Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro Early studies have shown that heat shock protein 90 (Hsp90) is enriched in dynamic complexes with co-chaperones in tumor tissues compared to normal tissue5. Consistent with this, recent findings revealed the existence of a more complex molecular rewiring of the Hsp70 and Hsp90 chaperone networks to form a highly integrated complex, referred to as the epichaperome, in cancer cells. This pathologic protein–protein interaction network has been shown to promote cancer cell survival and aggressiveness of certain tumors6. The epichaperome may also be amenable to drug targeting7.The chaperome is composed of chaperone and co-chaperone proteins that safeguard cellular proteostasis and thus plays a key role in determining cancer phenotypes. Cancer cells over-express molecular chaperones and co-chaperones, including Hsp90, Hsp70, Hsp90/Hsp70 organizing protein (HOP), Hsp40, p23, and CDC37, that are necessary to fold mutant proteins and other highly expressed proteins1. Extensive efforts have led to the discovery of multiple Hsp90 inhibitors. Compounds of a diverse chemical nature, including geldanamycin derivatives, resorcinol group-containing compounds, purine and purine-like analogs, and others, have been established as potent inactivators of Hsp90 through binding to the ATP binding pocket in the N-terminal domain. However, many of these agents induce the cellular heat shock response (HSR), which is characterized by the up-regulation of the anti-apoptotic proteins, Hsp70 and Hsp2712. Over-expression of Hsp70 and Hsp27 is thought to lower the efficacy of Hsp90 inhibitors as therapeutics18. PU-H71, a purine-based Hsp90 inhibitor, has shown improved preclinical and clinal efficacy and is a promising application as a theragnostic platform for precision medicine targeting the epichaperome19. Furthermore, preclinical and clinical data indicate that 4-benzamide TAS-116 (Pimitespib), a selective oral inhibitor of cytosolic Hsp90α and β, exhibits a potent anti-tumor activity in various cancers with minimal side effects, including visual disturbances23. Pimitespib has recently been approved for clinical use in Japan. However, to date, no Hsp90 inhibitor is approved by the FDA, and the full clinical potential of Hsp90 inhibition for treating cancers remains to be harnessed.Hsp90 has been recognized as a therapeutic target for cancer therapy for decades24. For example, Novobiocin, a C-terminal ATP binding inhibitor25, is currently being used as a chemical scaffold to develop more specific and potent inhibitors of Hsp9026. Interestingly, Hsp90 C-terminal inhibitors do not induce the HSR, and NCT-58 triggers the death of rapidly proliferating and stem-like cells of trastuzumab-resistant HER2-positive breast cancer models through down-regulation of HER2 and Heat Shock Factor 1 (HSF1). Alternatively, inactivation of Hsp90 could be achieved through inhibition of specific co-chaperone interactions or post-translational modifications of Hsp9027. Attempts to abrogate the HSR by developing inhibitors of Hsp27, Hsp70, and the HSF1 transcription factor are also being pursued30.To this end, alternative strategies of Hsp90 inhibition are being pursued to improve efficacy, including targeting the Hsp90 middle domain, which binds client proteins, and the C-terminal ATP binding domainCapsicum. Capsaicin is an agonist of transient receptor potential vanilloid-1 (TRPV1) that triggers both the sensations of burning and pain, but also promotes the desensitization of peripheral nerves to noxious stimuli31. Capsaicin is approved by the FDA for topical use in pain management of arthritis, diabetic neuropathy, and neuralgia and also to treat post-surgical neuropathic pain in cancer patients32. Numerous reports have shown that capsaicin induces cell cycle arrest and apoptosis in cancer cell lines33 and autophagy in anaplastic thyroid carcinoma cells34. Interestingly, co-treatment of cancer cells with capsaicin and capsazepine (a TRPV1 antagonist) showed that cells still die by apoptosis35, suggesting that cytotoxicity was independent of TRPV1 activation, which was confirmed in an oral cancer model36. Several mechanisms of action have been proposed for the cytotoxic properties of capsaicin, including NF-κB inactivation37, estrogen receptor (ER) stress-mediated apoptosis38, generation of reactive oxygen species (ROS), disruption of the mitochondrial transmembrane potential39, inactivation of the EGFR/HER-2 pathway40, cyclin-dependent kinase blockade41, anti-angiogenic activity by direct inhibition of Src kinase42 and the VEFG pathway43, and inactivation of the E2F-Rb pathway44, among others.Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the primary pungent ingredient of hot peppers of the genus 45. In this report, we demonstrate that Hsp90 is a critical target of capsaicin in cancer cells. Specifically, we show that capsaicin binds to the N-terminal domain of Hsp90 in a manner unique from classical Hsp90 N-terminus inhibitors, and rather than inducing Hsp70 and the HSR, capsaicin triggers the lysosomal degradation of Hsp70 in various cancer cell lines. Remarkably, capsaicin treatment did not affect Hsc70, which shares ~ 85% amino acid sequence identity with Hsp7046. Further, low concentrations of doses of capsaicin enhanced 17-AAG tumor-cell killing using two-dimensional and spheroid culture systems. These data support the use of capsaicin as a scaffold to develop novel N-terminal Hsp90 inhibitors that circumvent over-expression of the Hsp70 protein and/or use it as a co-therapeutic.Our data suggest that the cytotoxic effects of capsaicin in cancer cells may also involve the inactivation of Hsp90, and most of the inactivated pathways above are driven by protein clients of Hsp90. We have previously shown that capsaicin treatment destabilizes several Hsp90 client proteins2 was made by acylation of vanillylamine with 10-azidodecanoyl chloride with subsequent reduction of the azido functionality 52.Cell lysates from human breast cancer MCF7 cells were incubated with Cap-OH Fig. A or cont53. Human Hs578T breast cancer cells were treated with 200 µM capsaicin for 1 h. Cell protein extracts were heated in a thermocycler using a temperature gradient from 52.5 to 68.8 °C to cause protein melting and denaturation. Cell extracts were then centrifuged to remove precipitated proteins. If Hsp90 is a target of capsaicin in cells, binding would promote Hsp90 stability and resistance to temperature-induced denaturation. As shown in Fig. To validate Hsp90 as a cellular target of capsaicin, we used the cellular thermal shift assay (CETSA) to evaluate drug target engagement in cellsE. coli but yeast and human proteins were expressed in insect sf9 cells. As shown in Fig. To test capsaicin selectivity and direct binding to Hsp90, Cap-OH resin was used to pull down purified human cytosolic Hsp90α and β as well as the yeast (Hsp82) and bacterial (HtpG) orthologs. HtpG was expressed in 45, and reduction of Hsp70 protein levels.To determine the functional impact of capsaicin on Hsp90 in vitro, we tested the ability of capsaicin to inhibit the Aha1-stimulated ATPase activity of Hsp90. As shown in Fig. To delineate the capsaicin binding site on Hsp90, we used capsaicin resins to pull down full-length hsp90α and β as well as the N-terminal and C-terminal domains Fig. A. Intere3-OH on the vanilloid ring , glucocorticoid receptor (GR), and progesterone receptor (PR)), and kinases ted Fig. A. Howeveted Fig. A. Hsp70 Capsaicin-mediated down-regulation of Hsp70 Fig. A could rThe roles of the proteasomal and lysosomal degradation pathways in mediating capsaicin-induced Hsp70 degradation were then studied. MCF7 cells were treated with capsaicin in the presence of a proteasome inhibitor MG132) or autophagy inhibitor (3MA). As shown in Fig. 2 or auto57 and that dihydrocapsaicin  induces mito-autophagy in HCT 116 colon cancer cells58.Consistent with the paradigm above, light microscopic analysis of MCF7 cells showed that capsaicin-treated cells developed vacuoles Fig. B, which 60. We therefore further tested whether capsaicin-dependent inhibition of 17-AAG-induced Hsp70 expression would affect tumor cell killing. MCF7 and LNCaP cells were treated with 50, 100, and 200 μM capsaicin with and without 100 nM 17-AAG. In a concentration-dependent manner, capsaicin abolished the 17-AAG-induced over-expression of Hsp70 in the co-treated group compared to 17-AAG alone  cells, which were more sensitive to the cytotoxic effect of capsaicin than human hepatocarcinoma (HepG2) cells, which expressed less TRPV1 receptor63. Regardless, capsaicin can block the activity of many oncogenic signaling proteins including NF-κB, ER, EGFR/HER2, CDK4, Src, VEGF, and PI3K/Akt, among