The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains . In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes. Two evolutionarily conserved subfamilies of the SWI/SNF type chromatin-remodeling complexes, PBAF and BAF, play comprehensive and diverse roles in the transcriptional regulation of metazoans, which are being involved in transcription, development, and differentiation . These cDrosophila embryos and mammalian cell cultures , which replaces the canonical PBAF complex in adult mouse neurons. dcPBAF lacks the canonical BRD7 and PHF10A subunits but contains a PHF10D subunit. The expression of PHF10A, which is dominant in the neurons of the postnatal mouse brain, dropped down following the transition from the proliferative to the differentiation stage. At the same stage, the expression of PHF10D increased, and it became the only PHF10 isoform present in the terminally differentiated neurons of 4-month-old adult mice (P120). dcPBAF associates with promoters of actively transcribed housekeeping and neuron-specific genes. Moreover, we found that in human differentiated neurons, the PHF10D isoform is also dominantly expressed. This isoform replaces PHF10A isoforms upon the commencement of differentiation in SH-SY5Y immortalized human neuroblastoma cells and maintains transcription of the genes specific for differentiated neurons.Mouse and human PHF10 have a DPF-containing isoform and an isoform that lacks DPF . In addiWe also found that in both extracts (P1 and P56), the BRD7 subunit co-migrated with the high-salt peak, suggesting its association only with PBAF present in these fractions. Staining with antibodies against PHF10 indicated that PHF10A and PHF10D were associated with different peaks. PHF10A migrated with the high-salt peak, whereas PHF10B migrated with the low-salt peak. PHF10B and PHF10C were present in the high- and low-salt peaks, respectively. Significantly, PHF10D was dominant in the low-salt peak in the P56 extract. These results suggest the existence of a distinct type of the PBAF complex that mostly replaces canonical PBAF in differentiated neurons. We further refer to it as dcPBAF, the differentiated cell PBAF, to distinguish it from the canonical PBAF complex.Next, the MonoS fractions from P1 and P56 extracts containing PBAF or dcPBAF were collected and fractionated on a Superose 6 size exclusion column . The PBAThe strongest Coomassie-stained bands were cut out from the gel, and the proteins were identified by the MALDI-TOF MS, which indicated that they contained mostly PBAF subunits . PBAF puThe identified interactions were confirmed in co-immunoprecipitation experiments . We haveIn summary, our results demonstrated that dcPBAF differs from canonical PBAF in proliferating cells due to the absence of the BRD7 subunit and the presence of the PHF10D isoform.PHF10A transcript was dominant at the embryonic and early postnatal (P1-P7) stages but nearly completely disappeared between 7 and 23 days . This result was confirmed by qPCR  of the identified PHF10D peaks overlapped with promoter regions of genes (+/-200 bp from the transcription start sites) . The anap-value = 2 × 10−16, The analysis of the core promoter elements of the dcPBAF-occupied genes has shown that they contained TATA-box and Inr elements, and were enriched with CpG islands . The perAn analysis of the 238 PHF10D-bound genes for which the expression values were provided  has showThe gene set enrichment analysis (GSEA) using the GO database identified significantly enriched pathways among the dcPBAF target genes (FDR<0.05) . These wPHF10 isoforms are highly conserved between mice and humans . Human aBDNF, MAP2, and TrkB  . WhereasThe PHF10 PBAF subunit is highly expressed in mouse brain neurons. Two isoforms of PHF10, PHF10A, and PHF10D, are associated with two different states of the neuronal cell development program: immature neuroblasts and terminally differentiated mature neurons, respectively. The PHF10A/BAF45a isoform promotes the proliferation of neuroblasts . Our stuThe role of the replacement of PHF10A by PHF10D in terminally differentiated cells, for example, neutrophils and mature neurons, is not clear yet. However, previously we have demonstrated that PHF10A is a strong transcriptional co-activator, whereas PHF10D does not activate transcription . PHF10A/Our data demonstrate that dcPBAF does not contain Brd7 which is an important subunit of PBAF known as a tumor suppressor that inhibits growth and metastasis and initiates apoptosis in cancer cells . BRD7 anad libitum) at NRC Kurchatov Institute .We used 1- to 120-day-old male C57BL/6J mice . Animals were kept in identical conditions  and penicillin/streptomycin at 37°C with 5% CO2 in a humidified atmosphere.The human neuroblastoma SH-SY5Y cell line  was propagated in the Dulbecco modified Eagle’s medium  with 10% heat-inactivated fetal bovine serum (hiFBS)  supplemented with 2 mM L-glutamine; penicillin/streptomycin; and 10 μM all-trans retinoic acid (ATRA) (Sigma)). Three days later, the media were replaced by differentiation media 2 ; 1x B27 (Invitrogen); 2 mM GlutamaxI (Invitrogen); 10 μM ATRA (Sigma); and 50 ng/mL BDNF ), and cells were incubated for 7 days.Differentiation was performed according to the published protocol . For difThe knockdown of PHF10D was performed in the SH-SY5Y cells on the first day (before ATRA addition), fourth day, and sixth day of differentiation, by two different siRNA simultaneously, or control siRNA . MetafecFor Western blot analysis, the whole brain, cortex, or cerebellum was homogenized on ice in 10 volumes (W:V) of RIPA buffer; 50 mM Tris-HCl pH 7.4; 1% NP-40; 0.5% Na deoxycholate; 0.1% SDS; 150 mM NaCl; 2 mM EDTA; protease inhibitor cocktail ; and phosphatase inhibitor cocktail , and then 4× Laemmli buffer  was added, boiled for 10 min, and snap-frozen in liquid nitrogen for further Western blotting analysis.The SH-SY5Y cells were scrapped from the 60-mm Petri dishes, washed with PBS supplemented with PIC, centrifuged at 4°C, 2.5 krpm for 5 min, and resuspended in 10V of RIPA buffer, incubated for 10 min on ice, and centrifuged at 13.2 krpm at 4°C for 10 min. Protein concentration was measured using the Qubit Protein Assay Kit (Thermo Fisher Sci.). Then, 4× Laemmli buffer was added, probes were boiled for 10 min, and Western blotting was performed.Affinity-purified polyclonal antibodies against the PHF10, BAF155, BAF200, BRG1, and BAF250a subunits were described previously . The polFor the Fl-PHF10D-pSLIK expression vector, Fl-PHF10D cDNA was cloned from the previously described Fl-PHF10D-pcDNA  into the pSLIK plasmid (Addgene #25735 ) and mod2, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, and 0.35 M sucrose) supplemented with PIC (Roche) and PhIC (Sigma) on ice in Dounce loose (Millipore), and samples were incubated for 10 min on ice. After centrifugation at 4°C for 5 min at 100 g, the supernatant was transferred into new tubes and centrifuged at 4°C for 10 min at 2 kg. The pellet was washed with five volumes of NU-1 and centrifuged again in the same conditions. Next, the pellet was resuspended in an equal volume of NUS-1 , gently laid on an NUS-1 cushion, and centrifuged at 4°C for 20 min at 3.5 kg. Nuclei in the pellet were resuspended in NU-2 , supplemented with PIC and PhIC, and transferred in Dounce tight for nuclear destruction. 1/10 V NaCl (5M) and Triton X-100 up to 0.3% were added, mixed, and incubated for 15 minutes on ice. In the last step, the solution was ultracentrifuged at 4°C for 1 hour at 55 kg.Whole brains from P1 (1 g) or P120 (1 g) mice were homogenized in five volumes of NU-1  containing 150 mM NaCl (HEMG-150). Elution from MonoS HR 16/10 was performed by the gradient of increasing NaCl concentration, up to 1M (HEMG-1000). The immunoaffinity purification was performed on a column with immobilized anti-PHF10 antibodies, with the unbound protein being washed out with HEMG-1000 buffer containing 0.1% NP-40. HEMG-150 with 0.1% Nonidet P-40 was used for loading, HEMG-1000 with 0.1% Nonidet P-40 was used for washing, and 0.1 M glycine (pH 2.5) was used for elution. The eluted proteins were resolved via SDS/PAGE and visualized by silver staining. The protein bands were cut out and subjected to in-gel trypsin digestion. MALDI-TOF MS was performed using an Ultraflex II mass spectrometer . Protein spectra were internally calibrated using trypsin autolysis products, and the resulting peptide weights were searched against the non-redundant database maintained by the National Center for Biotechnology Information, using the MASCOT search engine. The Superose 6 column was calibrated with an HMW Calibration Kit . The void volume of the column was 7.0 mL, and the volume of each fraction was 0.5 mL.The nuclear extracts from the P1 and P56 whole mouse brains were used for the PHF10-containing complex purification, according to the flowchart . The col6 SH-SY5Y cells using TRI Reagent (MRC) according to the manufacturer’s protocol. The synthesis of cDNA was performed with an oligo (dT) primer and MMLV reverse transcriptase (Thermo Fisher Scientific). PCR primers are listed in RPLP0 housekeeping gene. At least three independent experiments were performed; values are presented as mean ± SD. Statistical analysis was performed using a two-way ANOVA with Holm-Sidak’s multiple comparison test and GraphPad Prism 6 software. p-values <0.05 were considered significant. More details are provided in Figure legends.RNA was isolated from 3 × 102O), 12.3 M formaldehyde, and 1 M formamide), heated at 65°C for 15 min, and immediately placed on ice. The formaldehyde electrophoresis in running buffer  was performed, and then the gel was equilibrated in 20x SSC buffer (3 M NaCl and 0.3 M Na3C6H5O7 x 5.5 H2O). The mRNA transfer on the Hybond-N membrane (Amersham) was also performed in 20x SSC buffer with a filter paper stack overnight, and then the mRNA was crosslinked by Stratalinker 1800 with UV light. Membrane was hybridized at 50°C overnight in HSB(f+) buffer  with a DNA probe against all PHF10 isoforms labeled by alpha [32P] dATP and then washed twice with 1x SSC buffer and 0.3x SSC buffer. Development was performed using Cyclone (Packard).Total RNA was isolated from 0.1 g of material ground with TRI Reagent in liquid nitrogen with a mortar and pestle. Then, the mRNA fraction was enriched with the Oligotex mRNA Kit (Qiagen). mRNA (1 mkg per lane) was mixed with the loading buffer  and PhIC (Sigma) on ice in Dounce loose (Millipore). Then, formaldehyde was added up to 1%, and samples were incubated for 10 min on ice. After centrifugation at 4°C for 10 min, 1000 g samples were washed three times with the same cold PBS and then resuspended in 1 mL of the sonication buffer  supplemented with PIC and PhIC in Dounce tight (Millipore) for nuclear destruction. The chromatin was sheared to ∼500 bp by sonication at Covaris and precipitated by 10 µL anti-PHF10 antibodies or 1 µg anti-Flag M2 antibodies and 1 µg control rabbit IgG (Invitrogen) on Mab-selected beads . Then, beads were washed with sonication buffer, Wash buffer A (sonication buffer with 500 mM NaCl), Wash Buffer B (20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% deoxycholate Na), and subsequently, TE. DNA was eluted by elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS, and 1 mM EDTA) and treated with RNase A for 30 min and protease K (Thermo Fisher Scientific) overnight, incubated at 65°C for 6 h, and extracted using the phenol-chloroform method. DNA from SH-SY5Y was dissolved in 100 μL TE buffer, and then qPCR was performed with primers listed in 7 reads were obtained per sample.For NGS library preparation, 30 ng of DNA and the NEBNext Ultra II DNA Library Prep Kit for Illumina were used according to the manufacturer’s protocol. Adaptor ligation was performed using the NEBNext Multiplex Oligos for Illumina (Index Primer Set #1) (NEB). The quality of the DNA was analyzed with Bioanalyzer DNA 1000 Chip Kits (Agilent). DNA yields were assessed using Qubit assay kits (Thermo Fisher Scientific). Libraries were sequenced on an Illumina NovaSeq 6000 with single-strand 100-bp-length reads. Approximately 3 × 10GSE199351 (reviewer token: uxyhwcseltcplqh). The average read length was 100 bp. Adapters, poly-N, poly-A, and low-quality read ends were removed using Cutadapt software (Mus musculus genome using Bowtie version 1 (http://broadinstitute.github.io/picard/). In addition, peaks overlapping with blacklist regions were discarded (https://sites.google.com/site/anshulkundaje/projects/blacklists). Peak calling was performed using MACS version 2 against pre-immune control . The IDR p-value threshold was set to 0.05. As the reproducibility was good according to IDR , we formed the final set of PHF10 sites by combining the peaks obtained for each replicate with a MACS2 p-value less than 1 × 10−5. Intersecting peaks were merged during this procedure. Examples of PHF10 peaks from both replicates and IgG control are provided in The raw data were represented as single-end reads from two biological replicates. Raw reads are deposited under accession number software   was obtained from the EPD database . CpG islhed data  using Tihed data  (17 tisshed data . Housekehed data .


