanohybrids was observed to be crystalline.The appearance of the 2,2-bonded nitrogen (C–NC), neutral nitrogen atoms (NH) and positively charged nitrogen atoms (N+) in Ppy further confirming the co-existence of Ppy in the nanohybrid.23 The Ag 3d3/2 and Ag 3d5/2 showed split doublet peaks at 367.8 eV, 368.5 eV, 373.5 eV and 374.8 eV respectively, + in AgFeO2, while the peaks at 368.5 eV and 374.8 eV were correlated to the presence of Ag0 on the surface of AgFeO2. These binding energies values were found to be in good agreement with the reported values.23 The XPS spectrum Fe 2p showed doublets at 710.5 eV (2p3/2) and 723.9 (2p1/2), 3+ ions.23 The peak at 531.5 eV observed in the XPS spectrum of O 1s, 2.23 The presence of the peaks associated with Ppy as well as AgFeO2 confirmed the formation of nanohybrid.X-ray photon spectroscopy (XPS) is an effectual tool for analyzing elements and their corresponding valence state. The XPS of 80%-Ppy/AgFeO2, 45.83 wt% degradation was recorded. Around 68.14% degradation was attained using 10%-Ppy/AgFeO2 after 140 min under visible light irradiation. Maximum degradation of TCP was found to be 99.6% in 120 min in the presence of 80%-Ppy/AgFeO2, while in presence of 30%-Ppy/AgFeO2, it was observed to be 82.23%. It can be seen that the photocatalytic efficiency of Ppy/AgFeO2 nanohybrid was higher than that of neat Ppy and AgFeO2. With the increase in the loading of Ppy in AgFeO2, the photocatalytic efficiency increased. The degradation kinetics data adequately fitted the pseudo-first order model in Ppy, AgFeO2, Ppy/AgFeO2 nanohybrids, with high correlation coefficients values of 0.998 and 0.981, 0.999, 0.999, 0.990 respectively, −l and 0.004 min−l for pure Ppy and AgFeO2 respectively. Incase of the nanohybrids the rate constant was observed to be highest for 80%-Ppy/AgFeO2 (k = 0.016 min−1).Degradation of 2,4,6-trichlorophenol (TCP) was performed by sonophotocatalysis and was monitored by UV-vis spectroscopy. The peaks associated with 2,4,6-trichlorophenol were observed at 242 nm and 310 nm, –(e)C/Co plots of the TCP degradation were studied by taking TCP solutions of 30 ppm, 50 ppm, 70 ppm concentrations in presence of 100 mg, 150 mg, 200 mg 80%-Ppy/AgFeO2 nanohybrid catalyst for a period of 40 min as shown in k) values using 100 mg, 150 mg, and 200 mg of the nanohybrid was calculated to be 0.037 min−1, 0.038 min−1, 0.072 min−1 respectively for 30 ppm TCP solution, 0.029 min−1, 0.056 min−1, and 0.067 min−1 for 50 ppm TCP solution and 0.013 min−1, 0.018 min−1, 0.025 min−1 respectively for 70 ppm TCP solution. The degradation was observed to follow pseudo first order kinetics in all cases.The Ka) value for TCP is reported to be 6 and therefore at pH < 6, the molecule will exist in its undissociated form while at pH > 6, it will exist as trichlorophenolate ion. Hence, the increase in the degradation efficiency in alkaline medium can be attributed to the presence of presence of excess hydroxyl ions which can form hydroxyl radicals (HO˙) by trapping the photo electrons produced during visible light illumination. These hydroxyl radicals can easily degrade the trichlorophenolate anions as compared to the undissociated TCP molecule.25The effect of pH on the degradation was analysed by taking 30 ppm, 50 ppm, and 70 ppm TCP solutions of pH 3.5, 5.5 and 12.5 respectively, as shown in 2 was tested upto five cycles and each cycle was carried out for a period of 40 min, 2 nanohybrid revealed 85% degradation upto four cycles while 80% degradation was noticed after fourth cycle. The nanohybrid was found to exhibit efficient reusability by maintaining its photocatalytic activity up to four recycles under visible light illumination.The recyclability of 80%-Ppy/AgFeOm/z values are shown in m/z values ranging from 160–70 were obtained. They were labelled as K1, K2, K3, K4, K5, K6, and K7. The first intermediate K1 (m/z 157) showed 100% abundance and was taken as the main degradation product.Degradation of TCP was confirmed by LCMS studies which revealed a variety of intermediate compounds formed during the course of the reaction, m/z values 151 (35%), 133 (50%), 130 (28%), 112 (33%), 108 (54%), 100 (19%), 76 (31%) and 73 (29%) were obtained. These intermediates revealed that degradation proceeded via substitution of Cl− with O−, through the attack of HO˙ free radicals. Interestingly the HO˙ radicals preferentially attacked the electron rich chloride functionality of the TCP molecule to form 6-chlorobenzene-1,2,4-tris(olate) . This fragment then degraded into 3-chlorocyclohexan-1-olate . 3,5-Dihydroxycyclohexan-1-one , was obtained from K2 fragment which was found to undergo oxidation to form cyclohexane-1,3-dione . The degradation product K5 was obtained by ring opening of cyclohexane via the attack of ˙OH free radicals, m/z 73).Intermediates with 2 nanohybrids were successfully synthesized via in situ chemical polymerization method. IR and UV studies confirmed the formation of the nanohybrid while XRD studies revealed the presence of mixed 2H and 3R polytype structures of AgFeO2 upon nanohybrid formation. TEM showed intense cluster formation upon higher loading of Ppy in AgFeO2. The photocatalytic activity of the nanohybrid was explored for the degradation of 2,4,6-trichlorophenol. The nanohybrid was found to exhibit high photocatalytic activity under visible light illumination and TCP was found to degrade within a short span of 40 min. The analysis of the degraded fragments revealed that the obtained products were diols which are potentially non-toxic. Hence, the synthesized hybrids can be safely used for the eco-friendly degradation of toxic organic pollutants.Polypyrrole/AgFeOThere are no conflicts to declare.RA-008-C8RA00754C-s001


High CO2 concentration generally inhibits algal growth, and previous studies have mostly focused on breeding freshwater algae with high CO2 tolerance. In this study, one marine algal strain Dunaliella salina  was grown under 0.03%-30 % CO2 and 3% NaCl conditions, and was evaluated to determine its potential for CO2 assimilation. The results showed that D. salina could tolerate 30% CO2, and its maximum biomass concentration could reach 1.13 g·L−1 after 8 days incubation, which was 1.85 times higher than that of incubation in air (0.03%). The phenomenon of high-CO2 tolerance in D. salina culture was discussed basing on transcriptome analysis. The results showed that D. salina was subjected to oxidative stress under 30% CO2 conditions, and the majority genes involving in antioxidant system, such as SOD, CAT, and APX genes were up-regulated to scavenge ROS. In addition, most of the key enzyme genes related to photosynthesis, carbon fixation and metabolism were up-regulated, which are consistent with the higher physiological and biochemical values for D. salina incubation under 30% CO2.Microalgae has been regarded as a promising method for reducing CO The highest biomass concentration (1.28gL−1) was obtained under 2% CO2. 5%–15% CO2 were harmful to microalgal cells and inhibited their growth. Chaetoceros muelleri in response to different CO2 levels (0.03%–30%). It was found that Chaetoceros muelleri showed maximum biomass concentration (1.06 g L−1) under 10% CO2. However, higher CO2 concentrations led to negative effect on microalgal growth, and the biomass concentrations sharply reduced to 0.59 and 0.31 g L−1 under 20% and 30% CO2, respectively. 2-tolerant microalgal strain isolated and identified as Thalassiosira weissflogii H1. There was no significant difference between the growth rates and maximum growth yields of this diatom under bubbling air, 5% CO2 and 10% CO2, but the growth rate and maximum growth yield under 20% CO2 markedly decreased. Isochrysis galbana and Nannochloropsis sp.in response to CO2 aeration. The results showed that maximum biomass concentrations of Isochrysis galbana and Nannochloropsis sp. were around 0.75 g L−1 under 10% CO2, and reduced to around 0.55 g L−1 under 15% CO2. 