others. Thus, there exists considerable pleiotropy with regard to the mechanism of capsaicin-induced cancer cell death64. Importantly, however, many of the signaling proteins inactivated by capsaicin are clients of Hsp90, which is consistent with capsaicin binding and inactivating Hsp90 as shown in this report. Hence, it is reasonable to propose that the seemingly pleiotropic effects of capsaicin may, in part, result from the inhibition of Hsp90 in cancer cells.Capsaicin has been shown to trigger a variety of signaling events in cancer cell lines. The mechanism by which capsaicin induces apoptosis in cancer cells is not well understood, but it appears to be independent of the TRPV1 receptor as neither capsazepine, a TRPV1 antagonist, nor intracellular Ca45. Pulldown experiments coupled with mass spectrometry analysis strongly suggest that cytosolic Hsp90α and β are crucial cellular targets of capsaicin and that capsaicin interacts with the N-terminus ATP binding domain of Hsp90 to promote cancer cell death through autophagy. This domain is the target of Hsp90 inhibitors including geldanamycin and its derivatives, 17-AAG, resorcinol compounds, and ATP-based structures such as PU and others. The apo N-terminal domain of Hsp90 has a highly flexible lid segment formed by residues 107 to 14165 and can be significantly affected by ligand binding66. Structural comparison of N-terminal domain interaction with ligands showed that the main structural variations are limited to the conformation of residues 104 to 111 located in the α-helix 3 domain67. This region can adopt a helical or more open conformation in yeast Hsp90, but it is structured as an extended loop that closes the binding site of human Hsp9068. The key residues for ligand binding in all Hsp90 orthologs are centered around residue Asp93. These include Asn51, Ser52 (Ala38 in yeast), Ala55, Met98, and Thr184. These structural changes are potentially important to the selective binding of capsaicin to Hsp90α and β compared to yeast and bacterial Hsp90. However, more structural studies are needed to better define the molecular motifs underlying this selectivity. It remains intriguing and potentially significant to understand how capsaicin binding the N-terminus of Hsp90 does not induce the HSR akin to classical N-terminal binding inhibitors of Hsp90. Indeed, It is well known that 17-AAG and other Hsp90 ATP competitors induce HSF169 through dissociation of the Hsp90:HSF1 complex and prolongation of Hsp70 promoter activation. However, cell treatment with capsaicin selectively down-regulated Hsp70 protein in various cancer models, including HeLa, MCF7, U2OS, and LNCaP cell lines , which show only 70.5% amino acid identity. These differences between Hsp70 and Hsc70 could impact their activities. For example, Menoret and coworkers measured the peptide-binding affinities of purified Hsp70 and Hsc70 using three different radiolabeled peptides under a variety of buffer conditions in vitro73. Several other reports have also documented that capsaicin disrupted mitochondrial function and induced ROS production in cancer cells, leading to death79. Importantly, Hsp90 impacts mitochondrial ETS function, and inhibition of Hsp90 causes mitochondrial dysfunction80.Our data show that capsaicin treatment induced autophagy and mitophagy in MCF7 cells Fig. C. In cap81. George and coworkers showed that inhibition of Hsp70 by the small molecule compound PES resulted in marked induction of autophagy82. These data seem to indicate that Hsp70 is necessary for the suppression of autophagy under normal physiological conditions. Hsp70, however, is not the only protein that suppresses autophagy, as Hsp70.1.3−/− mouse embryonic fibroblasts (MEFs) maintain basal levels of autophagy83. It is important to study how capsaicin induces autophagy and to further test how this leads to the degradation of Hsp70 without the degradation of the classical autophagic flux p62/SQSTM1 1  and potassium carbonate  were added to a solution of capsaicin  in DMF (13 mL). The reaction mixture was stirred at 75 °C overnight, cooled down to room temperature and extracted with diethyl ether (250 mL). The organic layer was washed with water (5 × 50 mL), brine (100 mL), and dried over MgSO4. The solvent was evaporated in a vacuum and the crude product was purified by chromatography (silica gel/ethyl acetate) to give 624 mg (80%) of capsaicin