Pemphigus is an autoantibody driven disease that impairs the barrier function of the skin and mucosa by disrupting desmosomes and thereby impeding cellular cohesion. It is known that the different clinical phenotypes of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are dependent on the autoantibody profile and target antigens that, amongst others, are primarily desmoglein (Dsg)1 and/or Dsg3 for PV and Dsg1 for PF. However, it was reported that autoantibodiesagainst different epitopes of Dsg1 and Dsg3 can be pathogenic or not. The underlying mechanisms are very complex and involve both direct inhibition of Dsg interactions and downstream signalling. The aim of this study was to find out whether there is target-epitope-specific Dsg3 signalling by comparing the effects of the two pathogenic murine IgGs, 2G4 and AK23.Dispase-based dissociation assay, Western Blot analysis, Stimulated emission depletion microscopy, Fura-based Ca2+ flux measurements, Rho/Rac G-Protein-linked immunosorbent assay, Enzyme-linked immunosorbent assay.The IgGs are directed against the EC5 and EC1 domain of Dsg3, respectively. The data show that 2G4 was less effective in causing loss of cell adhesion, compared to AK23. STED imaging revealed that both autoantibodies had similar effects on keratin retraction and reduction of desmosome number whereas only AK23 induced Dsg3 depletion. Moreover, both antibodies induced phosphorylation of p38MAPK and Akt whereas Src was phosphorylated upon treatment with AK23 only. Interestingly, Src and Akt activation were p38MAPK-dependent. All pathogenic effects were rescued by p38MAPK inhibition and AK23-mediated effects were also ameliorated by Src inhibition. The results give first insights into pemphigus autoantibody-induced Dsg3 epitope-specific signalling which is involved in pathogenic events such as Dsg3 depletion. The skin and mucosa provide an effective barrier against external hazards such as pathogens or harmful chemicals. Pemphigus is an autoantibody driven disease that impairs barrier integrity by disturbing the turnover of desmosomes. Desmosomes provide mechanical cohesion between neighbouring epithelial cells . As a reFor anti-Dsg1 autoantibodies, direct inhibition was not found in all studies , 15–17. in vivo  microscopy showed that both 2G4 and AK23 produced a Dsg3 staining pattern comparable to a commercial polyclonal IgG directed against the intracellular domain of Dsg3 , using STo check if there are additive and polyclonal effects potentially increasing the loss of adhesion, we used mixtures of AK23 and 2G4 in dispase dissociation assays. A mixture of AK23 and 2G4 at a concentration of 75 µg/ml each did not lead to a significant increase in the number of fragments, compared to AK23 alone at a concentration of 75 µg/ml . A mixture of AK23 and 2G4 at a concentration of 37.5 µg/ml for both led to a number of fragments similar to the number of fragments induced by 2G4 alone at a concentration of 75 µg/ml  and both IgGs induced a similar degree of p38MAPK phosphorylation. This effect was ameliorated upon treatment with the p38MAPK inhibitor SB202190 , 49, was2+-influx after treatment with either 2G4 or AK23 was monitored. 2G4 induced a rapid influx of Ca2+ into the cells whereas AK23, similar to a previous report, did not  (with BTP-2) blocked Ca2+-influx, indicating that 2G4 induced a different pathway of Ca2+-influx into the endoplasmic reticulum   with 10 % FCS  and 50 U/ml penicillin and 50 µg/ml streptomycin . They were passaged using a trypsin-EDTA solution . For experiments, the cells were pre-incubated with the mediators . Mediators (2(H2O)2, 0.5 mmol/l MgSO4, 0.5 mmol/l NaH2PO4(H2O)2, 2 mmol/l NaHCO3, 5 mmol/l HEPES and 5 mmol/l D+Glucose, pH 7.35) at 37°C. The cells were washed twice with measurement buffer. Measurements were performed using MetaFluor  on an Axio Observer A1  with a Polychrome V , a CoolSNAP-Hq2 digital camera  and a Fura-2 filter set. For each independent experiment, the signals from 8 out of 15 randomly selected cells were evaluated .Fura-2AM  was used to measure intracellular Caediators Table S22, pH 6.8, 0.1% pepstatin+aprotinin+leupeptin, 1% Phenylmethylsulfonylfluorid) for 10 min on ice under gentle shaking. The pellet was separated at 14.000 rpm for 10 min at 4°C and the supernatant was retrieved. The pellet was washed once and lysed with ultrasound in SDS lysis buffer ). The protein amount was determined with a commercial Pierce BCA protein assay kit. Western-blotting was performed, using a standard wet blotting protocol on nitrocellulose membranes . Membranes were blocked with ROTI®Block  1:10 in Tris-buffered saline with 0.05% tween (TBST) for 1 h. The Antibodies  and an EOS 600D camera .After incubation as described above, confluent cell monolayers were washed with Hank´s buffered saline solution (HBSS) and subjected to 2.4 U/ml dispase II  in HBSS for 20 min at 37 °C, 95% humidity and 5% COSamples were washed and fixed with ethanol (-20°C) shaking on ice for 30 min followed by acetone (-20°C) for 3 min. The samples were blocked with 3% bovine serum albumin (BSA) and 1% normal goat serum in PBS for 30 min. Primary antibodies were applied over 3 h at RT STAR-RED- or Alexa 594-coupled goat-anti-rabbit/mouse secondary antibodies  were incubated for 1 h and DAPI 1:10.000 for 15 min. The coverslips were mounted on glass slides using Prolong™ Diamond Antifade Mountant .The samples were imaged using an Abberior 3D Stimulated emission depletion (STED) confocal microscope with IMMOIL-F30CC  Star Red and Alexa 594 were excited at 638 nm and 594 nm respectively, using pulsed diode lasers . Fluorescent molecules were depleted at 775 nm with a pulsed fibre laser  and the emission was detected with an avalanche photodiode detector at 605-625 and 650-720 nm range.Data were analysed using two-way-ANOVA followed by Sidak-post-hoc-test for multiple comparison or one-way-ANOVA followed by Dunnett-post-hoc-test for multiple comparison, using Graphpad Prism . Error bars represent SEM. Significance was assumed with p ≤ 0.05. Data are shown as mean ± SEM. Each N represents one independent experiment, each n one technical replicate.The original contributions presented in the study are included in the article/TS: 65%, conceptualizing the project, performing experiments, performing data evaluation writing the manuscript, conceptualizing and designing the Figures, revising the manuscript. CH: 18%, providing materials, performing experiments, performing data evaluation, checking the manuscript, revising the manuscript. SM: 3%, performing experiments, performing data evaluation. MH: 2%, providing materials, checking the manuscript. RT: 2%, providing materials, checking the manuscript. JW: 10%, providing funding, conceptualizing the project, checking the manuscript, revising the manuscript. All authors contributed to the article and approved the submitted version.