2 concentrations to the growth of marine cyanobacterium Phormidium valderianum. The maximum biomass productivity was 83.33 mg L−1 d−1 was recorded under 3% CO2, and decreased to 51 mg L d−1 under 15% CO2, which meant higher CO2 concentration inhibited microalgal growth. The above results indicated high CO2 concentrations showed significant inhibition to the microalgal growth under high salt conditions. Therefore, it is urgent to develop microalgal strain with high-CO2 tolerance and high-salt tolerance.Compared with the chemical absorption and geologic sequestration of COemission . The CO2 than 5% . Only a ven 100% . Howevernd risks . To avoind risks . The usend risks . Therefo oculate , Thalassssflogii , Chaetocmuelleri , P. glacgicornis  and Phorderianum . Chiu et2 concentrations on the growth of one marine microalgal strain D. salina under high-salt conditions. Transcriptomic analysis was used to investigate the factors affecting microalgal tolerance for high CO2 concentration at a genetic level. These findings extend the knowledge which the key metabolic and biological pathways function in response to extremely high CO2 in marine microalgal strain.In this study, we investigated the effect of COD. salina (GY-H13) was purchased from Shanghai Guangyu Biological Technology Co., LTD, and maintained in petri dishes using BG11 solid medium containing 3% NaCl. D. salina cells were successively transferred from petri dishes to 250 ml flasks, and then cultivated in 400 ml bubble column photobioreactors  with 1% CO2 under 110 μmol m−2 s−1 and 25°C conditions. The cells of D. salina were harvested during their logarithmic growth phase by centrifugation, and then the harvested cells were resuspended into BG11 medium containing 3% NaCl with the required biomass density and used in the following experiments.one marine microalgal strain 2 concentration on the growth of D. salina, the microalgal cells were cultured at five CO2 levels, air (0.03%CO2), 1%CO2, 10%CO2, 20%CO2, and 30%CO2. The culture was incubated in 400 ml PBR (working volume 300 ml) under 110 μmol m−2 s−1 and 25°C conditions. The concentration of CO2 (v/v) was manipulated by adjusting the flow rates of pure CO2 and air with gas mass flow controllers, respectively. The mixed gases were filtered (0.22 µm) and then transferred into the bottom of PBR through one slender glass tube (inner diameter 0.25 cm) with an aeration rate of 0.2 L min−1. The initial cell density of the cultures was maintained at 0.1 g L−1. The temperature was maintained using constant temperature water bath.In order to investigate the effect of COD. salina were cultivated for 8 days at 0.03%–30% CO2. Samples were taken daily from the PBR to estimate microalgal growth and the pH of the culture medium. Generally, 12 ml of culture medium was collected in a clean glass tube, and the pH of the sample was immediately measured using a Five Easy pH meter (METTLER TOLEDO). The maximum quantum yield of photosystem Ⅱ was determined using 2 ml of the sample. The Fv/Fm ratio was measured using a fluorescence monitoring system  after the sample has been stored in dark conditions for 30 min , 1% and 30% CO2 were collected on day 1 and day 2, and denoted as C (0.03%) D1, C (1%) D1 and C (30%) D1; C (0.03%) D2, C (1%) D2 and C (30%) D2, respectively. Each sample was determined for two replicates. The sample treatment, transcriptomic determination, gene annotation and the related bioinformatics analysis have been described in previous studies  for multiple testing, and the corrected p-values are known as p-adjust. Differential folds FC (Fold change) for different CO2 concentration treatments were calculated based on the FPKM values of gene expression using RSEM software for differential analysis. Genes meeting the screening criteria of |log2 FC|≥1 & p-adjust <0.05 were considered as differentially expressed genes (DEGs). GO and KEGG enrichment analyses were performed on genes in the gene set using Goatools. Raw data was deposited at the NCBI Sequence Read Archive (SRA) with BioProject record PRJNA901516.Transcriptomic analysis was used to investigate and test potential CO studies . NCBI noD. salina was significantly influenced by the CO2 level. The biomass of microalgal cells was 0.61 g L−1 after 8 days of incubation in air (0.03%CO2). The cell biomass of the 1% CO2 group grew rapidly, reaching 1.7 g·L−1 on day 8, which was 2.79 times higher than that of incubation in air. Further increasing CO2 concentrations resulted in the lower biomass concentrations under 10%–30% CO2. However, the cell biomass of 30% CO2 group also reached 1.13 g·L−1, which was 1.85 times higher than that of incubation in air. The effect of different CO2 concentration treatments on the growth of D. salina is further evidenced by the picture of D. salina growth. , the combination of greater acidification and diminished photosynthesis resulted in the lower pH range.The pH of the culture medium affects the permeability of the cell membrane of microalgae cells, the uptake and utilization of ions in the culture medium, the utilization of COal cells . The staound 5.9 . It can ntration . At high2 concentration conditions can be detected by chlorophyll content . As a defense mechanism, organisms produce many endogenous antioxidants to eliminate harmful ROS, thus maintaining normal cellular function and organismal health . Compared with C (0.03%)D1, there were 807 differential genes in C (1%)D1 group, including 354 up-regulated genes and 453 down-regulated genes. Compared with C (0.03%)D1, C (30%)D1 group has 13408 differential genes, including 6342 up-regulated genes and 7066 down-regulated genes D1 and C(30%)D1 were subjected to GO analysis using the software Goatools (P-adjust<0.05). A total of 6039 DEGs were annotated in GO and divided into 225 functional subcategories, containing 137 groups of biological processes, 28 groups of cellular components and 60 groups of molecular functions. Within the category of biological processes, transmembrane transport, regulation of primary metabolic process, cell surface receptor signaling pathway, organic cyclic compound biosynthetic process, and cGMP biosynthetic process were prominent /D2 (PsbD) heterodimer, which can bind the central chlorophyll molecule and the primary electron acceptor and primary electron donor in the electron transfer process after photochemical reaction. The core antennas CP47 and CP43 of the optical system II (PSII) are combined at the side of the D1 (PsbA)/D2 (PsbD) core. CP47 and CP43 can not only function as core antennas, but also play an important role in maintaining the core structure of the PSII , A0, A1 and FX (PsbA), cp47 (PsbB), cp43 (PsbC), D2 (PsbD), PsaA, and PsaB were up-regulated in C(30%) D1 compared with C(0.03%) D1(PetB), cytochrome b6-f complex subunit 4 (PetD), apocytochrome f (PetA) and cytochrome b6-f complex iron sulfur subunit (PetC) in the cytochrome b6f complex were significantly up-regulated in C (30%) D1 compared with C (0.03%) D1. Compared with C (0.03%) D1, the gene expression level of F type ATPase coding subunit were up-regulated in C(30%)D1  to form NADPH. The transmembrane proton gradient generated in this process drives ATP synthase to form ATP . The corthe PSII . PsaA an1 and FX . The exp0.03%) D1. Cytb6f 0.03%) D1. The expC(30%)D1 . It show2 to produce organic sugars  is a key enzyme in the process of carbon fixation in the Calvin cycle, which catalyzes COc sugars . As showficiency .D. salina increased and the expression levels of fumarase and malate dehydrogenase decreased under the condition of short time exposure to high CO2, which may be due to the intermediate products in TCA cycle entering the amino acid metabolism and other pathways. Pentose phosphate pathway provides NADPH for biosynthesis, and its reversible non oxidized part is also an important source of carbon skeleton for the synthesis of nucleotides, aromatic amino acids, phenylpropanes and their derivatives. The key enzymes of pentose phosphate pathway are 6-phosphate glucose dehydrogenase and 6-phosphate gluconate dehydrogenase, which are involved in the pentose phosphate pathway to produce NADPH and ribonuclease 5-phosphate (Ru-5-P). Compared with C (0.03%) D1, we found that the gene expression levels encoding phosphogluconate dehydrogenase (1.1.1.49), phosphogluconate lactone (3.1.1.31) and phosphogluconate dehydrogenase (1.1.1.44) in C (30%) D1 were significantly up-regulated  through disproportionation reaction , Hsp family D gene (HspD1) and small Hsp genes   D1 . Compare (30%) D1. The higD. salina was cultured under 0.03%-30 % CO2 and 3% NaCl conditions. Results showed that D. salina produced a maximum biomass of 1.7 g·L−1 at 1 % CO2 and a biomass of 1.13 g·L−1 at 30% CO2, which was 2.79 and 1.85 times higher than that of incubation in air (0.03%). Under high CO2 stress, D. salina can eliminate ROS by synthesizing endogenous antioxidant carotenoids. In addition, the results of transcriptomic analysis also indicated that some key genes related to enzymatic antioxidant system and non-enzymatic antioxidant system antioxidant enzymes were up-regulated. Meanwhile, D. salina responded to high CO2 stress by promoting central carbon metabolism to produce more energy and enhancing photosynthesis to promote carbon fixation.In this study, one marine algal strain


Sclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata  of the fifteen isolate’s crude extracts showed anti-HIV-1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170 μg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2 ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile ; Pyrrololpyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4-dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2-Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds.Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungi were isolated from  The human immunodeficiency virus (HIV) is a public health issue worldwide, particularly in developing countries . In 2021S. birrea is well known for its antibacterial, antihelmintic, antifungal, and anticonvulsant effects. Hypoxis species has been documented to treat urinary infections and cardiac diseases. It increases immune function, prostate hypertrophy, burns, wounds, and ulcers .The biologically active secondary metabolites produced by fungi can be exploited for various activities such as anti-inflammatory, antimicrobial, anticancer, and antiviral, including HIV-1 . Since tompounds . Notablylication . Other c66 cells . Severaly others . These f. birrea (voucher no. 18234) and Hypoxis species (voucher no. 18233) were collected from the South Coast region of the Durban Municipality, KwaZulu-Natal. The voucher specimens were deposited at the UKZN School of Life Science Herbarium for taxonomic identification.SHypoxis species, eleven from S. birrea  were isolated following a protocol described by A total of fifteen endophytic fungi, four from the roots of ® PCR Purification kit, Invitrogen . After purification, the amplicons for each fungal isolate were quantified using a NanoDrop 2000c  and sent to Central Analytical Facility (CAF) at Stellenbosch University  for ITS sequencing. The quality assessment of the ITS sequences from CAF, Stellenbosch University, was performed using Snap Gene viewer (Version 6.1.2). The Basic Local Alignment Tool (BLAST) application of the nucleotide database of the National Centre for Biotechnology Information (NCBI) search program was used to generate consensus sequences from the forward and reverse ITS sequences. The consensus sequences were compared to other ITS sequences with GenBank databases through blast based on sequence similarity. The isolates PO2PL1, PO4PR1 and PO4PR2 were identified as A. Alternaria and deposited on the NCBI database under accession numbers OP648259, OP648254, and OP648252, respectively.Three of the 15 endophytic fungal isolates  were selected for molecular identification based on their anti-HIV activity profiles during pre-screening (data not shown). Genomic DNA was isolated using the Norgen Plant/Fungi DNA isolation kit  per the manufacturer’s instructions. The extracted fungal DNA was used for the amplification of the internal transcribed spacer (ITS) sequence region using primers: forward primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The polymerase chain reaction (PCR) was conducted using Phusion polymerase . The PCR products were then purified using the PureLink2) from 5-day-old malt extract agar  were cut. Each was inoculated into a 10 ml Potato Dextrose Broth  or Malt extract broth  in McCartney bottles and incubated in the dark at 25°C for 14 days. Each fungal crude extract was cultured in triplicates with fungal-free media as controls. After 14 days of incubation, an equal volume of absolute methanol  was added to each culture and placed on a rotatory shaker  to shake overnight at 150 rpm at 25°C. The extracts were separated from the mycelia using gauze as a filter on pre-weighed culture flasks. The mycelia were discarded, and the supernatants were dried at 40°C. The dried methanol crude extracts were stored at −80°C until further use. Before each use, the extracts were resuspended in distilled water to a concentration of 400 μg/ml.Three small fungal plugs . Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 50 μg/ml Gentamycin , and 25 mM Hepes buffer .env deleted pNL4.3luc (donated by Dr. Katlego Sojane) and 12 μl of both backbone recombinant plasmids (pCMV and pVSV-g) using X-tremeGENE™ HP DNA Transfection Reagent  according to the manufacturer’s protocol. The mixture of pNL4.3Δenv and pCMV, pVSV-g was incubated for 30 min at room temperature to promote DNA transfection complex formation. The DNA transfection complex was added dropwise into a 6-well plate and incubated at 5% CO2, 37°C, for 48 h. After that, supernatants were harvested using a syringe and 0.22 μm filter and were filtered into new tubes after 48 h for storage at −80°C freezer. The pseudoviruses’ 50% tissue culture infective doses (TCID50) were determined by serially diluting the virus and infecting the TZM-bl cell lines. The concentration that elicits a 50,000 relative luminescence unit was determined using the luciferase assay .Pseudovisuses were prepared by transfecting HEK 293T cells (10 million cells) in 10 ml growth medium  in a T75 flask with 12 μl of The cytotoxicity effect of the endophytic fungal crude extracts was assessed on TZM-bl cell lines using the MTT (3-[4.5-dimethylthiazol-2-yl]-25 diphenyl tetrazolium bromide) cell proliferation assay kit according to the manufacturer’s instruction  .2. Ten microliters of each crude extract were serially diluted 10-fold from 300 μg/ml in DMEM containing 10% heat-inactivated fetal bovine serum, 50 μg/ml Gentamycin, and 25 mM Hepes buffer in a 96-well plate. Azidothymidine (AZT) at 300 μg/ml was used as the positive control and uninfected cells as the negative control. The plates were covered with foil and incubated for 48 h at 37°C, 5%, CO2. After incubation, 10 µl MTT reagent (5 mg/ml in PBS) was added to each well and incubated for 4 h, 37°C, 5% CO2. Afterwards, the media was replaced with a fresh DMEM medium. Then, the formazan crystals were dissolved in 50 µl 0.2% DMSO and incubated for 10 min. The absorbance was measured using a Victor Nivo, multimode plate reader at 540 nm (PerkinElmer Inc. United States) (50) for each extract was calculated based on the non-fit regression curve on the GraphPad Prism Software (v.5.00.288). The percentage cell viability was calculated using the formula:Briefly, TZM-bl cells were seeded  in a 96-well plate and incubated at 37°C, 5% CO States) . The res2 in DMEM medium   was seeded  in a 96-well plate and incubated at 37°C, 5% CO2 for 72 h. Positive control was set up using a known reverse transcriptase inhibitor, azidothymidine (AZT), at a 300 μg/ml starting concentration. Negative control was set up using uninfected cells. Cells infected with the virus without treatment were also included in the experiment (virus control). After incubation, the DMEM medium was replaced, and 100 µl BrightGlo luciferase reagent  was added to each well under low light conditions and incubated at room temperature for 2 min to allow complete cell lysis. All the contents were transferred to a corresponding 96-well bottom flat black plate . Luminescence was read immediately in a Victor Nivo multimode microplate reader at 540 nm . The level of viral replication was expressed as a percentage of the HIV-1 inhibition following this equation:The luciferase-based antiviral assay was performed according to  buffer) . In this50) was calculated using GraphPad Prism Software (v.5.00.288).The half-maximal inhibitory concentration  were isolated from the blood of HIV-1 uninfected donors at the HIV Pathogenesis Programme (HPP) sample repository. Peripheral blood mononuclear cells were isolated from 10 ml of blood using the Ficoll-Histopaque gradient method. The cells were cultured in R-10  supplemented with 10% heat-inactivated fetal bovine serum (FBS) plus 100 μg/ml Penicillin/Streptomycin  and 1 M HEPES buffer.+ T cells were isolated from PBMCs using EasySep™ Human CD4+ T Cell Isolation Kit according to manufacturer instructions . A total of 10 × 106 cells/ml [in interleukin-2 (IL-2) growth medium] were stimulated with 10 μg/ml phytohaemagglutinin (PHA)  in a cell culture T25 flask at 37°C, 5% CO2 for 3 days. After the incubation period, the cells were centrifuged at 1,000 rpm for 10 min, and the cell pellet was washed twice with PBS. The final stimulated cell pellet was resuspended in an RPMI-1640 supplemented with an IL-2 growth medium.Human CD4+ T cells and PBMCs were prepared in RPMI-1640 (Sigma) medium growth supplemented with penicillin/streptomycin plus 10% fetal calf serum (2 for 48 h. Azidothymidine (AZT) at 300 μg/ml was used as a positive control, and uninfected cells were used as negative controls. After incubation, the plate was centrifuged at 2,400 rpm for 10 min, and 1,000 μl of culture supernatant was collected every 3 days up to day 12. Old media was replaced with fresh RPMI-1640 media (Sigma) supplemented with penicillin/streptomycin plus 10% fetal calf serum and used for determining the p24 antigen levels. The HIV-1 viral load in the culture supernatant was measured using the Quicktiter Lentiviral Quantification kit  following the manufacturer’s instructions. The multimode microplate reader measured the p24 level at 480 nm (PerkinElmer Inc. United States). The viral titer was calculated as per the manufacturer’s instructions as follows:The CD4lf serum . They weThe average genome size of lentivirus is 8 kbp, therefore.