derivative 2 as a white amorphous solid.2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl 4-methyl benzenesulfonate , 6.77–6.80 , 5.83 , 5.04 , 4.36–4.37 , 4.16–4.18 , 3.86–3.89 , 3.83 , 3.73–3.75 , 3.60–3.68 , 3.50–3.53 , 3.26–3.28 , 2.18–2.22 , 1.63–1.67 , 1.43 , 1.36–1.29 , 0.85–0.89 .13C-NMR: 173.12, 156.19, 149.98, 147.89, 131.99, 120.26, 114.09, 111.96, 79.35, 71.04, 70.82, 70.75, 70.45, 70.38, 69.84, 68.90, 56.12, 43.56, 40.55, 37.04, 32.00, 29.53, 29.52, 29.35, 28.62, 26.00, 22.83, 14.28.2, 510 mg, 0.9 mmol) in DCM (5 mL) at room temperature. The reaction mixture was stirred overnight, quenched with saturated sodium bicarbonate (50 mL), and extracted with DCM (200 mL). The organic layer was washed with brine and dried over anhydrous sodium sulfate; the solvent was removed in a vacuum, and the crude product was purified by chromatography  to give 328 mg (78%) of CAP-OH as a white powder. M.P. 73–75 °C.Trifluoroacetic acid  was added to a solution of the capsaicin derivative , 6.07 , 4.34–4.35 , 4.31 , 4.14–4.17 , 3.83–3.86 , 3.70–3.72 , 3.64–3.68 , 3.58–3.60 , 2.91–2.93 , 2.17–2.21 , 1.62–1.65 , 1.25–1.28 , 0.84–0.88 .13C-NMR: 173.31, 149.28, 147.01, 132.43, 120.00, 113.49, 111.62, 70.72, 70.51, 70.39, 70.02, 69.53, 68.55, 55.78, 43.00, 40.90, 36.56, 31.78, 29.33, 29.13, 25.83, 22.59, 14.06.3 as a colorless oil.A solution of vanillylamine hydrochloride  in DMF (2 mL) was placed in a flame-dried round bottom flask under Ar atmosphere. 5 M aqueous solution of NaOH  was added drop-wise at room temperature. The reaction mixture was stirred for 30 min at 35 °C, cooled to 0 °C, and 10-azidodecanoyl chloride  was added dropwise. The ice bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction was quenched with water (5 mL) and extracted with ethyl acetate (3 × 75 mL). The organic layer was dried over anhydrous sodium sulfate, solvents were removed in a vacuum, and the crude product was purified by chromatography  to afford 287 mg (26%) of compound 1H-NMR: 6.81–6.83 , 6.76 , 6.70–6.72 , 6.19 , 6.08 , 4.29–4.31 , 3.82 , 3.20–3.24 , 2.15–2.19 , 1.54–1.63 , 1.26–1.31 .13C-NMR: 173.21, 146.91, 145.26, 130.35, 120.74, 114.57, 110.86, 55.97, 51.52, 43.55, 36.80, 29.37, 29.32, 29.30, 29.13, 28.87, 26.74, 25.85.3, 276 mg, 0.792 mmol) in tetrahydrofuran (THF) (2 mL). After 10 min, deionized water  was added to the flask, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in a vacuum, and the residue was purified by chromatography  to yield 182 mg (71%) of CAP-NH2 as a light brown oil.Triphenyl-phosphine  was added to a solution of 10-azido-N-(4-hydroxy-3-methoxybenzyl) decanamide , 6.74–6.76 , 5.79 , 4.34–4.36 , 3.86 , 3.16 , 2.66–2.70 , 2.17–2.21 , 1.62–1.65 , 1.40–1.44 , 1.25 .13C-NMR: 173.07, 147.35, 145.65, 130.42, 120.91, 115.06, 111.12, 56.06, 43.68, 42.22, 37.02, 33.56, 29.57, 29.48, 29.44, 29.37, 26.97, 26.01.Anti-β-actin was from Santa Cruz Biotechnology (sc-47778); anti-Hsp70 from Enzo Life Sciences ; anti-Hsc70 from StressMarq Biosciences Inc  or anti-Hsp70 ; anti-Hsp90β ; anti-LC3B ;, anti-cleaved LC3A (LC3AII) ; anti-GAPDH ; and p62/SQSTM1 from Santa Cruz Biotechnology, Inc .17-AAG (Selleckchem), Capsaicin  and Ethidium Homodimer-1 (Thermo Fisher).E. coli and purified as previously described88. Yeast Hsp90 (Hsp82), and cytosolic human Hsp90α and β were expressed in insect sf9 cells and purified as previously described89.Bacterial Hsp90 (HtpG) was expressed in Cell proliferation was monitored using The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega #G3580). Cells were grown to 40–60% confluency on 96-well tissue culture plates  followed by treatment with capsaicin or DMSO control. For protein analysis by Western blot, cancer cell lines were grown for at least 48 h to be at 40–60% confluent in 6-well plates (Corning #3516) before drug treatment was started. Cells were then harvested at specified times, and cell lysates were made. For Western blotting, 15 μg of protein lysate was analyzed using the following specific antibodies.HeLa. MCF7, Hs578T, U2OS, LnCaP and PC3 cell lines were purchased from American Type Culture Collection  within the last 5 years. Large stocks were made and stored in liquid nitrogen. All cell lines are regularly tested at least quarterly for mycoplasma using the Genetica Inc mycoplasma test and MycoAlert PLUS Mycoplasma Detection Kits.g for 10 min. Clarified lysates (15 μg) were