Chromatin is dynamically reorganized spatially and temporally, and the post-translational modification of histones is a key component of this regulation. The basic subunit of chromatin is the nucleosome core particle, consisting of two copies each of the histones H2A, H2B, H3, and H4 around which ∼147 base pairs of DNA wrap. The intrinsically disordered histone termini, or tails, protrude from the core and are heavily post-translationally modified. Previous studies have shown that the histone tails exist in dynamic ensembles of DNA-bound states within the nucleosome. Histone tail interactions with DNA are involved in nucleosome conformation and chromatin organization. Charge-modulating histone post-translational modifications (PTMs) are poised to perturb the dynamic interactions between histone tails and DNA. Arginine side chains form favorable interactions with DNA and are sites of charge-modulating PTMs such as citrullination. Our current focus is on the H3 tail, the longest histone tail. Four arginine residues are relatively evenly spaced along the H3 tail sequence, suggesting multivalent interactions with DNA poised for regulation by PTMs. In this study, we use NMR nuclear spin relaxation experiments to investigate the contribution of arginine residues to H3 tail dynamics within the nucleosome core particle. By neutralizing arginine via mutation to glutamine, we begin to work towards a comprehensive understanding of the contribution of individual residues to H3 tail dynamics. We find that neutralization of arginine residues results in increased regional mobility of the H3 tails, with implications for understanding the direct effects of arginine citrullination. Altogether, these studies support a role for dynamics within the histone language and emphasize the importance of charge-modulating histone PTMs in regulating chromatin dynamics, starting at the level of the basic subunit of chromatin. Chromatin is dynamically reorganized during development and in response to stimuli. Dynamic regulation occurs at many length scales, and it is appealing to consider a model wherein dynamic regulation of chromosome organization and transcriptional activity originates at the level of the basic subunit, the nucleosome. The nucleosome core particle (NCP) is comprised of an octamer of histones with two copies each of the histones H3, H4, H2A, and H2B wrapped by ∼147 base pairs (bp) of DNA. The histone tails are the intrinsically disordered termini of the histones, which protrude from the nucleosome core. Histone tails have indirect and direct mechanisms of regulation, which are both modulated by post-translational modifications (PTMs). Indirect mechanisms involve the readout of histone tails and their PTMs by “reader,” “writer”, and “eraser” domains that are components of regulatory proteins and complexes . Direct The histone tails dynamically interact with DNA in a broad ensemble of states. Experimental characterization of nucleosomal H3 tail dynamics began with seminal work by Is the modulation of histone tail dynamics by PTMs a regulatory mechanism within the histone language? Charge-modulating histone PTMs, which include acylation of lysine, citrullination of arginine, and phosphorylation of serine and threonine, are prime candidates for modulating histone tail interactions with DNA and thus histone tail dynamics. Our overall goal is to decipher the role of dynamics in the histone language. This involves characterizing the dynamics of the multivalent network between histone tail residues and DNA by cataloging each tail residue and the effect of mutating or modifying the residue. It is interesting to consider whether there is long-range communication between residues via dynamics, which would be a form of histone PTM crosstalk. Histone tails are rich in both arginine and lysine, but the arginine side chain is particularly favorable for interactions with DNA, especially within a narrowed minor groove . The H3 In this study, we use NMR spin relaxation to measure the ps-ns timescale motions of the H3 tail within the NCP. Neutralization of arginine via mutation to glutamine leads to an increase in dynamics on this timescale with neutralization of all four H3 tail arginines resulting in a global increase across the tail. Neutralization of each individual arginine leads to a regional increase with the extent of the effect dependent on the arginine position. Notably, the increase in dynamics is purveyed across ∼60%–70% of the tail for R8Q, R17Q, and R26Q at 150 mM KCl. In general, the largest effect is observed for neutralization of R26, adjacent to the putative H3 tail ‘hinge’. This study provides insight into mechanisms of histone PTM crosstalk and tail accessibility and additionally has implications for the regulatory mechanisms of citrullination.15N-M9 minimal media. To confirm histone masses, electrospray ionization mass spectrometry was performed. 147 bp Widom 601 DNA was amplified and purified as previously described . Final samples for data collection were 100 µM 15N-H3 NCP in 20 mM MOPS pH 7 (with 8 mM NaOH to pH), 1 mM EDTA, 1 mM DTT, and 5% D2O. Samples additionally contained 150 mM KCl where noted.Purified NCPs were exchanged into NMR buffer using consecutive rounds of concentration and dilution in 10k MWCO centrifugal filtration devices. NCP concentration was determined spectrophotometrically via absorbance at 260 nm. Samples were first diluted into 2 M salt, and concentrations were determined using the calculated extinction coefficient for the DNA  × 400 (15N) total points, acquisition times of 98 ms (1H) and 82 ms (15N), and spectra widths of 13 ppm (1H) and 30 ppm (15N). All nuclear spin relaxation experiments were collected with 2,048 (1H) × 300 (15N) total points, acquisition times of 98 ms (1H) and 84 ms (15N), and spectra widths of 13 ppm (1H) and 22 ppm (15N). {1H}-15N steady-state heteronuclear nuclear Overhauser effect (hnNOE) data were collected using the Bruker pulse sequence hsqcnoef3gpsi with the reference and saturated spectra interleaved. Parameters included 96 scans with an interscan delay of 5 s. Longitudinal (R1) 15N relaxation experiments were collected with total relaxation loop lengths of 0.033 s, 0.195, 0.390 s (×2), 0.813 s (×2), 1.301 and 1.951 s in randomized order. Experiments were collected with 32 scans and an interscan delay of 2 s. Transverse (R2) 15N relaxation experiments were collected with an effective CPMG field of 500 Hz and total relaxation CPMG loop lengths of 0.0088, 0.031 s (×2), 0.053, 0.079, 0.110 s (×2), 0.150 s, with the exception of the wild-type H3-NCP in 150 mM KCl, which was collected with delay times of 0.0044, 0.018 s (×2), 0.040, 0.062 s, 0.084 s (×2), and 0.110 s. Experiments included temperature compensation blocks and were collected in randomized order, with 32 scans, and with an interscan delay of 2.5 s. HSQC spectra were collected for each sample between relaxation series to confirm sample integrity.Data were collected on a Bruker Avance Neo 800 MHz spectrometer with cryogenic probe at 304 K and running Topspin 4.0.7. For chemical shift perturbation analysis, 1H-15N HSQC spectra of all mutant H3-NCP. Questionable assignment transfers at 0 mM KCl are as follows: K9 and K18 for 15N-R17Q-H3-NCP and L20 and K23 for 15N-R17Q- and R2/8/17/26Q-H3-NCP. Questionable assignment transfers at 150 mM KCl are as follows: K9 and K18 for 15N-R2/8/17/26Q-H3-NCP and L20 and K23 for all constructs. The assignment for R2 was used from 15N-H3-NCP were calculated from the differences in amide proton  from the saturated and reference spectra, and errors were calculated via standard error propagation of the spectral noise as determined by CcpNmr Analysis 2.5.2. Relaxation rates (R1 and R2) were determined by fitting peak heights from the relaxation series to a single-exponential decay without offset, and errors were determined from the covariance matrix within CcpNmr Analysis 2.5.2. The R2/R1 ratio was calculated from the fit values for R1 and R2, and error was determined using standard error propagation. As is common with intrinsically disordered regions, amide spectra suffer from low chemical shift dispersion. These residues were analyzed in the same manner described and are included in the overall analysis with the caveat that the relaxation rates determined from the analysis are likely influenced by convolution with neighboring peak(s). In order to test the robustness of the analysis of overlapping peaks, a separate analysis was conducted for 15N-WT-H3-NCP at 0 mM KCl using PINT v2.1.0 , was calculated for each mutant with respect to WT. For calculation of ΔhnNOE and Δ(R2/R1) with peak doublets, if the peak for a given residue in WT-H3-NCP was a singlet, then the ΔhnNOE or Δ(R2/R1) of both mutant peaks was calculated with respect to the single WT peak. Both values are plotted in the corresponding figures.15N-labeled WT-H3 along with unlabeled H2A, H2B, and H4 and 147 bp Widom 601 DNA. Consistent with previous results, H3 is only visible in amide spectra through K36  . The H3 ough K36 . A1, P1615N-WT-H3-NCP was subjected to NMR spin relaxation experiments, which provide insight into nucleosomal H3 tail mobility on the ps-ns timescale  shows a decrease in hnNOE values for residues S28-K36 (average = 0.16 ± 0.05) as compared to residues T3-K27 (average = 0.41 ± 0.04), supporting a putative flexible ‘hinge’ region. As noted initially by 15N-WT-H3-NCP across residues T3-K27 (average hnNOE = 0.30 ± 0.08) as compared to at 0 mM KCl when considering the standard deviation. The putative ‘hinge’ region is maintained for S28-K36 but is less distinct than at 0 mM KCl (average hnNOE = 0.13 ± 0.04). Altogether, these data support a model, in agreement with the literature, wherein the H3 tails are neither conformationally rigid nor unrestrained but rather are conformationally restrained by dynamic interactions with DNA.imescale . HnNOE v15N-labeled mutants of H3, either one of four single-arginine mutants of H3  or a quadruple-arginine mutant of H3 (R2/8/17/26Q). Amide assignments were transferred from WT to each of the four single-arginine mutants of H3 via inspection, and the single-arginine mutants were together used to transfer amide assignments to R2/8/17/26Q-H3.In order to catalog the effects of neutralizing the four H3 tail arginine residues, both individually and all together, on H3 tail dynamics within the NCP, we used NMR spectroscopy. We reconstituted NCPs with A subset of residues is split into two peaks, which is dependent on the H3 construct used, and additional peaks appear broadened . At 304 1H-15N HSQC spectra were used for an initial evaluation of the effects of the arginine-neutralizing mutations on the conformational ensemble of the H3 tail within the NCP  determined from  the NCP . CSPs of the NCP . While t the NCP , moving 15N-WT-H3-NCP and 15N-R2/8/17/26Q-H3-NCP. NMR spin relaxation experiments reveal differences between H3 tail dynamics on the ps-ns timescale with neutralization of all four tail arginine residues. The hnNOE value for the H3 tail within R2/8/17/26Q-H3-NCP varies between 0.04 and 0.37 (average = 0.24 ± 0.09) at 0 mM KCl, an average decrease from WT of 31% (15N R1 of 17% (average R1 = 1.25 ± 0.05 s−1), decrease in 15N R2 of 54% (average R2 = 10 ± 3 s−1), and decrease in R2/R1 ratio of 62% (average R2/R1 = 8 ± 3) across the H3 tail as compared to WT. Together, these trends support an overall increase in H3 tail dynamics on the ps-ns timescale as a result of neutralizing mutations to all four arginine residues. Despite the overall increase in mobility as compared to WT, hnNOE values for H3 tail residues S28-K36 within R2/8/17/26Q-H3-NCP remain depressed (average = 0.11 ± 0.04) as compared to residues T3-K27 (average = 0.28 ± 0.05), a trend that was observed for WT. Similarly, the R2/R1 profile shows smaller values for S28-K36 and additionally around the first TGG repeat at residues 11–13 , and whether there is a positional effect within the tail . NMR nuclear spin relaxation experiments collected on NCPs reconstituted with While less extensive than with neutralization of all four tail arginine residues together, nuclear spin relaxation experiments reveal differences in H3 tail dynamics on the ps-ns timescale with neutralization of each individual H3 tail arginine . Each muEach of the single-arginine mutations affects the dynamics of at least one other H3 tail arginine residue. R2Q and R17Q influence the R8 position while R8Q and R26Q influence the R17 position. While the R2 position could not