−9)g/ X 6 × 1,023 = 1.1 × 108 VP1 ng lentiviral RNA = (1 × 10Virus Titer (VP/ml) = Amount of lentiviral RNA (ng) X 1.1 × 108 VP X (20 μl/5 μl)Viral sample volume (ml)Virus Titer (VP/ml) = Amount of lentiviral RNA (ng) X 4.4 × 108 VP/ng.Viral sample volume (ml).The p24 core protein concentration was calculated using GraphPad Prism (v.5.00.288).Solid phase extraction (SPE) was performed following a method described by Analysis of fractions was carried out using a Hewlett Packard 6890 (United States) GC-MS fitted with a Zebron ZB-5MSplus column  following a modified protocol based on 50 and IC50 values.The results of all the experiments were the average of three independent experiments. Analyses of concentration-response data were performed with a non-linear curve-fitting program in GraphPad Prism to determine CCA. alternata  sequencing with 350–400 bp. The fungal ITS sequences  were trimmed and analyzed using the SnapGene viewer, version 6.1.2 . The forward and reverse sequences were aligned to produce consensus sequences and submitted to National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLASTN) for identifying similar sequences . The reslternata . Phylogelternata . Per they 99.81% . PO4PR2 y 99.80% , and PO2t 99.81% .A. alternata PO2MS2B strain had a cell viability of less than 50%, followed by A. alternata (PO4PR2), which was above 50%. According to the United States National Cancer Institute Plant Screening Program, a crude extract is generally considered to have in vitro cytotoxic activity when the CC50 value is < 30–40 μg/ml  out of 15 screened endophytic fungal crude extracts showed antiviral activity against HIV  ranging from 0.017 to 1.170 μg/ml. Alternaria tenuissima. Peganum harmala seeds with 50% inhibitory concentration at 1.5 µM. Alternaria sp. displayed high inhibition on three viral enzymes, HIV-1 Reverse Transcriptase, which was inhibited by 82.81% , integrase enzyme activity which was inhibited by 98% and protease activity was inhibited by 78%. Most studies have tested A. alternata against bacterial strains for activity. Hence there are not many published reports on anti-HIV activity. A. alternata had antimicrobial effects against all tested bacteria with minimum inhibitory concentrations between 2.5 and 5 μg/ml, slightly higher than our observed activity. From CC50 and IC50, the selective index of the three isolates, PO4PR1, PO4PR2, and PO2PL1, were determined to be 38.82, 2558.82, and 59.71, respectively. Among all three endophytic extracts, PO4PR2 showed the most significant virus inhibition as it had the highest selective index. These results demonstrated that crude extracts of A. altenata have a broad spectrum of biological activities and provide an opportunity for further anti-HIV drug development from natural products.In our study, crude extracts of A. alternata in all three isolates showed effective antiviral activity compared to the standard drug AZT in HIV p24 antigen inhibition assay. A. alternata PO4PR1 showed the lowest p24 concentration at 22.1 ng/ml, revealing that there is strong anti-HIV activity. On the other hand, A. alternata PO4PR2 showed a higher p24 concentration at 50.2 ng/ml in PBMCs, meaning that the HIV-inhibition activity is low. The PO2PL1 extract showed inhibition at 24.6 ng/ml after 3 days of infection. Methanol extracts of all tested fungi, A. alternata showed a weak HIV-1 inhibition of less than 50 ng/ml in CD4+ T cells after infection. The inhibition rate was less than 50 ng/ml after infection. All the methanol extracts revealed a gradual decrease of p24 antigen expression in each, indicating potential denaturation of virus  or HIV-1 inhibitory activity. Perhaps this decrease might also be caused by the instability in the cell culture medium, which was replaced after every collection of the supernatant. Similar results were reported by Padina tetrastromatica inhibited HIV-1 by more than 50% and showed a significant reduction in HIV-1 p24 levels after 7 days of incubation. The methanol extract of Alternaria species inhibited the activity of HIV-1 Reverse Transcriptase (82.81 ± 1.0)  . The lowactivity .A. alternata crude extracts identified different compounds which might be responsible for the anti-HIV activities. The major bioactive compounds with a similarity index of more than 80% are listed in Bacillus subtilis and Clostridium tetani  and elucidate a mode of action to establish a precise HIV-1 inhibition mechanism of the bioactive compound(s) from A. alternata crude extracts in this study.In the current study, our results showed that


This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4+ levels. Relative to 1 mM NH4+, a high (50 mM and 75 mM) NH4+ load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH4+ load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K+ “salt-in” strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent mK value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2− and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4+ exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH4+.A high NHIMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4+ concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4+ load. An understanding of the physiological and molecular response mechanisms of Methylocystis spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to an increasing NH4+ load. The pMMO is an integral part of an extensive intracytoplasmic membrane system (ICM), which is a particular characteristic of proteobacterial methanotrophs  and type II (Alphaproteobacteria) methanotrophs. Besides phylogeny, the carbon fixation pathway, however, remained the only major feature of the above-mentioned criteria that validly differentiates between type I and type II methanotrophs. As suggested by Knief (Gamma- and Alphaproteobacteria. The methanotrophic Alphaproteobacteria were further divided into type IIa (Methylocystaceae) and type IIb (Beijerinckiaceae) methanotrophs , although neither is able to grow on the alternative substrate (–3 to hydroxylamine (NH2OH)  . Ammonia of pMMO , 22. Whe habitat –25. Addi10.1128/msystems.00403-22.1TEXT S1Text S1, DOCX file, 0.03 MB.Environmental effects of ammonium on the methanotrophic activity. Download Copyright © 2022 Guo et al.2022Guo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3 oxidation by pMMO on methanotrophic activity occur through toxic nitrogen products such as NH2OH and nitrite (NO2−). Although the affinity of pMMO for NH3 is generally lower than that for CH4  to determine the NH4+ threshold level to which strain SC2 is able to acclimatize and (ii) to assess the cellular adjustment processes triggered by this threshold level. We expected to observe a dual response of strain SC2, with the first one being a general response to increasing ionic-osmotic stress and the second one being a methanotroph-specific response to hydroxylamine stress. Recently, we developed a new analytical proteomics workflow for strain SC2 which captures 62% of the predicted SC2 proteome under standard growth conditions (4+ load (1 to 100 mM) with global proteomics, analysis of intracellular amino acids (metabolomics), and measurement of nitrogen oxides compounds . Finally, we quantified NO2− and nitrous oxide (N2O) production by strain SC2 in relation to both an increasing NH4+ load and incubation time.The following subsections describe the effects of an increasing NH4-air mixing ratio of 20:80 (vol/vol) (600]) and the headspace concentrations of both CH4 and CO2 were regularly measured during the complete incubation period of up 336 h (14 days), ranging from early lag phase to late stationary phase  and 125 h (75 mM NH4+). This was linked to a significant decrease in the growth and CH4 consumption rates (4+), all SC2 cultures grew to the same final OD600 of about 0.45  . Cell dery phase . The addrain SC2 . Supplemon rates . Howeverout 0.45 . Accordiresponse . There w mM NH4+ .10.1128/msystems.00403-22.4TABLE S24, aqueous NH3, and CH4/NH3 ratio across the NH4Cl treatments. Download Table S2, PDF file, 0.08 MB.Growth medium: ionic strength, pH, dissolved CHCopyright © 2022 Guo et al.2022Guo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4-air mixing ratios adjusted to 10:90 and 20:80 (vol/vol) showed no significant difference in SC2 growth response when supplemented with 10 mM, 30 mM, and 50 mM NH4+. No increase in cell density (OD600) was observed in SC2 cultures supplemented with 50 mM NH4+ after the CH4-air mixing ratio was adjusted to below 10:90 (vol/vol) .The CH4-air mixing ratios and increasing NH4+ concentrations revealed that relative to the control (20:80 [vol/vol]), ratio values of 5:95 and 2.5:97.5 (vol/vol) reduced the CDW production, growth rate, CH4 consumption, and CH4 consumption rate. This was significant (P < 0.001) across all physiological growth parameters for SC2 cultures supplemented with 50 mM NH4+ ] for CH4 oxidation, being 0.17 μM, 1.20 μM, and 1.40 μM under growth conditions with 10 mM, 30 mM, and 50 mM NH4+, respectively (4 to NH3 dissolved in the liquid growth medium (mK(app) value for 75 mM NH4+ could not be calculated, because growth of strain SC2 was completely inhibited when incubated under a headspace of 2.5% and 5% CH4.The SC2 growth parameters determined for the different CHectively . This coh medium . The Km. Download Table S1, PDF file, 0.1 MB.Physiological parameter of Copyright © 2022 Guo et al.2022Guo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4+ treatments (https://www.uniprot.org/taxonomy/187303). Neither the PmoCAB1 subunits of low-affinity pMMO1 nor the PmoB2 subunit of the high-affinity pMMO2 showed a differential regulation. The PmoC2 and PmoA2 subunits of pMMO2 were not detectable at any of the NH4+ treatment concentrations (4+ conditions (https://doi.org/10.6084/m9.figshare.20750236.v3). The heat map of sample-to-sample distances showed high similarities between the DRP profiles of the 1 mM and 10 mM NH4+ treatments, but in particular between those of the 50 mM and 75 mM NH4+ treatments. More specifically, the DRPs grouped into three distinct clusters comprising a total of 141, 65, and 232 proteins, respectively. The 232 proteins of DRP cluster III were significantly upregulated only under 50 mM and 75 mM NH4+ conditions  .Global proteomics led to the detection of 2,206 proteins, of which 438 proteins were identified to be differentially regulated proteins (DRPs) in at least one of the NHeatments . The 438trations . Hierarc10.1128/msystems.00403-22.5DATA SET S14+ treatment. Download Data Set S1, XLSX