run on SDS-PAGE (10% gel) and transferred to PVDF membranes. Proteins were detected by Western blotting using indicated antibodies against specific proteins. All primary antibodies were used at 1/1000 dilution with overnight incubation at 4 °C.Cells were lysed with buffer A  supplemented with a protease inhibitor cocktail  on ice for 30 min, shaking every 5 min. The lysate was centrifuged at 16,000×90. Stable LNCaP and DU145 cell lines containing control or UNC45A shRNA were seeded in a low-adhesion 96-well plate at a density of 5 × 103 cells/well, while stable control and UNC45A sh RNA PC3 cells were seeded at a density of 1 × 104 cells/well. The cells were cultured in serum- and antibiotic-free medium supplemented with B27, N2, insulin, transferrin, selenium, hEGF, and FGF for 8 days. After this time, cells were stained with 0.5 µM EthD-1 and (1:5000 dilution) of Hoechst dyes to distinguish between dead and live cells. Confocal microscopy was used to capture images of the cell spheroids. t-test was used for unpaired analysis comparing average expression between conditions. p values < 0.05 were considered statistically significant90.The general protocol described by Edner et al. was generally followed4; and 3% paraformaldehyde, then permeabilized with 0.1% triton X-100, blocked with 2% fetal bovine serum, 5% goat serum and 5% glycerol, and stored at 4 °C. The primary antibodies against LC3B, as well as mouse and rabbit secondary antibodies, were prepared in the blocking buffer.The indicated cells were grown in 24-well plates (Corning #3337) on micro-cover glasses (Electron Microscopy Sciences) to about 50% confluency in MEM, 1X (Cellgro #10–010-CV) medium supplemented with 10% fetal bovine serum. Cells were treated with indicated concentrations of 17-AAG, capsaicin, or DMSO control for 24 or 48 h. Cells were fixed with 0.1 M PIPES, pH 6.95; 1 mM EGTA, pH 8.0; 3 mM MgSOIn each well of a 6-well tissue culture plate, 100,000 MCF7 cells were grown to 60% confluence. Cells were treated with 200 μM capsaicin or DMSO control  for 15 h. Cells were fixed and imaged using a light microscope (Carl Zeiss). Mr. Brendan Marshall from the Electron Microscopy & Histology Core Laboratory at Augusta University performed the electron microscopic study of MCF7 cells.Monoclonal Hsp70 antibody (BB70) was incubated with 30 μL slurry of protein A/protein G-agarose beads  for 2 h at room temperature. Following incubation, cell extracts (200 μg of protein) from MCF7 cells were added to each sample and incubated overnight at 4 °C with gentle rotation. The samples were then washed three times with 1X RIPA buffer along with protease inhibitors , 10 mm NaF, 2 mm sodium pyrophosphate, 2 mm β-glycerophosphate, and 1 mm sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and eluted with 2× sample buffer at 95 °C for 5 min. Protein complexes were eluted using SDS-PAGE sample buffer and analyzed by Western blot.7 Hs578T cells were seeded in 10 cm culture dishes. After 24 h, cells were treated with DMSO (1%) or capsaicin (200 μM) for 1 h. Following treatment, cells were trypsinized and collected with PBS containing protease inhibitors and transferred to PCR tubes. All samples were heated at 52–60 °C for 2 min using a Bio-Rad C1000 thermal cycler and lysed with 3–4 freeze–thaw cycles using liquid nitrogen. For each freeze–thaw cycle, the cells were exposed to liquid nitrogen for 1 min and placed in a water bath at 30 °C for 1 min. Precipitated proteins and cell debris were then pelleted by centrifugation at 16,400 rpm for 15 min at 4 °C and the cleared cell lysates were analyzed by Western blotting.To analyze the engagement of capsaicin and Hsp90 protein, a cellular thermal shift assay was performed. 1 × 1047. The Cap-NH2 immobilization was implemented at pH 10, and Cap-OH immobilization was done at pH 6. Sepharose resin subjected to Divinyl sulfone activation and blocking with ethanolamine was used as a control. To test the binding of Hsp90 and its fragments to capsaicin derivative resins, 15 µL of resin and 10 µg of protein were used in a final reaction volume of 200 µL of incubation buffer . Samples were incubated for 30 min at 30 °C and washed four times with 1 ml of buffer. Bound proteins were extracted with sample buffer, resolved by SDS–PAGE, and stained with Coomassie Blue.Capsaicin derivatives solubilized in 5% DMSO were covalently linked to Sepharose 