be measured directly, R8Q influences through to the T3 position, suggesting the potential of influencing R2. The R26 position is not significantly affected by individual neutralization of the three other arginine positions. Together, this speaks to crosstalk between arginine residues.As already mentioned, the quadruple mutant has a global effect on the H3 tail. To gain insight into whether the effects of each arginine neutralization are additive or show indications of cooperative effects, the ΔhnNOE and Δ(R2/R1) were summed for all four single-arginine mutations and compared with the data for R2/8/17/26Q-H3-NCP  leads to a global increase in H3 tail mobility on the ps-ns timescale. Comparison of the single-arginine mutants to the extremes of WT- and R2/8/17/26Q-H3-NCP reveals that the hnNOE and R2/R1 profiles are depressed from WT levels toward R2/8/17/26Q levels regionally, up to ∼70% of the tail, rather than simply locally around the site of mutation. Monovalent salt concentrations of 150 mM KCl broaden the regional increase in ps-ns dynamics. The effect on slower timescale motions (µs-s) remains untested. Altogether, these studies support an emerging model wherein histone tail dynamics are an important part of the histone language.H3 tail mobility is constrained, but not rigid, within the NCP; the H3 tails exist in a dynamic ensemble of DNA-bound conformations within the NCP . We propThe data reported here are interpreted as reporting on ps-ns timescale dynamics of the H3 tail. The effective CPMG field strength used in the R2 experiments (500 Hz) may not fully suppress any µs-ms timescale dynamics within the H3 tail. Thus, the reported R2 rates may have contributions from µs-ms timescale motions, which could also be differentially affected by the arginine mutations. Future experiments will be required to measure dynamics across a wider range of timescales. In addition, the experiments reported in this study were only conducted at a single concentration of NCP. We interpret the data as though we are only monitoring histone tail dynamics from intra-nucleosomal interactions with DNA. However, we cannot distinguish between intra- and inter-nucleosomal tail-DNA interactions at this point. Using salt-dependent data from nucleosome arrays , we specα target genes, which is associated with a local opening of chromatin and activation of gene expression  . Citrullpression . In addipression . It is a15N-R2 relaxation experiments revealed an important arginine anchor point in H4 R3 (In this study, we only perturb H3 tail arginine residues. All the lysines remain, spaced relatively evenly along the length of the tail as potential multivalent interaction points with DNA . In genein H4 R3 . Other Min H4 R3 . Ongoing15N-R1 and R2 data show reduced ns-timescale dynamics for the H3 tails within nucleosome as compared to NCP, which is further reduced upon the binding of linker histone H1 (In recent years, a model has been emerging wherein histone tail dynamics are an important part of the histone language. A variety of nuclear factors can perturb the ensemble of DNA-bound histone tail conformations. We previously demonstrated that the conformational ensemble of the H3 tails is sensitive to the assembly state of the NCP, with the H3 tails more dynamic within tetrasome than within NCP . In addistone H1 , but thestone H1 . Charge-stone H1 . These mstone H1 . Interesstone H1 . This sustone H1 . The reg


Mitochondrial fission and fusion are essential processes underlying mitochondrial homeostasis in health and disease. We examined the role of mitochondrial fission and fusion in PM2.5-induced alveolar epithelial cell damage and lung injury. Key genes in these processes include dystrophin-related protein 1 (DRP1) and optic atrophy 1 (OPA1) respectively.Exposure to particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5 (32 µg/ml) in the presence and absence of Mdivi-1  or BGP-15 . Results were validated using DRP1-knockdown (KD) and OPA1-overexpression (OE). Mice were injected intraperitoneally with Mdivi-1 (20 mg/kg), BGP-15 (20 mg/kg) or distilled water (control) one hour before intranasal instillation of PM2.5 (7.8 mg/kg) or distilled water for two consecutive days.Alveolar epithelial (A549) cells were treated with PM2.5 exposure of A549 cells caused oxidative stress, enhanced inflammation, necroptosis, mitophagy and mitochondrial dysfunction indicated by abnormal mitochondrial morphology, decreased mitochondrial membrane potential (ΔΨm), reduced mitochondrial respiration and disrupted mitochondrial fission and fusion. Regulating mitochondrial fission and fusion pharmacologically using Mdivi-1 and BGP-15 and genetically using DRP1-KD and OPA1-OE prevented PM2.5-induced celluar damage in A549 cells. Mdivi-1 and BGP-15 attenuated PM2.5-induced acute lung injury in mice.PM2.5-induced alveolar epithelial cell damage in vitro and lung injury in vivo.Increased mitochondrial fission and decreased mitochondrial fusion may underlie PMThe online version contains supplementary material available at 10.1186/s12989-023-00534-w. RecombiApoptosis denotes a genetically programmed pathway involving caspase activation, while necrosis is induced by extreme physical or chemical stress . Recent 2.5 exposure disrupts the mitochondrial fission and fusion balance in alveolar epithelial cells and lungs, leading to ROS responses and pro-inflammatory responses, which may be attenuated by regulating mitochondrial fission and fusion. We therefore examined these effects by PM2.5 on alveolar epithelial (A549) cells in vitro and in murine lung in vivo and whether PM2.5 actions were prevented by pharmacological and genetic modulation of mitochondrial fission and fusion.We hypothesized that PM2.5 have been described in a previous study [2.5 samples were collected on quartz filters  using a high flow volume PM2.5 Sampler  at a flow rate of 1.13 m3/min, located on the top of a building in Xuhui District in Shanghai, China, from September 2017 to April 2018. The filters were cut into small fragments (1 cm × 1 cm), then immersed into ultrapure deionized water and eluted with an ultrasonic cleaner , followed by freeze-drying with a vacuum freeze dryer . PM2.5 solid particulates were preserved at -80 °C until required.The collection, extraction and component analysis of PMus study . PM2.5 s2.A549 cells  were cultured in F12K medium  with 10% fetal bovine serum, 100U/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% CO2 PM2.5 correspond to 0, 8, 16, 32, 64 and 128 µg/mL, respectively. We mainly use six-well plates, confocal dishes and 96-well plates for our experiments. 3 ml of medium is added per well for six-well plates (9.6 cm²) and confocal dishes (9.6 cm²) and 100 µl of medium per well for 96-well plates (0.32 cm²).In this study, 0, 2.5, 5, 10, 20 and 40 µg/cm2.5 to induce cell injury. Cells were pretreated with 10µM of Mdivi-1 (a DRP1 inhibitor)  or 10µM BGP-15 (an OPA1 activator)  for 2 h and then were cultured with vehicle or PM2.5 for 48 h  . All the mice were housed in a specific pathogen-free facility where the circulating temperature is 22 °C with 50–60% humidity, equal light-dark cycle, and with access to standard food and water ad libitum. All experimental studies involving animals were approved by the laboratory animal ethics committee of the institute. According to a preliminary study, mice were administered intraperitoneally with Mdivi-1 (20 mg/kg) or BGP-15 (20 mg/kg) , 40% polyethylene glycol 300 (PEG 300) and 5% Tween 80), one hour before intranasal instillation of 50µL of PM2.5 on cell proliferation was measured by BeyoClick™ EdU Cell Proliferation Kit with Diaminobenzidine (DAB) staining . Cells were cultured in a 6-well plate (3ml per well of culture medium) with a density of 5 × 104cells/ml overnight at 37 °C before exposure to vehicle or PM2.5. After 48 h of co-culture, cells were cultured in the presence of EdU for 2 h, after which they were fixed with paraformaldehyde and permeabilized with a detergent, which allows the detection reagents to access the EdU-labeled DNA. The incorporated EdU was detected with a horseradish peroxidase (HRP) -conjugated antibody that binds to an azide group on the EdU molecule. This was followed by addition of the DAB substrate, which produces a brown precipitate at the site of EdU incorporation. The samples were measured under microscopy to visualize the brown precipitate at the site of EdU incorporation, as well as the counterstained nuclei.The effect of vehicle or PM4cells/ml. Total RNA was extracted from A549 cells using TRIzol and then its concentration and purity were assessed. One µg total RNA was reverse transcribed into cDNA and quantitative real-time PCR was performed with ChamQ Universal SYBR qPCR Master Mix  in an ABI ViiATM 7 System . The reaction conditions included 95 °C for 30s, followed by 40 cycles of 95 °C for 10s and 60 °C for 30s, with a final cycle of 95 °C for 15s, 60 °C for 60s and 95 °C for 15s. The primer sequences of the cytokines  and β-actin are shown in Table Cells were cultured using 6-well plates (3ml per well of culture medium) with a density of 5 × 104 /ml with 6 parallel wells in each group. Cells were stained with 10µM DCFH-DA at 37 °C in the dark for 15 min. Then cells were washed with serum-free F12K for three times, the level of ROS was determined using a fluorescence plate reader  at 488/525nm. Mitochondrial ROS(mtROS) level was measured using MitoSOX Red . Briefly, the cells were seeded in 96-well black plates with a density of 5 × 104 /ml with 6 parallel wells in each group. Cells were incubated with 5mmol/L MitoSOX Red probe for 10 min at 37 °C. The cells were washed twice with PBS, and red fluorescence was determined at 510/580nm using a fluorescence plate reader (Molecular Devices).Intracellular ROS generation was detected using 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) . Briefly, the cells were seeded in 96-well black plates(100 µl per well of culture medium) with a density of 5 × 104cells/ml, and incubated with 500 µl of 200nM Mito-Tracker Green FM (Invitrogen) 30 min at 37 °C in the dark and washed twice with serum-free medium. 100 µl of Hoechst 33,342 (Beyotime) was added and incubated for 30 min at 37 °C. The mitochondrial morphology was assessed using a confocal microscopy . Cell area and mitochondrial area were measured by ImageJ software (https://imagej.nih.gov/ij/). Mitochondrial fragmentation was judged to occur if > 90% of mitochondria in the cytoplasm outside of the perinuclear compaction were punctate or circular, and mitochondrial perinuclear compaction was judged to be present if > 90% of mitochondria accumulated in the perinuclear area[Mitochondrial morphology was measured with Mito-Tracker Green FM assay (Invitrogen) according to the manufacturer’s instructions. Cells were seeded in confocal dishes (3ml of medium per well) with a density of 5 × 10lear area.5 cells/well, 3ml of medium per well) and cultured with 1mL JC-1 staining working solution  for 20 min. The cells were later washed twice using PBS. Cells were photographed under an inverted fluorescence microscope(Leica). Red fluorescence was determined at 579/599nm and green fluorescence was determined at 490/516nm. The relative ΔΨm level was evaluated by comparing the red fluorescence cells to green fluorescence cells in the groups.Cells were seeded in 6-well plates (2 × 10Cells were seeded on Seahorse XFe96 plates (100 µl of medium per well) at a density of 10,000 cells/well in complete F12K. The OCR was measured using an XFe96 Extracellular Flux Analyzer  according to the manufacturer’s instructions. The following inhibitors were added sequentially: 1.5µM oligomycin (an adenosine-triphosphate (ATP) uncoupler), 1µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an electron transfer chain (ETC) accelerator), 0.5µM antimycin A (a complex III inhibitor), and 0.5µM rotenone (a complex I inhibitor). OCR was calculated using the standard XFe96 Extracellular Flux Analyzer protocol.The cell culture medium was discarded without washing and then quickly 2.5% glutaraldehyde as fixative was added to the cells. The cells were gently scraped along one direction and then collected (to avoid damaging the cells). The cells were centrifued and added with the fresh fixative and then fixed at room temperature for 2 h. Dehydration was accomplished with increasing concentrations of ethanol (30–100%) and then replaced with propylene oxide for 10 min. The dehydrated cells were immersed in a mixture of propylene oxide and SPI-pon812 embedding agent  overnight at room temperature for soaking and embedding. Ultrathin Sect. (70-80 nm) were obtained by using a diamond knife  and Leica EM UC7 ultramicrotome . Finally, the ultrathin sections were stained with uranyl acetate and lead citrate. The samples were treated and analyzed for transmission electron microscopy imaging. The morphology was qualified by Image J analysis .After anesthesia with an intraperitoneal injection of 0.2mL 1% pentobarbital, mice were tracheostomized and placed in a plethysmograph before inhaling of aerosolised acetylcholine (ACh) for the measurement of airway resistance and compliance . Lung resistance (RL) was recorded and presented as the percentage change from baseline RL of nebulizing PBS. The concentration of ACh required to increase lung resistance by 100% from baseline was calculated as PC100, and -logPC100 was taken as a measure of airway responsiveness.The whole lung was removed, and the right lung was dissected and snap-frozen in liquid nitrogen for later analysis. The left lung was inflated with 4% paraformaldehyde under 25 cm of water pressure and then embedded in paraffin. Paraffin blocks were sectioned to expose the maximum surface area of the lung tissue in the plane of the bronchial tree. Four µm sections were cut and stained with hematoxylin and eosin (H&E). The extent of lung inflammation was evaluated in the H&E-stained lung sections as described previously  using thThe localization and expression of pulmonary surfactant-associated protein C (SFTPC) was examined by immunofluorescence staining. Lung sections were incubated with anti-SFTPC primary antibody  at 4 °C overnight, and the secondary antibody (Goat anti-rabbit IgG (Alexa Fluor 488) ,1:500, Abcam, Cambridge, MA, USA) at room temperature in the dark for 60 min. Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI) (Beyotime) staining solution, and imaging was obtained.Total protein from A549 cells cultured in 6-well plates and mice lung tissues were respectively homogenized with a RIPA lysis buffer (Beyotime), and protein concentrations were quantified by a Bicinchoninic Acid (BCA) assay kit . 30 µg of protein per lane was separated through 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: DRP1 , MFF , MFN2 , OPA1 , phosphorylated (phospho) DRP1 (p-DRP1), PINK1 , PARK2 , SQSTM1/p62 , LC3 , MLKL , RIPK1 , RIPK3 , overnight at 4 °C. Membranes were then incubated with a HRP-conjugated anti-rabbit secondary antibody  and then visualized by chemiluminescent detection.Data were presented as mean ± SD. GraphPad Prism 8 was used to compare multiple groups by One-way ANOVA with Bonferroni’s post hoc test  or Dunnett’s T3 post hoc test .Two-way ANOVA was performed for comparisons of %change in lung resistance between individual groups. P < 0.05 was considered significant.EdU was used to evaluate cellular proliferation, while DCFH-DA and MitoSOX Red were employed to measure levels of intracellular and mitochondrial ROS, respectively.2.5 concentrations of 32, 64 and 128 µg/ml -1β, IL-6, IL-8/CXCL8 and IL-18 can be stimulated by PM2.5 exposure . Red fluorescence represents normal ΔΨm, while green fluorescence represents decreased ΔΨm and the early stage of apoptosis [2.5 exposure compared with the control cells and were significantly improved by pretreatment with Mdivi-1 or BGP-15 , which is indicative of the basal respiration, maximal respiration, ATP production and spare respiration capacity.2.5 for 48 h before measuring the OCR. PM2.5 significantly inhibited mitochondrial basal respiration , and secondly, the changes of mitophagy-related proteins PINK1, PARK2, SQSTM1/P62 and LC3. We inferred that mitochondria were in a state of excessive mitochondrial autophagy. Inhibition of mitochondrial fission and promotion of mitochondrial fusion can prevent or inhibit excessive mitochondrial autophagy.The protein expression of PINK1 Fig. F and SQS2.5 exposed A549 cells. Pretreatment with Mdivi-1 or BGP-15 inhibited the expression of MLKL . Pretreatment with Mdivi-1 inhibited RL at 64, 128 and 256 mg/mL of ACh  injury was performed by SFTPC immunofluorescence assay.2.5 are shown in Fig. 2.5-instilled mice compared with distilled water-instilled mice  injury. Mdivi-1 or BGP-15 and DRP1-KD or OPA1-OE alleviated the damage and abnormal mitochondrial function in PM2.5 exposed A549 cells by inhibiting the pro-inflammatory response, redox-responses and necroptosis, regulating mitochondrial dynamics and mitophagy. Similar results were found in murine lung tissue, where Mdivi-1or BGP-15 demonstrated protective effects by regulating mitochondrial dynamics, necropotosis, and pro-inflammatory responses.In the present study, we confirmed the damaging effects of PM2.5 have been reported previously [2.5. A recent study in Shanghai suggested that PM2.5 constituents from the combustion of fossil fuel  may have an appreciable influence on the health impact attributable to PM2.5. OC concentrations correlated significantly with adverse health effects, such as cardiopulmonary diseases, which require emergency hospitalization [2.5 collected in Seoul induced neutrophilic inflammation, cellular aging and enhanced macro autophagy in primary lung epithelial cells [2.5 rely on its multiple components and this is similarly likely for its adverse health effects which may vary depending on its chemical characteristics and where it is generated.Biochemical analyses of PMeviously . Organiclization , 25. In al cells . The bio2.5 can induce pro-inflammatory responses and redox-responses in cells and in the lung. For example, PM2.5 can increase the expression of pro-inflammatory cytokines and their receptors [2.5 exposure, and inhibition of mitochondrial fission and promotion of mitochondrial fusion reduced the pro-inflammatory response caused by PM2.5. Short-term exposure of PM2.5 to mice induced inflammatory cell infiltration and lung tissue congestion, increasing the expression of pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β, and oncogenes such as c-Fos and c-Jun [2.5 exposure aggravates AHR and allergic airway responses [2.5-induced AHR in mice represents, to some extent, an enhanced pro-inflammatory response and oxidative stress in mice in the respiratory tract, leading to a narrowing of the airways and the stiffening of the alveoli and lung tissue. Administration of Mdivi-1or BGP-15 significantly inhibited PM2.5-induced lung inflammatory cell infiltration and prevented PM2.5-induced AHR. These results demonstrate that imbalance of mitochondria fission and fusion may be needed for the expression of pro-inflammatory mediators.PMeceptors . Our expnd c-Jun . AHR connd c-Jun , 29. PM2esponses , 31 simi2.5-induced redox-responses, characterized by an altered redox regulation with increased ROS, could lead to disruption of redox homeostasis and sustained cell or tissue damage [2.5-stimulated cells produced more intracellular and mitochondrial ROS, while Mdivi-1, BGP-15, DRP1-KD and OPA1-OE reduced this cellular redox-responses.PMe damage . Regulate damage , 33, 34.e damage . Extramie damage . Alterate damage . In our 2.5 exposure, we inferred that mitochondria were in a state of excessive mitophagy. Inhibition of mitochondrial fission and promotion of mitochondrial fusion can inhibit excessive mitophagy.Mitochondria adapt to homeostatic perturbations induced by cellular stress. This includes changes in biogenesis or remodeling of the mitochondrial network by fission or fusion events and elimination of damaged mitochondria by mitophagy . Mitopha2.5 were accompanied by PINK1-dependent mitophagy. In our study, PARK2 decreased after PM2.5 exposure confirming previous results [2.5-exposed alveolar epithelial cells, mitochondrial dysfunction was associated with mitochondrial fission and fusion as well as mitophagy, and that this process depended on the stability of PINK1. By inhibiting mitochondrial fission and promoting fusion, PINK1-dependent mitophagy was surpressed and mitochondrial respiratory functions such as basal respiration, maximal respiration, ATP production, and spare respiratory capacity were restored.The enhanced mitochondrial fission and diminished mitochondrial fusion by PM results , 40. How results . Here, w2.5 suggest that the total mitochondrial content of a cell is in a dynamic equilibrium between mitochondrial biogenesis and mitochondrial degradation, including mitophagy and other forms of mitochondrial recycling [Changes in mitochondrial dynamics observed in response to PMecycling . In geneecycling , 43. Difecycling .This may2.5 emphasizes the effect on mitochondrial and cellular damage.Fusion can also be beneficial by permitting “functional complementation”: if specific mitochondria carry a high level of damaged components or mutated mitochondrial DNA (mtDNA), the deleterious effects of these dysfunctional components may be compensated for by functional components from other mitochondria . Fission2.5 exposure in A549 cells and mice, increased mRNA expression of pro-inflammatory factors and inhibition of A549 cell proliferation suggested that the necroptosis pathway was activated after PM2.5 exposure. Necroptosis in A549 cells and murine lung was controlled after inhibition of mitochondrial fission and promotion of mitochondrial fusion. Previous studies have also suggested that Beas2B cells undergo mitophagy-dependent necroptosis mediated by PINK1-induced mitophagy, and that Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro by reducing the phosphorylation of MLKL [2.5-induced changes in molecules related to mitochondrial dynamics can regulate the necrosome.The increased expression of necroptosis-related proteins MLKL and RIPK1 observed following PM of MLKL . These d2.5 could be restored by modulating mitochondrial fission and fusion. Pulmonary resistance and compliance are used as a measure of pulmonary edema and an indication of AECII impairment. Our results suggested that BGP-15 reduced the AECII apoptosis under PM2.5 exposure. PM2.5-induced apoptosis of AECII was accompanied by increased expression of MLKL, RIPK1 and RIPK3, so we assume that necropotosis plays an important role in AECII apoptosis, which can be controlled by modulation of mitochondrial fusion.AECII in lung tissues play an important role in timely repair of damaged cells to prevent invasion by external pathogens, while SFTPC, surfactant-associated protein A (SFTPA) and surfactant-associated protein D (SFTPD) proteins can improve lung compliance and play an immunomodulatory role , 48. The2.5[2.5 effects were due to the presence of endotoxin in our experiments, the low levels presence (0.68EU/ml) suggest that this is unlikely. Therefore, our experimental results may only be applicable to the PM2.5 exposure of A549 cells under submerged conditions at a single time point.There are several limitations in the present research. First, as our results show, there are some differences between the effects of drugs versus KD/OE in cells and in vivo. We consider that it may be due to the existence of other compensation mechanisms. Second, due to the limitations of experimental conditions, we used A549 cells as a model of primary AECII in this study. Third, to better stimulate the physiological state of lung cells in vivo, 3D air-liquid interface (ALI) culture can be applied in later studies. Fourth, increased cell permeability and impa2.5, 53, whi2.5-induced cells and lung injury. Inhibition of mitochondrial fission and promotion of mitochondrial fusion can restore PM2.5-induced cellular and lung damage by suppressing pro-inflammatory responses and ROS responses, regulating the expression of mitophagy-related proteins , and decreasing the expression of necroptosis-related proteins . Future studies should examine these effects in primary cells at various time points after PM2.5 exposure.In summary, we have demonstrated that an imbalance of mitochondrial fission and fusion plays an important role in PMBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