file, 0.1 MB.Complete list of all 438 proteins that were differentially regulated in at least one NHCopyright © 2022 Guo et al.2022Guo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4+ concentration. A total of 18 DRPs (10 mM NH4+), 57 DRPs (30 mM NH4+), and 95 DRPs (50 mM NH4+) were co-upregulated under the 75 mM NH4+ condition (4+), 24 DRPs (30 mM NH4+), and 39 DRPs (50 mM NH4+) being co-downregulated under the 75 mM NH4+ condition (4+ condition. This corresponds to more than 30% (140/438) of the total identified DRPs  .A certain number of DRPs were coregulated regardless of the initial NHondition . A similied DRPs . The numhttps://doi.org/10.6084/m9.figshare.20750236.v3 and Data Set S3 at https://doi.org/10.6084/m9.figshare.20556633.v1). A protein-protein interaction (PPI) network analysis revealed 121 proteins to be highly interactive  . Nitrogen metabolism included proteins involved in NH4+ transport and assimilation and in hydroxylamine detoxification . In particular, the intracellular concentration of glutamate significantly increased to 2,438.69 μmol/g CDW under the 50 mM NH4+ condition but slightly decreased to 2,020.59 μmol/g CDW under the 75 mM NH4+ condition (https://doi.org/10.6084/m9.figshare.20750203.v2). Glutamine also showed the greatest intracellular accumulation at 50 mM NH4+, with 235.69 μmol/g CDW. Unlike with glutamate and glutamine, the intracellular concentration of proline significantly decreased from 1 mM to 50 mM NH4+ but showed a sharp and highly significant increase to 84.12 μmol/g CDW at 75 mM NH4+  (https://doi.org/10.6084/m9.figshare.20750203.v2). Concurrently, the intracellular concentration of ornithine was significantly increased and was greatest (167.20 μmol/g CDW) at 75 mM NH4+ (P ≤ 0.001) (https://doi.org/10.6084/m9.figshare.20750203.v2). Arginine and lysine were also most enriched at 75 mM NH4+, with 1,189.02 and 2,004.68 μmol/g CDW, respectively.Of the 16 amino acids detected, 15 amino acids showed a significant change in their intracellular concentrations across the five NH2− and N2O were detectable across all five NH4+ conditions (https://doi.org/10.6084/m9.figshare.20750236.v3). The production of NO2− significantly increased at 1 mM NH4+ to 50 mM NH4+ but did not further increase at 75 mM NH4+. The maximum concentrations of NO2− that accumulated in the growth medium during the incubation experiments were 4.44 μmol/L (1 mM NH4+), 16.99 μmol/L (10 mM NH4+), 23.19 μmol/L (30 mM NH4+), 59.95 μmol/L (50 mM NH4+), and 54.32 μmol/L (75 mM NH4+) (https://doi.org/10.6084/m9.figshare.20750236.v3 and Table S3 at https://doi.org/10.6084/m9.figshare.20559417.v1). The production of N2O significantly increased at 1 mM NH4+ to 75 mM NH4+. The maximum headspace concentrations of N2O that accumulated during the incubation experiments were 0.65 μmol/L (10 mM NH4+), 1.85 μmol/L (30 mM NH4+), 4.93 μmol/L (50 mM NH4+), and 5.84 μmol/L (75 mM NH4+) (https://doi.org/10.6084/m9.figshare.20750236.v3 and Table S4 at https://doi.org/10.6084/m9.figshare.20589000.v1).NO4+ levels, the NO2− production rate was highly correlated with the greatest SC2 growth activity. Compared to NO2− production, the accumulation of N2O was time shifted. Strong N2O accumulation occurred only after NO2− had nearly reached its peak concentration (https://doi.org/10.6084/m9.figshare.20750236.v3).At high (50 mM and 75 mM) NHMethylocystis sp. strain SC2 to increasing NH4+ concentrations. The ionic medium strength to which strain SC2 is able to acclimatize differs between NH4Cl and NaCl as the stressor. It is lower for NH4Cl (between 114 and 139 mM [present study]) than for NaCl . This mnditions , 33. Thecriptome , 34 or tcriptome  level. I+ “salt-in” strategy, the uptake and/or synthesis of compatible solutes, and the induction of the glutathione metabolism pathway. The stress-responsive proteins upregulated in response to a high NH4+ load were the DNA-binding protein (Dps), the general stress response protein (CsbD), and heat shock proteins. Dps has a significant role in protecting the chromosome from oxidative damage but also from UV radiation, iron toxicity, heat, and pH stress  NH4+ load  follows a widely distributed response pattern that is also known to occur in Escherichia coli and Bacillus subtilis  under a high NH4+ load. These enzymes have been shown to be expressed when cells are exposed to oxidative stress and hyperosmotic shock conditions. In particular, their activity is involved in detoxifying reactive oxygen (ROS) and nitrogen (RNS) species such as, for example, metal-bound NO·. The latter results in the formation of nitrosothiols (RSNO) and nitrosamines (RN2O), which are regarded as nonradical RNS (J7QHL1 and J7Q532) (4+ load. Their increase in expression level may have been induced by the increased production of both hydroxylamine (The stress-triggered induction of the glutathione metabolism pathway involved a significant upregulation of both glutathione peroxidase (GPX) and glutathione ical RNS . Notably J7Q532)  were spexylamine  and RNS xylamine , 51.https://doi.org/10.6084/m9.figshare.20750215.v2). On pBSC2-1, single-stranded DNA-binding protein (SSB), the three-component CzcCBA complex, and subunits of the FoF1 ATPase complex were among the proteins significantly upregulated under a high NH4+ load. SSB was the most greatly enriched (3.95-fold) plasmid-encoded protein during exposure of strain SC2 to 75 mM NH4+ (see Data Set S6 at https://doi.org/10.6084/m9.figshare.20750215.v2). It plays a major role in DNA replication, recombination, and repair. On pBSC2-2, multicopper oxidases and the type IV secretion system (T4SS) were among the proteins most significantly upregulated under a high NH4+ load, with the latter having functions in conjugation, DNA exchange with the extracellular space, and delivery of proteins to target cells  among all differentially regulated proteins in response to a high NH4+ load   value could not be experimentally determined for the SC2 exposure to 75 mM NH4+ due to methodological constraints  were detectable in the SC2 proteome, with seven Mxa-MDH subunits (MxaFJCKLDH) being the only CH4 oxidation pathway proteins significantly enriched at a high (75 mM NH4+) load. The Mxa-MDH-associated cytochrome cL (MxaG) was highly expressed constitutively. It is reasonable to assume that the significant enrichment of these seven Mxa-MDH subunits (including MxaF) is a proteomic response to compensate for the inhibitory effect of hydroxylamine. In contrast, the expression response of Xox-MDH varied, with XoxF being significantly downregulated at NH4+ loads of 30 and 50 mM  , 53. Thi4+ load . This fin of MDH . All subnd 50 mM .4+ load and the accumulation of NO2− and, with a delay, N2O  and hybrid cluster proteins (Hcps). NorB is encoded on pBSC2-2 but was not detectable in the SC2 proteome. Previous transcriptome research had shown, however, that relative to 10 mM NH4+, the transcript expression of the plasmid-borne norB significantly increased after a 10-h exposure of SC2 cells to 30 mM NH4+ (J7Q787) is one of the most highly expressed proteins in strain SC2 and is significantly upregulated in response to increasing NH4+ levels , which has been established as physiologically relevant , thereby suggesting that N2O may be further reduced to N2. The nos operon is located on a 20-kb region of pBSC2-2, which also contains the genes encoding NorB and two Hcp proteins . This proximity of nitrogen-cycling genes , but also involving those encoding Hcps, further substantiates the functional relevance of pBSC2-2 for strain SC2. One of the two Hcps (UniProt ID I4EBE8) was also significantly enriched in response to an increasing NH4+ load, but its overall expression level was 1,000-fold lower than that of the chromosome-encoded Hcp protein  is constitutively expressed at a high level  was in the same range as previously shown for Methylosinus sporium (71 mM NH4+)  . The neetrations .Methylocystis sp. strain SC2 was found to comprise a 3.77-Mb chromosome and two large plasmids (HE956757 (chromosome) and FO000001 and FO000002 (plasmids). Genomic analysis revealed the presence of a complete denitrification pathway in strain SC2  and a single plasmid-borne pmoC gene (PspmoC)  .600 of 0.25 ± 0.05  medium on its cell density, CH4 consumption, and CO2 production. A 1-mL aliquot of NMS-precultured SC2 cells was inoculated into 120-mL serum bottles containing 40 mL AMS medium. The initial OD600 was 0.01 ± 0.003. The composition of AMS was the same as that of NMS, with the exception that 1 g of KNO3 (10 mM) was replaced by increasing amounts of NH4Cl. This resulted in treatment concentrations of 1, 10, 30, 50, 75, and 100 mM NH4Cl in the medium (4Cl) to 139 mM (100 mM NH4Cl) (see 4 and air at a 20:80 (vol/vol) ratio. The serum bottles were sealed with rubber stoppers and incubated on a rotary shaker at 130 rpm and 25°C. Both OD600 and changes in the headspace concentrations of CH4 and CO2 were regularly monitored during the whole incubation period  medium in 120-mL serum bottles and grown to an OD5 ± 0.05 . The com5 ± 0.05 , containe medium , correspn period .600 was determined using an Eppendorf BioPhotometer UV/Vis spectrophotometer . Cell dry weight (CDW) was calculated based on the following relationship: biomass (g CDW) = OD600 × 0.261 × volume . The methane consumption rate is indicated as millimoles of CH4 consumed/g of CDW/day. All rate calculations are based on parameter values measured during exponential growth. The production of N2O was monitored using an N2O microsensor with a piercing needle. The microsensor was connected to a microsensor multimeter . The O2 concentration in the headspace was monitored with a Fibox 4 trace meter using SP-PSt3 sensor spots. This yielded an oxygen detection limit as low as 0.002% (by volume) . The production of NO2− was determined using the Griess reagent system by following the manufacturer`s instruction .The OD× volume . Biomass4+ on CH4 oxidation, SC2 cells were grown at the following CH4-air mixing ratios: 20:80, 15:85, 10:90, 5:95, and 2.5:97.5 (vol/vol). Each CH4-air mixing ratio was tested in triplicate incubations under three different ammonium concentrations . Cell density (OD600) and CH4 concentration in the headspace were regularly measured over the whole incubation period. Cell densities were converted into cell numbers as described previously  and the maximum apparent rate of metabolism [Vmax(app)] of SC2 cultures using nonlinear regression with the Michaelis-Menten equation. Multiplication by the Oswald constant (0.03395 at 25°C) gave the mK(app) as the methane concentration in water (To test for the inhibitory effect of NHensity OD00 and CHensity OD00 and CHin water .600 of 0.01 ± 0.003) supplemented with 1, 10, 30, 50, or 75 mM NH4+ (600 = 0.25 ± 0.02) and then collected by centrifugation at 7,000 × g and 4°C for 20 min. The cells were thoroughly washed twice with 1× phosphate buffer (5.4 g Na2HPO4 · 7 H2O and 2.6 g KH2PO4 per L of distilled H2O) to remove medium traces. The washed cell pellets were transferred to 2 mL sterile Safe-Lock microcentrifuge tubes (Eppendorf) and stored at –80°C for subsequent protein extraction. Each NH4+ concentration involved the analysis of triplicate cultures.Samples for proteomics were collected from the same cultures. Strain SC2 was inoculated into 300 mL mineral salts medium . The peptides were first loaded onto a C18 precolumn (preconcentration setup) and then eluted in backflush mode using a gradient from 96% solvent A (0.15% formic acid) and 4% solvent B  to 30% solvent B over 115 min. The flow rate was set to 300 nL/min. The data acquisition mode for the initial label-free quantification (LFQ) study was set to obtain one high-resolution MS scan at a resolution of 60,000 (m/z 200) with a scanning range from 375 to 1,500 m/z, followed by MS/MS scans of the 10 most intense ions. To increase the efficiency of MS/MS acquisition, the charged-state screening modus was activated to exclude unassigned and singly charged ions. The dynamic exclusion duration was set to 30 s. The ion accumulation time was set to 50 ms (both MS and MS/MS). The automatic gain control (AGC) was set to 3 × 106 for MS survey scans and 1 × 105 for MS/MS scans  or less than or equal to −1 (downregulated) and a q value of  ≤0.01 were submitted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for enrichment function analysis.Discovery-LFQ was done using Progenesis QI software  as described before  were pipetted into the quenching solution, and the quenched cells were immediately pelleted in an Eppendorf 5430R centrifuge for 10 min at 10,000 × g and −10°C, using a fixed-angle rotor. After centrifugation, the supernatant was removed and the cell pellets were stored at −80°C until further extraction of the endometabolome.SC2 cells were grown to mid-exponential phase in 120-mL serum bottles containing 40 mL mineral salts medium supplemented with 1 mM, 10 mM, 30 mM, 50 mM, and 75 mM NH4+ . Aliquot600 of 1. The extraction fluid consisted of 50% (vol/vol) methanol at LC-MS grade and 50% (vol/vol) TE buffer . The resulting cell suspension was incubated in a ThermoMixer C shaker (Eppendorf) at 4°C for 2 h , followed by a two-phase separation of the suspension in an Eppendorf 5430R centrifuge for 10 min at 12,000 × g and −10°C, using a fixed-angle rotor. The upper phase was filtered through a 0.20-μm polytetrafluoroethylene (PTFE) membrane filter (Phenomenex) into 2-mL sterile Safe-Lock microcentrifuge tubes (Eppendorf). The metabolite extracts were stored at −80°C until downstream analysis.The endometabolome was extracted by suspending the frozen cell pellets in equal volumes of extraction fluid (−20°C) and chloroform (−20°C). The extraction volume was adapted to sample biomass, using 1 mL of extraction fluid and an equal volume of chloroform per 1 mL of sample at an ODQuantitative determination of amino acids was performed using LC-MS/MS. The chromatographic separation was performed on an Agilent Infinity II 1260 high-performance liquid chromatography (HPLC) system using a SeQuant ZIC-HILIC column  connected to a ZIC-HILIC guard column  (Merck KGaA), with a constant flow rate of 0.3 mL/min with mobile phase A being 0.1% formic acid in 99:1 water-acetonitrile  and phase B being 0.1% formic acid in 99:1 water-acetonitrile  at 25°C.2, and drying gas of 250°C at 11 L/min. Compounds were identified based on their mass transition and retention times in comparison to standards. Chromatograms were integrated using MassHunter software . Absolute concentrations were calculated based on an external calibration curve prepared in a sample matrix.The injection volume was 1 μL. The mobile phase profile consisted of the following steps and linear gradients: 0 to 8 min from 80% to 60% B; 8 to 10 min from 60% to 10% B; 10 to 12 min constant at 10% B; 12 to 12.1 min from 10% to 80% B; 12.1 to 14 min constant at 80% B. An Agilent 6470 mass spectrometer was used in positive mode with an ESI source and the following conditions: ESI spray voltage of 4,500 V, nozzle voltage of 1,500 V, sheath gas of 400°C at 12 L/min, nebulizer pressure of 30 lb/inhttps://www.bioinformatics.com.cn/. The volcano plots were created using VolcaNoseR  Sigmaplot version 14.0, (ii) OriginPro 2020, (iii) GraphPad Prism 9.0.2, (iv) commercial software TIBCO Spotfire, and (v) and Adobe Illustrator 2020.https://figshare.com/projects/Methylocystis_sp_Strain_SC2_Acclimatizes_to_Increasing_NH4_Levels_by_a_Precise_Rebalancing_of_Enzymes_and_Osmolyte_Composition_-_supplementary_files/147147. The MS proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE , tables (S3 and S4), and data sets (S2 to S6) are available at he PRIDE  partner


The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically relevant manner, an air–liquid interface culture of mono and cocultures of respiratory cells and assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference particles applied in simulated lung fluid was studied in bronchial  and alveolar (A549 +/−THP-1 and EpiAlveolar™ +/−THP-1) models. Cytotoxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and ciliary beating frequency were used as readout parameters. With the exception of increased secretion of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and alveolar models were noted, suggesting no marked differences between the models. Since the doses for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in the EpiAlveolar™ tissues has physiological relevance. Nanoparticles are used in industry, consumer products, health care and medicine, but the safety of some particles is still a matter of discussion . One par6 are thought to be due not only to differences in cells, assays, exposure conditions and sample preparation but also to different production of the MWCNTs [2 NM-403 for A549 alveolar epithelial cells in conventional testing [Reported cytotoxicity of multi-walled CNTs (MWCNTs) varied from 5 ng/mL to 10 mg/mL and based on a systematic review, A549 and HUVEC were the most appropriate cells for comparison among research groups [e MWCNTs ,8,9, whe testing .2. The limitation of Calu-3 cells is that effects of toxicants on ciliary function, the important clearance mechanism of the upper airways, cannot be assessed because the cells form only immature and not motile cilia [2 [Air–liquid interface culture (ALI), where cells grow on transwell membranes and are exposed to medium with nutrients only from the basal side and the apical parts face the air, is the physiologically relevant culture for respiratory cells. Using this technique, Calu-3 cells, established models for the bronchial epithelium, produce mucus and A549 as models for the alveolar epithelium surfactant ,13. Calule cilia . Reconstcilia [2 ,16. EpiAcilia [2 .https://www.vitrocell.com/inhalation-toxicology/exposure-systems/vitrocell-cloud-system/vitrocell-cloud-alpha-6 accessed on 2 May 2021), indicating that each well of a 12-well plate will receive ~17 µL of saline together with the particles. This solution differs in volume and composition from the lung lining fluid in the lung. The surface of the lung is covered by a dipalmitoyl-glycero-3-phosphocholine (DPPC)-rich lung lining fluid of 70–300 nm thickness [To mimic inhalation of MWCNTs, some researchers have used the VITROCELL Cloud system ,18,19. Ahickness ,23. To tSauer et al. compared the cytotoxicity of respiratory toxicants in A549 and 3T3 monolayers, MucilAir™ and EpiAirway™ tissues , but the2+ and Mg2+  to reach a concentration of 0.02% (w/v) DPPC.