The high calcium (Ca) and phosphorus (P) diet was modified by increasing both high calcium and phosphorus by 30% (2:1) for the first 12 wks and then only increasing P for 12–17 wks to reduce the Ca to P ratio. At 17 wk, growth performance was measured, whole body composition was measured by dual energy x-ray absorptiometry (DEXA), and femur bones were scanned using Micro-computed tomography (Micro-CT) for bone 3D structure analyses. The data were subjected to a one-way ANOVA using the GLM procedure, with means deemed significant at p < 0.05. There was no significant outcome for growth performance or dual energy x-ray absorptiometry parameters. Micro-computed tomography results indicated that the H25D + Ca/P treatment had lower open pore volume space, open porosity, total volume of pore space, and total porosity in the cortical bone compared to the D + Ca/P. It also showed that a higher cortical bone volume/tissue volume (BV/TV) in the H25D + Ca/P than in the D + Ca/P. Furthermore, the H25D + Ca/P treatment had the lowest trabecular pattern factor and structure model index compared to the other treatments, which indicates its beneficial effects on trabecular structural development. Moreover, the H25D + Ca/P had a higher trabecular percentage compared to the D and 25D, which suggests the additional high calcium and phosphorus supplementation on top of 25D increased trabecular content in the cavity. In conclusion, the combination of 25D with higher levels of high calcium and phosphorus could improve cortical bone quality in pullets and showed a beneficial effect on trabecular bone 3D structural development. Thus, combination of a higher bio-active form of vitamin D3 and higher levels of high calcium and phosphorus could become a potential feeding strategy to improve bone structural integrity and health in pullets.Bone issues such as osteoporosis are major concerns for the laying hen industry. A study was conducted to improve bone-health in pullets. A total of 448 one-day-old Hyline W36 pullets were randomly assigned to four treatments  until 17 weeks (wks). Dietary treatments were: 1) vitamin D The ever-increasing improvement in laying hen nutrition and genetics has resulted in vastly more efficient hens capable of laying more than 340 eggs by 72 weeks of age . With adOsteoporosis is described as a progressive decrease in the amount of mineralized structural bone and leads to bone fragility and susceptibility to fracture . These aNumerous pullet studies have addressed the importance of early bone development, and its prolonged effects on bone health during laying periods . However3 (H25D) is an intermediate form of vitamin D3 and is now readily available for commercial use in the poultry industry since 2006  and available phosphorous (P) level and ratio in the diets which were not ideal to provide sufficient Ca and P to induce proper mineral deposition in increased bone size by H25D Dg period , maintai by H25D . Thus, iad libitum from 0 to 17 wks. There were two nipples per cage. The pullets received an intermittent lighting program during the first 7 days with 4 h of light followed by 2 h of dark circles. The lighting management was customized by Hy-line North America lighting program throughout 2–17 wks .The study was conducted at the research facility of the Department of Poultry Science at the University of Georgia. The trial was conducted in accordance with the Institutional Animal Care and Use Committee at the University of Georgia. A total of 448 one-day-old Hyline W36 chicks were randomly assigned to 4 dietary treatments (8 replicates and14 birds/replicate) and raised until 17 wks. Chicks were obtained from at Hy-line North America hatchery . Birds were housed in colony cages for 17 wks [(90 cm (L) × 46 cm (W) × 38 cm (H)]. Water and experimental diets were offered 3 at IU/kg (D), 2) vitamin D3  + 62.5 mg 25-(OH)D3/ton (H25D), 3) vitamin D3  + 62.5 mg 25-(OH)D3/ton + high Ca and P (H25D + Ca/P), and 4) vitamin D3  + high Ca and P (D + Ca/P). The high Ca and P diet was modified by increasing 30% of both Ca and P (2:1) for the first 12 wks, and then only increasing P for 12–17 wks to reduce the Ca to P ratio (3 or 25-(OH)D3 in the diets.The diets were designed based on the Hy-Line W36 guide (2020). The formulation of corn-soybean based diet is shown in  P ratio . The exp3 level was determined using a mass spectrometry procedure .Blood samples (8 birds/treatment) were collected from the wing vein at 17 wks. After the blood was clotted, the blood samples were centrifuged at 1,500 g in a refrigerated centrifuge  for 12 min. The serum was collected and transferred into a clean polypropylene tube. The samples were maintained at −80°C until analysis. The serum 25-(OH)DBody weight (BW), body weight gain (BWG), and feed intake (FI) were recorded at 0, 3, 6, 12, 15, and 17 wks. The phase periods for recording are 0–3 wks (Starter 1 period), 4–6 wks (Starter 2 period), 7–12 wks (Grower period), 13–15 wks (Developer period), and 16–17 wks (Peak period). However, growth performance was reported for 0–17 wks.®, Bone Densitometer, General Electric Company, 41 Farnsworth Street, Boston, MA 02210, United States) was used for whole bird body composition analyses, and the whole bird was defined as a region of interest (2) (BMD), bone mineral content (g) (BMC), bone area (cm2), fat weight (kg), fat percent (%), muscle weight (kg), muscle percentage (%), and total body weight (kg). Each sample bird was placed chest up on the scanner at the same position and orientation during the measurement. All scans were obtained at a scan speed of 2.5 mm/s, with a voxel resolution of 0.07 mm × 0.07 mm × 0.50 mm.At 17 wks, two birds per cage (2 birds × 8 replicates = 16 birds per treatment) were euthanized by cervical dislocation. Dual energy x-ray absorptiometry  at 17 wks of age. After the soft tissue was removed completely, the samples were wrapped with PBS-soaked cheesecloth to keep the bones moist. The femur bones were scanned with micro-computed tomography  which was used for 3- dimensional image acquisition . The femYij = µ +αi +εij. Significant differences among the treatments were determined using GLM procedures. Data were considered significantly different at p ≤ 0.05, and trends (0.05 ≤ p ≤ 0.1) were also presented  of SAS . One-way ANOVA was used with model resented .p > 0.05)  along with higher Ca and P levels did not have an impact on growth performance parameters ( > 0.05)  or whole > 0.05) .3 with higher Ca and P levels (H25D + Ca/P) had significantly lower open pore volume space, open porosity , total volume of pore space, and total porosity  in the cortical bone compared to vitamin D3 with higher Ca and P levels (D + Ca/P) (p < 0.05)  compared to the D + Ca/P . 25-(OH) < 0.05) .p = 0.009) and structure model index (SMI) (p = 0.031) among the treatments (p < 0.05).The H25D + Ca/P treatment had the lowest trabecular pattern factor (TPF) D3 level in the serum compared to the other treatments (p < 0.0001) (The 25-(OH)D 0.0001) .3 , which is later converted in the kidney and other tissues including bone by 1α-hydroxylase to generate the active form 1,25-hydroxyvitamin D3 2D3, calcitriol) , inducing a broad range of biological responses  . 1,25 and phosphorus (30% throughout) provided in the diet. This was further observed by the trending volume of closed pores and closed porosity for the H25D + Ca/P treatment, which complements the findings with the lower open pore space. Therefore, the nutritional strategy with H25D + Ca/P during rearing periods have potential to create a more compact and structurally sound bone.Inside the cortical cortex, the coupling between osteoblast and osteoclast activities is carried out in the matrix of cortical pores which provide a conduit for the movement of neurovascular structures . CorticaIn the present study, higher content of trabecular bone in the H25D treatment was found during the rearing period. The trabecular bone corresponds to 20%–30% of the bone mass, which is in the internal section of the bone . It has 3 and higher levels of Ca and P during rearing periods may become a promising feeding strategy to enhance pullet bone quality.This study suggests that early supplementation of the combination of H25D with higher level of Ca and P could improve the cortical bone microstructure and have beneficial effects on trabecular bone 3D structural development in pullets. The combination of a higher bio-active forms of vitamin D


Unexpectedly, it migrated the negative effect of SO2 on mercury removal performance, while also enhancing Hg0 adsorption. Colloidal copper sulfides demonstrated the superior Hg0 adsorption rate of 306.9 μg·g−1·min−1 under 6% SO2 + 6% O2 atmosphere with a removal efficiency of 99.1%, and the highest-ever Hg0 adsorption capacity of 736.5 mg·g−1, which was 277% higher than all other reported metal sulfides. The Cu and S sites transformation results reveal that SO2 could transform the tri-coordinate S sites into S22− on copper sulfides surfaces, while O2 regenerated Cu2+ via the oxidation of Cu+. The S22− and Cu2+ sites enhanced Hg0 oxidation, and the Hg2+ could strongly bind with tri-coordinate S sites. This study provides an effective strategy to achieve large-capability adsorption of Hg0 from non-ferrous smelting flue gas.This paper reports on a novel method to remove Hg Therefo0), oxidized mercury (Hg2+), and particulate mercury (Hgp) [p would be captured by the electrostatic precipitator (ESP) [2+ has strong water solubility and can be removed by wet scrubber, whereas the capture of Hg0 is difficult due to its strong stability, high volatility, and low solubility [0 removal promote the conversion of Hg0 to Hg2+, such as the Boliden–Norzink and advanced oxidation process, thereby improving mercury removal efficiency [0 removal in the wet scrubbing of non-ferrous smelting flue gas [There are various forms of mercury in non-ferrous smelting flue gas, including elemental mercury (Hgry (Hgp) ,5,6. In or (ESP) ,8,9. Hg2lubility . Wet metficiency ,12. Hencflue gas .2 (>6% vol) in the flue gas, which is thousands of times higher than Hg0 concentration [0 poses a great challenge since the standard oxidation potential of Hg0 (0.85 V) is higher than that of SO2 (0.17 V) [2 is preferentially oxidized over Hg0, bringing about the depletion of oxidants and making it difficult to oxidize Hg0. Therefore, the efficient oxidation of Hg0 from high-SO2 flue gas is key to overcoming the bottleneck of Hg0 removal in wet scrubbing.Oxidative demercurization remains a major bottleneck in the application of wet scrubbing from non-ferrous smelting flue gas ,15. Therntration ,17. Sele(0.17 V) ,19. Cons0 capture due to the strong affinity of sulfur sites with Hg0 [2 could react with defective S2− on TMSs to generate highly active Sn2−, which might promote Hg0 oxidation [2 dissolves into aqueous phase and forms dissolved oxygen, which has a high oxidation activity due to hydrogen bonding and Van Der Waals force with water molecules, providing an oxidative environment for TMSs to capture Hg0 [2 on the removal of Hg0, which could enhance the ability of TMSs to oxidize mercury, thereby greatly improving the adsorption capacity of mercury. However, the mechanism of SO2 and O2 in wet scrubbing for mercury capture by TMSs has not been reported.Transition metal sulfides (TMSs) have become a hot material for Hgwith Hg0 ,21,22. Pxidation . O2 dissture Hg0 . Therefo0 from high-SO2 flue gas. The optimal mercury adsorbent was prepared by sulfide precipitation and optimized systematically. The morphology and structure of the adsorbents were characterized. The excellent Hg0 removal performance of colloidal copper sulfides (c-CuS) was examined in the flue gas wet scrubber. The effects of SO2 and O2 on Hg0 removal by c-CuS were investigated, and the mechanism of the activation of oxidizing sites (S22− and Cu2+) by SO2 and O2 was proposed after identifying the structural changes of c-CuS under different atmospheres.Based on the above considerations, this paper involves a novel method of using metal sulfides in the wet scrubbing process for large-capability adsorption of Hg2), copper nitrate (CuNO3·6H2O), Copper sulfate (CuSO4·5H2O), sodium sulfide (NaS·9H2O), sodium hydroxide (NaOH), sodium chloride (NaCl), and nitric acid (HNO3). They were purchased from Sinopharm Chemical Reagent Co., Ltd.  All the agents were directly used without any further purification. Ultrapure water was used in all experiments unless otherwise stated.The chemicals in analytical grade were used in this study, including copper chloride  fluorescence spectroscopy. 3D-EEM spectra were generated by scanning in the range of 200 nm to 600 nm, while excitation and emission sampling wavelengths were 10 nm, and the scanning speed was 12,000 nm·min−1. Finally, temperature-programmed desorption (TPD) was employed to identify the form of mercury species adsorbed on c-CuS. The TPD tests were carried out under pure nitrogen at the flow rate of 500 mL·min–1 from 50 °C to 600 °C at a rate of 5 °C·min−1.These suspensions and colloids were filtered with nanopore filter membranes. The filtered residues were washed three times with ultrapure water and then dehydrated at −89 °C in a vacuum freeze-dryer for 8 h. The obtained filtered residues of TMSs suspensions and c-CuS were analyzed for crystalline structure by X-ray diffraction  with Cu-Kα radiation. High-resolution transmission electron microscopy  was used to observe the morphology of c-CuS particles. The scanning electron microscope  of c-CuS was characterized at the accelerating voltage of 200 kV after splashing a thin Au layer. The structure information of samples under different atmospheres was characterized by X-ray photoelectron spectroscopy . The ultimate vacuum degree of the sample analysis room was 5 × 100. Pure N2 was used as the carrier gas for the Hg0 generator (VICI Metronics) with a flow rate of 200 mL·min−1. The simulated flue gas (SFG) containing N2, SO2, and O2 at a flow rate of 300 mL·min−1 was mixed with the Hg0 carrier gas. The mixture gas was then blown into a bubbling reactor containing the scrubbing solution. Then, the SFG was treated with 5 mol·L−1 NaOH and quartz wool before the mercury detection to reduce the influence of SO2 and water vapor. The mercury analyzer  was used to monitor the outlet Hg0 concentration. All data were obtained by averaging three measurement results. Finally, the remaining Hg0 in the tail gas was absorbed by KMnO4 solution and activated carbon. The operating conditions in the experiment are listed in 0 removal efficiency and adsorption capacity were calculated according to Equations (1) and (2), respectively.0 removal efficiency, and inC and outC (μg·m−3) are the Hg0 concentration at the inlet and outlet of the experimental system, respectively. −1) is the adsorption capacity of Hg0, M (mg) is the mass of adsorbent, t1 and t2 (min) are the start and end times, Δt (min) is the scrubbing time, and u is the gas flow rate (m3·min−1).An experiment was conducted in a bubbling reactor to remove HgadE) was calculated using Equation (3), based on the optimized structure.HEg is the state energy of a free Hg atom, CuSE represents the total energy of the CuS of different configurations, and (Hg+CuS)E is the total energy of the Hg atoms being adsorbed on CuS.Perdew–Burke-Ernzerhof (PBE0) functional was used to characterize the exchange-correlation effects in the density functional theory (DFT) by CP2K ,26. The 0 removal performance was evaluated by adding TMSs suspensions into a bubble reactor as the scrubbing solution. 0 in wet scrubbing. Especially, copper sulfides suspension was the preferred Hg0 removal scrubbing agent, with the highest efficiency of 87.6%. Furthermore, the effect of regulating feeding methods on the dispersibility of detergent was investigated to improve the Hg0 removal performance of copper sulfides. 0 capture performance.Hg0 removal. As shown in −1, the efficiency of Hg0 removal was only 65% due to the insufficient content of active components in the solution. Increasing the concentration of CuCl2 and Na2S fourfold to synthesize c-CuS could immediately improve the removal efficiency of Hg0 to 93%. When the concentrations were optimized to 1/2 mmol·L−1 and 2 mmol·L−1, the efficiencies were higher than 96.3%. Unfortunately, a large amount of precipitation appeared in the solution of 2 mmol·L−1 concentration, which was not conducive to sufficient contact with Hg0. Finally, the Cu/S ratio of copper sulfides was optimized, as shown in 0, which was achieved by reaching 98.1% when the Cu:S ratio was 1:1. However, as shown in 2− ratio caused possible instability of colloid, which would have a negative effect on the Hg0 removal. In summary, the c-CuS was prepared with the concentration of 1/2 mmol·L−1 and the Cu:S raw material ratio of 1:1, which was the optimal mercury capture agent.Optimization of the raw material concentration and ratio to synthesize copper sulfides is crucial for enhancing Hg4(S2)2(CuS)2 (JCPDS card NO. 74-1234), with overlapping diffraction peaks that could be divided into lattice planes such as (101), (102), (103), (110), and (116) [0 [Ic is the crystal diffraction intensity and Ia is the amorphous scattering intensity.nd (116) . The dif(116) [0 ,30.(4)X2 is typically considered to be a negative effects gas that inhibits Hg0 removal in industrial flue gas [2 resistance has a crucial impact on the mercury capture performance of adsorbents. SO2-contained simulated flue gas was inputted into a bubbling reactor with 10 mL of c-CuS to investigate the effect of SO2 on the Hg0 removal performance of c-CuS. As shown in 2. The efficiency significantly increased to 95.1 ~ 97.4% under 3 ~ 9% vol SO2. Furthermore, the performance of c-CuS for Hg0 removal was compared with and without SO2 pretreatment, as shown in 0 was about 10 μg·m−3 after passing through c-CuS solution pretreated without SO2, but it decreased immediately to ~0 μg·m−3 after turning on SO2. When c-CuS solution was pretreated with SO2, the outlet Hg0 concentration was about 3 μg·m−3 after opening gas pipeline of Hg0. Meanwhile, the outlet concentration exhibited almost no fluctuation after supplying SO2 and Hg0 simultaneously. This might mean that SO2 did not occupy the adsorption sites, and SO2 could interact with c-CuS to enhance adsorption with Hg0. The 3D-EEM was used to compare the fluorescence fingerprint spectra of c-CuS under N2 + 6% vol SO2 atmospheres. As shown in 2 was smaller than the region I area of c-CuS under N2 atmosphere, confirming that SO2 leads to the formation of more vacancies. The formation of vacancies meant that low-active S2− had been converted to more active S22− [SOflue gas ,32. SO2 ive S22− ,34. The 2. This was attributed to the fact that c-CuS solution could dissolve only 0.076 mmol·L−1 oxygen, which was much higher than the amount of mercury and much lower than the O2 proportion in the flue gas. Therefore, we conducted further investigations to explore the influence of dissolved oxygen on Hg0 oxidation. As displayed in −1 to 7.6 mg∙L−1. The redox potential of the c-CuS solution gradually increased from 166 mV to 222 mV, which meant enhancing the oxidation ability of the scrubbing solution. It led to a significant increase in the removal efficiency of mercury from 45.0% to 95.3%. As shown in 2+ of binding energy at 568.1 eV and Cu+ at 565.8 eV [+ spectral peak of c-CuS in oxygen-free water was visible, while it was weakened for c-CuS in oxic water, conversely indicating the oxidation of Cu+ to Cu2+ by O2. It could be inferred that dissolved oxygen in an aqueous solution could increase the Cu2+ content of c-CuS and strengthen the oxidation of Hg0.In 565.8 eV . The Cu+0 adsorption capacity and rate of c-CuS were investigated and calculated by penetration experiments. The prepared 10 mL c-CuS (containing 0.48 mg CuS particles) was input with 6% vol SO2 + 6% vol O2 flue gas containing an inlet Hg0 concentration of 890.0 µg·m−3. As shown in 0 gradually increased from 303.2 µg·m−3 to 835.5 µg·m−3 after 2400 min of scrubbing experiment. The Hg0 adsorption capacity of c-CuS was calculated to be 736.5 mg·g−1, and the average adsorption rate was 306.9 μg·g−1·min−1. 0 adsorption capacity of c-CuS is exceptionally high, exceeding them by an impressive 277%, such as nano-CuS, Co3S4, S/FeS2, ZnS, and so on. Additionally, the Hg0 adsorption rate of c-CuS is much higher than that of other mineral sulfides, which is due to the abundant adsorption sites under the positive effect of SO2 and O2 [The Hg2 and O2 ,38,39,402 and O2 have positive effects on c-CuS for Hg0 capture. To determine the mechanism of Hg0 adsorption under SO2 and O2 atmospheres, as shown in 2, 6% vol SO2, 6% vol SO2 + 6% vol O2, and 6% vol SO2 + 6% vol O2 + Hg0, respectively. The forms of S on the c-CuS surface are multiple, such as tri-coordinated S2−(CN=3), tetra-coordinated S2−(CN=4), and sulfur–sulfur-coordinated S22− [2−(CN=3), while the peaks at 161.9 eV and 163.0 eV were attributed to S2−(CN=4). The peaks at 163.2 eV and 164.4 eV were attributed to S22−, while the peaks at 167.9 eV and 168.9 eV belonged to SO42− [As mentioned above, SOted S22− . After pto SO42− ,38,41.2 atmosphere, the ratio of S and Cu total amount  on c-CuS was found to be 1.01:1, with S 2p consisting of 26.8% S2−(CN=3), 39.0% S2−(CN=4), 33.5% S22−, and 0.7% SO42−. Upon treatment with 6% vol SO2, the St:Cut ratio increased to 1.79:1, with a decrease in S2−(CN=3) content to 10.4%, an increase in S22− to 49.5%, and a slight change in other forms of S, indicating the formation of new S22− by the combination of S2−(CN=3) with SO2. Additionally, the Cu 2p peak in 2+ to Cu+ by SO2. Similarly, under 6% vol SO2 + 6% vol O2 conditions, St:Cut decreased with the oxidation of Cu+ into Cu2+ by O2 and inhibition of S2−(CN=4) combination with SO2 adsorbed on the c-CuS surface. The S2−(CN=3) content decreased with an increase in S2−(CN=4), which led to less S22−. The further supply of Hg0 resulted in a decrease in the ratio of S22− and S2−(CN=4), but an increase in S2−(CN=3), indicating the transformation of S22− and S2−(CN=4) into S2−(CN=3) after capture of Hg0. The results of Cu 2p shifting towards higher binding energy confirmed that Cu2+ was also an active site for Hg0 oxidation. It suggests that S22− and Cu2+ serve as oxidation sites, while S2−(CN=3) is the binding site with Hg after adsorption. The TPD results in Under Nad at each site on the c-CuS (110) crystal was analyzed at the molecular level using DFT calculations. The result in −1. It means that the tri-coordinate sulfur site might be the main binding site for mercury, which is consistent with XPS results.The experimental results were inconclusive about the main binding site of the adsorbed mercury. Therefore, the E0 capture under SO2 and O2 atmospheres could be described by Equations (5) ~ (10). After the adsorbing of Hg0 on the c-CuS surface, Cu2+ and S22− sites oxidized Hg0ad to Hg2+ and binded with S2−(CN=3) to form HgS. As described in Equations (9) and (10), SO2 and O2 respectively activate S2−(CN=3) and Cu+ transformed into S22− and Cu2+ as Hg0 oxidation sites.In summary, the mechanism for Hg−1 were synthesized by a double-jet liquid-phase sulfidation precipitation method. Defect-rich c-CuS with low crystallinity was used as a scrubbing agent to remove mercury from flue gas in wet scrubbing. It migrated the negative effect of SO2 on mercury removal performance, while also enhancing mercury capture. The Hg0 removal efficiency of c-CuS was 99.1% under 6% vol SO2 + 6% vol O2 atmosphere. The Hg0 adsorption capacity of c-CuS reached 736.5 mg·g−1, and the average adsorption rate was 306.9 μg·g−1·min−1, which was far better than other reported metal sulfides adsorbents. Based on structural characterization and DFT calculation, it was found that SO2 and O2 can enhance the formation of Cu2+ and S22− sites from Cu+ and S2−(CN=3) to promote the oxidation of Hg0 to Hg2+, and then Hg2+ could strongly bind with S2−(CN=3). However, reusability of the material should be developed in future studies.In this paper, the optimal conditions for preparing c-CuS with a Cu–S ratio of 1:1 and a concentration of 0.5 mmol·L