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine  is dissolved in ethanol and added dropwise to prewarmed (37 °C) phosphate-buffered saline with Ca2/g surface area with average length of 443 nm and average diameter of 12 nm [w/v) BSA and in the same way in SLF to find out if the same protocol works also for SLF. Briefly, 15.36 mg of CNT4003 powder was prewetted with 30 µL absolute ethanol. Subsequently, 0.97 mL of SLF or of 0.05% BSA in distilled water was added while slowly rotating the glass vessel. To prepare the stock solution, additional 5 mL of SLF or of 0.05% (w/v) BSA was added. Sonication time and amplitude can be optimized, e.g., 16 min at 10% amplitude or 12 min at 20% amplitude can be used [CNT4003 was obtained from the Joint Research Center . According to the list of representative nanomaterials status June 2016 of the JRC, CNT4003 are MWCNTs with 189 mof 12 nm . Suspensof 12 nm  in 0.05%Measurements were performed at 25 ± 0.5 °C with a particle concentration of 25.6 µg/mL in triplicates. Hydrodynamic size and polydispersity index (PDI) were measured via photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS  equipped with a 532 nm laser. The scattering angle chosen was 173°, and the refractive indices for the CNTs and the dispersion medium were 2.50 and 1.330, respectively. The zeta potential was measured by electrophoretic light scattering  considering the same optical properties of the CNTs and the dispersion media. The calculation was based on the electrophoretic mobility using Henry’s equation. The zeta potential was −9.9 ± 0.7 mV for CNT 4003 in BSA and 1.4 ± 0 mV for CNT 4003 in SLF. The hydrodynamic size of particles suspended in 0.05% BSA determined by PCS was 2327 nm with a PDI of 1.0, indicating that PCS was not suitable for accurately determining particle size. In addition, SLF contained DPPS, which formed micelles of 58 ± 7 nm and interfered with the size measurements. Therefore, the sizes of CNT 4003 were additionally measured by laser diffraction  by adding the CNT suspension to 18–20 mL SLF or 0.05% BSA until a laser obscuration of 4–6% was achieved. Volume based values considering d (0.1), d(0.5) and d (0.9) were determined in triplicate at 25 °C and CNTs showed d(0.1) 974 ± 32, d(0.5) 2254 ± 44 and d(0.9) 5399 ± 71 nm in BSA and d(0.1) 1290 ± 8, d(0.5) 3829 ± 38 and d(0.9) 6990 ± 43 nm in SLF. These data indicate that the protocol using SLF leads to a particle distribution similar to that observed with BSA.Calu-3 cells were obtained from LGC Standards GmbH  and cultured in minimum essential medium (MEM), 2 mM L-glutamine, 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. A549 cells obtained from Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH  were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 2 mM L-glutamine, 1% penicillin/streptomycin (P/S) and 10% FBS. THP-1 was purchased from Cell Line Services  and cultured in RPMI, 2 mM L-glutamine, 1% penicillin/streptomycin and 10% FBS. Cells were passaged at regular intervals.6 Calu-3 cells were seeded in 500 µL MEM, 2% L-glutamine and 1% PS + 10% FBS on 12-well polyethylene terephthalate transwell inserts  with 1500 µL of the same medium in the basolateral compartment. Medium in the apical compartment was removed after 24 h, and the medium amount in the basolateral compartment was reduced to 500 µL, which was changed every 2 or 3 days. Cells were used for the experiments when they had reached a transepithelial electrical resistance (TEER) value of >300 Ω × cm2.For the exposures with the CNTs, 0.5 × 105 in DMEM + 10% FBS on 12-well polyethylene terephthalate transwell inserts  with 1500 µL of the same medium in the basolateral compartment. Medium in the apical compartment was removed after 24 h as reported previously [A549 was seeded at a density of 0.8 × 10eviously . Accordieviously  is obtai6 THP-1 cells/mL were seeded in RPMI 1640 containing 10% FBS, 1% L-glutamine and 1% P/S in flasks. Differentiation to macrophages was induced by addition of 10 nM phorbol 12-myristate 13-acetate  to the media for 48 h. The stimulation medium was changed to medium without PMA for another 24 h before use in the cocultures. Cells were harvested by treatment with trypsin/EDTA (0.05%). For addition to EpiAlveolar™ tissues , THP-1 cells were stimulated with 8 nM PMA for 72 h, harvested by treatment with trypsin/EDTA and added immediately to the tissues.For coculture with A549 cells, 1.0 × 10EpiAirway™ 3D human respiratory epithelial tissues (AIR-100 PC6.5/PE6.5) were obtained from MatTek Corporation. The tissues were cultivated at the air–liquid interface and medium (MatTek Corporation) changed every other day according to the instructions of the producer. EpiAlveolar™ 3D tissues with and without THP-1 macrophages (MatTek Cooperation) were maintained at the air–liquid interface with medium (MatTek Corporation) changes every other day. Since EpiAlveolar™ 3D tissues with THP-1 macrophages did not tolerate the long delivery, they were added to the EpiAlveolar™ 3D tissues in the laboratory in Graz following the protocol of the company in which 25,000 THP-1 macrophages were added per insert. The cells were seeded in 75 µL media into the apical compartment during feeding and after 24 h the culture was switched back to air-liquid interface condition.® cell culture inserts (Sigma-Aldrich) in a humidified incubator . The culture medium was changed every 2–3 days.MucilAir™ tissues were purchased from Epithelix Sárl  and maintained in MucilAir™ culture medium (Epithelix Sárl) in 24-well format Transwell2 tissues and 30 µL for the models with 1.12 cm2. They were applied on the surface of the cell monolayers or reconstructed tissues. The entire observation time was 14 d. After initial addition of the CNT dose for seven days, cell and tissue surfaces were rinsed with PBS, Transepithelial Electrical Resistance (TEER) measured and new particles or SLF added for another seven days.CNT4003 in different doses were suspended in SLF. A total of 10 µL of the suspension were used for the 0.336 cm2) =  xmembrane area, given in cm2. Blank resistance is defined as the resistance of the membrane without cells. Membrane area is indicated for EpiAirway™ as 0.336 cm2 and for EpiAlveolar™ as 1.12 cm2.TEER values were determined with an EVOM STX-2-electrode . A total of 0.4 mL MEM was added to the apical and 1.2 mL MEM to the basolateral compartment for TEER measurements for EpiAirway™ and 0.5 mL in the apical and 1.5 mL in the basolateral compartment for EpiAlveolar™ tissues. TEER values were calculated as follows: TEER  was used according to the manufacturer’s instructions. To avoid interference with CNT4003 absorption, inserts were first rinsed with cell culture medium. Subsequently, 100 µL of the combined MTS/PMS solution + 500 µL medium was added in each insert. Plates were incubated for up to 2 h at 37 °C in the cell incubator. The supernatant was transferred into a new plate to remove remaining CNT4003 particles and absorbance was read at 490 nm on a plate reader .Viability was assessed at the end of the exposure time (14 d). CellTiter 96Medium from the basolateral compartments of all cultures was collected, and the re-lease of IL-6 and IL-8 was determined using the human IL-6 ELISA set  and the human IL-8 ELISA set (BD OptEIA™) according to the protocol given by the producers. To induce IL-6 and IL-8 secretion, 24 h prior to the harvesting, 20 µL containing 1, 5 and 10 µg lipopolysaccharide from Escherichia coli 055:B5  as proinflammatory stimulus was added. The concentration of LPS, which caused the maximum release of the cytokines by A549 cells was already determined in a previous study . Absorba5 yellow-green, fluorescent carboxyl polystyrene particles  was added and migration of the beads monitored for 10–30 s using Ti2-E/confocal system  equipped with heated stage (37 °C) and incubation chamber at 10× magnification. Object tracking was performed by NIS Element software (Nikon CEE GmbH).Prior to the measurements, tissues were rinsed in PBS and 10 µL of PBS containing 4 × 10Prior to the measurements, tissues were rinsed in PBS. Tissues were cut out from the insert and stripes of epithelium were placed on a glass slide and put into the incubation chamber (37 °C) on a fully motorized Ti2-E/confocal system (Nikon CEE GmbH) mounted on an antivibration table. Peripheral and central parts of the stripes were imaged. In pilot experiments, propranolol hydrochloride  was included as inhibitor and 5 µM forskolin (Sigma-Aldrich) + 100 µM 3-Isobutyl-1-methylxanthin  as stimulator of CBF. Background noise was determined by measurement of samples fixed with 4% paraformaldehyde. The background was negligible at 0.15–0.75 Hz. Further, the reaction to 1, 5 and 10 µg/insert LPS was recorded. Specimens were examined using 20× magnification. Beating ciliated edges were recorded using a digital high-speed video camera  at a rate of 100 frames per second with a frame size of 512 × 512 pixel. Five regions of interest (ROI) were analysed per sample. CBF was calculated by NIS Element software (Nikon CEE GmbH) and indicated as beats/second (Hz). In the later experiments, only propranolol was used because no prominent increase in CBF was seen.