Endocrine therapies such as, selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs), and selective estrogen receptor downregulators (SERDs) are used to treat ER+ breast cancer. However, despite their effectiveness, severe side-effects and resistance are associated with these endocrine therapies. Thus, it would be highly beneficial to develop breast cancer drugs that are as effective as current therapies, but less toxic with fewer side effects, and less likely to induce resistance. Extracts of Cyclopia species, an indigenous South African fynbos plant, have been shown to possess phenolic compounds that exhibit phytoestrogenic and chemopreventive activities against breast cancer development and progression. In the current study, three well characterized Cyclopia extracts, SM6Met, cup of tea (CoT) and P104, were examined for their abilities to modulate the levels of the estrogen receptor subtypes, estrogen receptor alpha and estrogen receptor beta (ERβ), which have been recognized as crucial to breast cancer prognosis and treatment. We showed that the Cyclopia subternata Vogel (C. subternata Vogel) extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, reduced estrogen receptor alpha protein levels while elevating estrogen receptor beta protein levels, thereby reducing the ERα:ERβ ratio in a similar manner as standard of care breast cancer endocrine therapies such as fulvestrant (selective estrogen receptor downregulator) and 4-hydroxytamoxifen (elective estrogen receptor modulator). Estrogen receptor alpha expression enhances the proliferation of breast cancer cells while estrogen receptor beta inhibits the proliferative activities of estrogen receptor alpha. We also showed that in terms of the molecular mechanisms involved all the Cyclopia extracts regulated estrogen receptor alpha and estrogen receptor beta protein levels through both transcriptional and translational, and proteasomal degradation mechanisms. Therefore, from our findings, we proffer that the C. subternata Vogel extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, selectively modulate estrogen receptor subtypes levels in a manner that generally supports inhibition of breast cancer proliferation, thereby demonstrating attributes that could be explored as potential therapeutic agents for breast cancer.Breast cancer is the most diagnosed type of cancer amongst women in economically developing countries and globally. Most breast cancers express estrogen receptor alpha (ERα) and are categorized as positive (ER The bur+ BC . The mos via AIs .via endocrine and paracrine mechanisms , Cyclopia species (honeybush tea) and Athrixia phylicoides (bush tea) are considered medicinal herbal teas , and the standard of care endocrine therapies (SOCs), (2)-4-hydroxytamoxifen (4-OHT) as a SERM control , and P104 were obtained from cultivated and commercially harvested plant material and were previously prepared and characterized  and stored at −20°C until use see . Stock s5 cells/well into 12 well plates and steroid starved in steroid starving media (phenol red-free DMEM (low glucose) supplemented with 1.5 g/L sodium bicarbonate, 3.5 g/L glucose, 1% penicillin-streptomycin and 5% heat-inactivated doubly dextran-coated charcoal-stripped FCS (2xDCCFCS) for MCF7 cells and 10% 2xDCCFCS for T47D cells for 24 h. MCF7 and T47D cells were then washed in pre-warmed phosphate-buffered saline (PBS) and treated a with increasing concentrations of the test panel for 24 h in the treatment medium.MCF7 and T47D cells were plated at 1.0 × 10Following treatment, the treated MCF7 and T47D cells were washed in 1 mL ice-cold PBS and lysed in 100 μL Radioimmunoprecipitation assay (RIPA) buffer . The lysates were transferred into Eppendorf tubes and 5x SDS reducing buffer  was added to enhance cell lysis. Thereafter the cell lysates were boiled at 95°C for 20 min.To separate the proteins, the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique was employed, where 10 μL of the lysate was loaded onto a 15 well 10% acrylamide gel containing 0.9% (v/v), 2,2,2- trichloroethanol (TCE), both procured from Sigma-Aldrich. A protein molecular weight marker  was loaded alongside the lysates to verify the sizes of ERα and ERβ protein. The gel was set to run at 75 V for 15 min and at 150 V for 60 min.The separated proteins on the acrylamide gel were imaged under UV light and the image acquired using the BioRad molecular imager, Gel DocTM XR+ with Image LabTM software. The acquired image of the total protein content was utilized for normalization (as detailed below). The proteins were then transferred to a Hybond-ECL nitrocellulose membrane (Separation Scientific) under 0.18 A electric current for 2 h. To ensure a successful protein transfer, the nitrocellulose membrane and the gels were imaged and acquired under UV light with the BioRad molecular imager, Gel DocTM XR+ with Image LabTM software after the transfer see . The gel50). To quantify the intensity of the ERα and ERβ protein bands, MyImage Analysis software was used. Equal protein loading was ensured by normalization to the total protein content of each lane. The total protein content was obtainable by adding a sedative agent, TCE , to the To study the effects of proteasomal and translational inhibition on the modulation of ERα and ERβ protein levels by the test panel the proteasomal inhibitor, MG132  and the # and $), as specified in the figure legends. Non-significant results are denoted by “ns.” For all Figures Average ± SD is of three independent biological experiments analyzed as such.Statistical analysis was carried out using GraphPad Prism software version 5. Details of the individual statistical analysis used, including post-tests are described in the figure legends. Statistical difference is expressed as either a different letter or using symbols  different  lower than the upregulation of ERβ protein levels by SM6Met (p > 0.05) different.ERβ protein levels were upregulated by the two y SM6Met . In contBoth SM6Met and CoT downregulated ERα protein levels while simultaneously increasing ERβ protein levels in both cell lines, resulting in a decreased ERα:ERβ ratio . However−9 mg/mL) than in T47D (1.2 × 10−12 mg/mL) cells as was the potency of SM6Met to increase ERβ proteins levels (1.7 × 10−12 mg/mL in MCF7 compared to 4.4 × 10−13 mg/mL in T47D cells). Similarly, the potency of CoT in decreasing ERα protein levels was lower in MCF7 (2.5 × 10−9 mg/mL) than in T47D (1.1 × 10−11 mg/mL) cells. However, in contrast, the potency of CoT in upregulating ERβ protein levels in MCF7 (1.0 × 10−13 mg/mL) was slightly higher than in T47D (9.9 × 10−13 mg/mL) cells. Additionally, the potency of P104 in decreasing ERα protein levels was significantly (p < 0.05) lower in MCF7 (4.6 × 10−7 mg/mL) than in T47D (7.1 × 10−15 mg/mL) cells, while the potency of P104 in downregulating ERβ protein levels in MCF7 (1.3 × 10−10 mg/mL) and in T47D (4.7 × 10−10 mg/mL) cells were similar. The potencies of the Cyclopia extracts in modulating either ERα or ERβ protein levels were not statistically different across the two cell lines, except for the potency of P104 in downregulating ERα protein levels in T47D cells.The potency  of SM6MeCyclopia extracts with those elicited by the ER subtype specific ligands , which includes the SOCs and the Cyclopia extracts, except for P104 in T47D cells, and those that had a major effect on the ratio (ERα:ERβ ratio below 0.2) such as liquiritigenin only in MCF7 cells. Overall comparison of the effects of the Cyclopia extracts with that of the SOCs indicates that the C. subternata Vogel extract, SM6Met, was slightly less effective than fulvestrant, but as effective as 4-OHT, in reducing the ERα:ERβ ratio, however with a markedly higher potency in increasing ERβ protein levels, while CoT was slightly less effective than both the SOCs and P104 was the least effective Cyclopia extract at reducing the ERα:ERβ ratio.In comparing the relative effects of the full test panel in modulating the ERα:ERβ ratio , we can Cyclopia extracts in MCF7 and T47D BC cells, translation was inhibited using a protein synthesis inhibitor, cycloheximide (CHX)  , while dr, MG132 .p < 0.01) so only at the higher concentration of SM6Met where the increase in protein levels was between 1.3 and 1.4-fold. Similarly, CHX also reversed the downregulation of ERα by P104 to basal levels in both cell lines (p < 0.05) was only observed in the T47D cells where the increase in protein levels was between 1.3 and 1.5-fold. In contrast, the addition of CHX had little effect on the downregulation of ERα by CoT (p < 0.05).SM6Met and CoT downregulate ERα and upregulate ERβ protein levels in MCF7 and T47D cells, while P104 downregulates the protein levels of both ER subtypes , 5. Addill lines , 5H, howα by CoT , 5G and α by CoT  and CoT α by CoT  in T47D p < 0.05), increase in the protein levels of ERα and ERβ upon the addition of CHX to MPP compared to MPP alone  increased the protein levels of ERα and ERβ modulated by fulvestrant in T47D cells (p < 0.0001) reversed (1.7 to 1.8-fold) by the addition of CHX.Similarly, the addition of the translational inhibitor, CHX, had no significant effect on the downregulation of either ERα or ERβ protein levels by E2 in either cell line , 5A. Lik7D cells  by 1.3 t7D cells  was signCyclopia extracts, SM6Met, CoT and P104, on ERα protein levels while enhancing the stabilization of ERβ protein levels by SM6Met and CoT  reversed (1.1 to 1.5-fold) by proteasomal inhibition in MCF7 cells  by the addition of MG132, except in the case of 4-OHT in MCF7 cells. Interestingly, the highest fold-change in ER levels due to the addition of MG132 (1.4 to 1.7-fold) was observed with 4-OHT in T47D cells, where addition of MG132 stabilized ERα protein levels to above that of basal levels. Upregulation of ERβ protein levels by the SOCs, fulvestrant (p < 0.05).Proteasomal inhibition with MG132 largely counteracts the effects of E2 , 7A in dvestrant , 7B and vestrant , 7C, was+ BC include SERDs such as fulvestrant that target and reduce the expression of ERα -induced rat mammary gland carcinogenesis model  is similar to the 86.0% shown by Cyclopia extracts upregulates ERβ protein levels in MCF7 cells. The efficacy of the upregulation of ERβ protein levels in MCF7 cells by SM6Met is slightly higher at 145.4% in the current study than the 130.8% shown by Visser, as is the efficacy of CoT at 124.5% in the current study compared to the 110.9% shown by Visser. In contrast to that seen by Regarding the Cyclopia extracts revealed in the current study with that of other botanicals or plant extracts suggest some similarities. For example, the citrus plant-derived flavanone naringenin had been shown to have little effect on ERα , and is 7.9 to 12.1-fold higher than the levels in the C. subternata Vogel extracts, SM6Met and CoT, respectively, and although no work has been done on its effect on ERα or ERβ protein levels it has been shown to inhibit MCF7 cell proliferation and to suppress tumor growth in a mouse breast cancer mouse xenograft model using MDA-MB-231 cells , but is 1.9 to 3.6-fold lower than the levels in the C. subternata Vogel extract, SM6Met and the C. genistoides extract, P104, respectively, have been shown to activate transcription via ERα but not via ERβ (Mangiferin , which i via ERβ , to inhi via ERβ  and to i via ERβ . It is tvia ERβ but not via ERα in SK-N-BE neuroblastoma cells (Cyclopia extracts and is thus unlikely to alone be responsible for the effects of the Cyclopia extracts. In fact, we have previously shown that activity-guided fractionation does not retain all the desirable estrogenic attributes of the original SM6Met in one fraction (Furthermore, luteolin and protocatechuic acid downregulate DHT-induced ERα protein expression and upregulate DHT-suppressed ERβ protein expression in a human prostatic epithelial cell line, BPH-1 , while lma cells , but notma cells , inhibitma cells , binds pma cells  and dispma cells  but inhima cells . Luteolifraction  and thusfraction . Thus, pCyclopia extracts, CoT and P104, appear to equally favor proteasomal, and transcriptional and translational mechanisms, while fulvestrant and SM6Met generally favor proteasomal degradation.Taken together, our findings show that the molecular mechanism involved in the regulation of ERα and ERβ protein levels may be organized into several types. Those primarily regulated through proteasomal degradation such as E2 and liquiritigenin and those such as MPP primarily regulated through transcriptional and translational mechanisms. Other types involve a mixture of mechanisms, either equally or preferentially leaning towards one of the mechanisms. Specifically, 4-OHT and the via proteasomal degradation by E2 and fulvestrant in MCF7 cells agrees with the findings of via proteasomal degradation by E2 agrees with the findings of via both protein synthesis and proteasomal degradation. Additionally, although not in BC cells, via protein synthesis nor transcription in lactotrope cells, PR1. Our report is the first on the molecular mechanism of regulation of ER subtypes by the Cyclopia extracts and to our knowledge also by the ER subtype selective ligands, liquiritigenin and MPP.The regulation of ERα protein levels Cyclopia extracts has in contrast explicitly evaluated selectively in terms of ER subtype levels. In conclusion, the current study indicates that the C. subternata Vogel extracts, SM6Met and CoT, rather than the C. genistoides extract, P104, display favorable attributes by degrading ERα while stabilizing ERβ. Coupled to the proven oral bioavailability of the C. subternata Vogel extracts (C. subternata Vogel extracts may be of therapeutic benefit for BC prevention and treatment and provide the underpinning for the development of an ER-targeted phytopharmaceutical product from Cyclopia.Despite many studies investigating the selective degradation of the ER for BC treatment , these hextracts  the curr