®VIP™ 5 . Radial sections of 2–5 µm were cut at a rotary microtome, stained with hemalaun and viewed with an Olympus BX51 microscope. Additional sections were cut for immunocytochemical staining.MatTek tissues were fixed in 4% paraformaldehyde and embedded in paraffin using Tissue-TekSections were incubated with DakoCytomation Target Retrieval Solution pH 6.0  in decloaking chamber  for 10 min at 110 °C and 20 s at 85 °C. Sections remained in the solution for cool down for 30 min. Three rinses with PBS were performed prior to the incubations with blocking serum (1% goat serum in PBS for 30 min at RT) and between each staining step. Antibodies were diluted in antibody diluent . Incubation with anti-α-tubulin antibody  or control mouse IgG  for 1 h at RT was followed by incubation with anti-mouse Alexa 488 antibody  in antibody diluent (DAKOCytomation) for 30 min at RT and counterstain with Hoechst 33342  for 15 min at RT. After three final washes in PBS, sections were mounted with fluorescence mounting media.Antigen retrieval was performed with DakoCytomation Target Retrieval Solution pH 9.0 (DAKO) in decloaking chamber  for 10 min at 110 °C and 20 s at 85 °C. Sections remained in the solution for cool down for 30 min. Three rinses with PBS were performed, cells permeabilized with PBS plus 0.2% Triton X-100 for 2 h at RT. Unspecific antibody binding was blocked with 10% normal goat serum for 30 min at RT. Incubation with anti-mucin 5AC  antibody was performed for 1 h at 37 °C, incubation with anti-mouse Alexa 488 antibody  for 1 h at 37 °C and with Hoechst 33342  in PBS for 15 min at RT.After deparaffinization, cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 60 min at RT and incubation with APC-CY7 anti-CD45  for 1 h at RT and counterstain of nuclei with Hoechst 33342  for 30 min at RT.EpiAlveolar™ tissues were fixed for 15 min with 4% paraformaldehyde. After three washes with PBS, the tissues were permeabilized with 0.1%Triton X100 in PBS, washed three times in PBS again and subsequently incubated with Alexa Fluor™ 488 Phalloidin  and Hoechst 33342  for 30 min at RT.Images were taken at a Ti2-E/confocal system (Nikon CEE GmbH) with ex/em of 395 nm/414–450 nm for Hoechst 33342 and 470 nm/500–530 nm for α-tubulin, mucin 5AC and phalloidin, and 640 nm/660–850 nm for CD45.p-values < 0.05 were considered to be statistically significant.Experiments were performed in triplicates and repeated at least two times. Data from all experiments were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons (SPSS 28 software). Results with In all evaluations, CNT4003 was applied in SLF and the following parameters and assays used as readout: MTS assay for cytotoxicity, TEER measurements for epithelial barrier tightness, interleukin 6 and 8 levels for proinflammatory effects, movement of polystyrene marker particles for mucociliary clearance and high-speed video microscopy for ciliary beating frequency. IL-6 is a marker for acute and chronic inflammation and IL-8 a marker for acute inflammation and a chemoattractant for neutrophils . Doses aCalu-3 cells, EpiAirway™ and MucilAir™ tissues were used as models for the bronchial part and A549 and EpiAlveolar™ tissues with and without macrophages as models for the alveolar part. The models were characterized regarding the stability and ability to react to LPS as an inflammatory stimulus. Evaluation of CNT4003 effects over 14 d corresponded to the recommendation of Behrsing et al. for analysis in EpiAirway™s and MucilAir™ tissues .No cytotoxicity of CNT4003 was seen up to 50 µg with a viability of 86 ± 15% of untreated controls .As seen in the immunocytochemical staining, Calu-3 monolayers do not form cilia but contain mucus-producing cells . Alpha tStability of the Calu-3 model has been shown previously . In thisStimulation with 10 µg LPS increased IL-6 and IL-8 secretions at all time points significantly . Interleα-tubulin staining  increased CBF at all concentrations significantly from 9.9 ± 1.7 Hz (untreated controls) to 15.3–17.8 Hz at 7 d and from 12.7 ± 1.4 Hz (untreated controls) to 15.1–16.2 Hz at 14 d. Basal CBF was significantly lower in this batch of tissues than the frequencies measured in the batch where CNT4003 were tested and no LPS was included. CBF of untreated tissues was highest at 1 d and significantly lower at all time points . The posMucilAir™ tissues are composed of two to three rows of cells. Cilia are already visible in the hemalaun staining A. They f2 at 1 d to 282 ± 24 Ω × cm2 at 21 d. Therefore, tissues were studied for only 14 d of exposure with LPS and CNT4003. While TEER values of the LPS-treated tissues were significantly decreased compared to 1 d, CNT4003 particles did not affect TEER values over the entire observation time of 14 d  and werEpiAlveolar™ tissues consist of alveolar epithelial cells and fibroblasts at the apical site and endothelial cells at the basal site of the membrane . In the Exposure to 25 µg CNT4003 particles did not decrease viability in EpiAlveolar™ tissues with and without THP-1 cells .2  were noted. TEER values may be a good parameter to screen the quality of the tissues because after one delayed delivery, TEER values of the tissues were 97 ± 137 Ω × cm2.EpiAlveolar™ tissues formed tight epithelial barriers with TEER values > 850 Ω × cm2 . TEER vaEpiAlveolar™ tissues reacted to stimulation with 1–10 µg LPS with variable and moderate increases in IL-6 and IL-8 secretion. Significant increases in cytokine secretion were mainly seen after stimulation with 5 and 10 µg LPS . In geneThe tightness of the epithelial barrier and low basal levels of proinflammatory cytokines, are crucial properties of the healthy lung, and the used models should be able to assess these parameters. In this comprehensive study, CNTs were evaluated for potential adverse effects on the bronchial and alveolar part of the respiratory system. Further, not only acute cytotoxicity but also epithelial barrier properties, release of cytokines and CBF were used as readout parameters.2, which is within the range  reported in the literature [2 to >900 Ω × cm2 [2. Similar to the EpiAirway™ tissues, there were prominent variations depending on the batch ranging from 346 ± 14 to 638 ± 81 Ω × cm2 [The suitability of Calu-3 cells as a model for the respiratory barrier and identification of inflammatory effects has already been reported. In this study, TEER values were 210–365 Ω × cmterature ,35,36. V Ω × cm2  and valu Ω × cm2 . The difSimilar to the values in this study, IL-6 and IL-8 values in EpiAirway™ tissues were reported as 100–350 pg/mL and 14,500 ± 3000 pg/mL, respectively . InterbaNormal CBF in human bronchi was reported as 12–15 Hz , but gre2 for the EpiAlveolar™ tissues with THP-1 and 1071 ± 111 Ω × cm2 for EpiAlveolar™ tissues without THP-1 at 7 d. These data are within the range of the reported values and indicate intact barrier properties. In addition to differences in equipment and operations for the measurements, the shipment of the tissues may impair their viabilities resulting in decreased TEER values compared to those at the producer. TEER values at 7 d and 14 d in the laboratory of the producer were ~1400–1000 Ω × cm2 and in another laboratory ~400–600 Ω × cm2 [The suitability of A549 cells in mono and in coculture with THP-1 macrophages for the assessment of acute and prolonged effects of particles was shown previously . Assessm Ω × cm2 .IL-6 levels released by EpiAlveolar™ tissues were reported to vary between 500 pg/mL and 3000 pg/mL in the tissues with THP-1 and between 1500 and 2500 pg/mL in the tissues without THP-1 macrophages . Levels 2 lung surface [2) MWCNT exposure of human lung explants [All models for the bronchial tract were stable over 14 d of evaluation and reacted to the proinflammatory stimulus LPS with significant cytokine increases. They were, therefore, regarded as suitable to detect potential toxic effects of CNT4003. The administered dose was 2 × 25 µg CNT4003, which is in the range of the lifetime lung exposure of workers to CNTs (12.4–46.5 µg/cm surface ). Exposu surface . Lack ofexplants . They obexplants . Also duThe two models for the alveolar part of the respiratory tract indicated adverse effects only in the EpiAlveolar™ tissues with and without THP-1 macrophages by increased IL-6 secretion upon exposure to 12.5 and 25 µg CNT4003 with no dose dependency. The greater cytokine release induced by CNT4003 in the EpiAlveolar™ tissues than in the A549 model is in contrast to the more pronounced response of the A549 model to LPS. Greater toxicity of SDS in A549 than in MucilAir™ tissues has been reported and can be explained by the lack of a mucus layer . ProtectCytotoxicity, TEER values, interleukin secretion and CBF show no indication of adverse effects of CNT4003 in the bronchial part of the respiratory tract. The increased IL-6 secretion of the EpiAlveolar™ tissues may indicate adverse effects of CNT4003 on the alveolar part of the lung. Since the doses for whole-life exposure of workers were applied over a shorter time, the physiological relevance is not clear. Although the reconstructed tissues are produced in a standardized way, considerable variations in basal parameters between batches were seen. In addition to the interdonor differences, the duration of the delivery from the producer to the laboratory of the user has an effect on the viability and reaction of the tissues.