4B as described previously by Cornillot et al.91. Rabbit reticulocyte lysate (RRL) was used to refold the PR. Briefly, approximately 0.05 μM PR was incubated with 1.4 μM Hsp70, 0.8 μM Hsp90β, 0.2 μM Hsp40 (Ydj), 0.08 μM HOP, and 2.6 μM p23 in reaction buffer . After incubation for 30 min at 30 °C, 0.1 μM [3H]-progesterone  was added. Samples were incubated on ice for 3 h at 4 °C. Complexes were then washed three times with 1 ml of reaction buffer and assessed for bound progesterone by liquid scintillation counting using a PerkinElmer Microbeta plate reader. PR activity is expressed in radioactive count per minute (CPM).Purified PR was adsorbed onto PR22 antibody-protein A-Sepharose resin beads and was assembled into complexes, as described previously68. The protein and ligand were optimized with force field: CHARMm, Partial charge: MMFF94, and the radius of receptor active site employed: 4.030776, 10.9669, 24.5626, 9.0. Our results showed that the superimposition of the docked poses with the bioactive ligand conformations yields a low root mean square deviation (RMSD) of 0.095 Å, indicating that molecular docking can accurately reproduce the ligand conformation in the Hsp90 crystal structures.Discovery Studio 2.5 software was used to carry out docking simulations using the Hsp90 crystal structure co-crystalized with the selective inhibitor PU3 (PDB accession number: 1UYM)Supplementary Information.


Prolactin (PRL) has been reported to be associated with oxidative stress, which is an important contributor leading to cell apoptosis. However, little is known about the mechanisms underlying the effects of PRL on cytotoxicity and oxidative stress in ovine ovarian granulosa cells (GCs).Ovine ovarian GCs were treated with 0, 4, 20, 100 and 500 ng/mL of PRL. Then, the cytotoxicity, cell viability, malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of GCs were detected. Additionally, 500 ng/mL PRL was chosen as the high PRL concentration (HPC) due to its high cytotoxicity and oxidative stress. Proteomic and metabonomic were performed to examine the overall difference in proteins and metabolic pathways between C (control: 0 ng/mL PRL) and P groups (500 ng/mL PRL).P < 0.05) the cytotoxicity, ROS and MDA, increased (P < 0.05) the cell viability, SOD and T-AOC, and the GCs treated with 500 ng/mL PRL showed the opposite trend (P < 0.05). Supplementation with 500 ng/mL PRL significantly increased the proteins of MT-ND1, MAPK12, UBA52 and BCL2L1, which were enriched in ROS and mitophagy pathways. Pathway enrichment analysis showed that the pentose phosphate pathway was significantly enriched in the P group.The results indicated that GCs treated with 4 ng/mL PRL significantly decreased (A low concentration of PRL inhibited cytotoxicity and oxidative stress. HPC induced oxidative stress in ovine ovarian GCs via the pentose phosphate pathway by modulating the associated proteins MT-ND1 in ROS pathway and UBA52, MAPK12 and BCL2L1 in mitophagy pathway, resulting in cytotoxicity. ROSs includes superoxide anion  is a protein hormone synthesized and secreted by several cells and tissues in the body, such as anterior pituitary, mammary glands, T-lymphocytes and hypothalamus . The norIn this study, we investigated the impact of varied concentrations of PRL on GCs cytotoxicity and oxidative stress, intending to establish a theoretical foundation for enhancing GCs growth and addressing functional irregularities. It will provide a basis for future applications of PRL in ruminant reproduction.Abnormal follicle development can lead to decreased fecundity of sheep and affect production . At the in vitro culture. The growth of GCs was observed under a microscope, and non-adherent cells were removed. All procedures used in this study were approved by the Laboratory Animal Ethics Committee of Hebei Agricultural University .Fresh ovaries from twelve sexually mature ewes aged between 1 and 1.5 years were collected at the local cooperative abattoir , kept at 37 °C, then transported to the laboratory in a buffered saline solution supplemented with streptomycin/penicillin mixture (1%). Dominant follicles with 3–7 mm diameter were isolated from the ovaries after 3 times sterile Dulbecco’s Phosphate Buffered Saline (DPBS) cleaning. Then GCs were collected from the interior of the follicles with a one mL injector and filtered with a 100 mm filter. The filtrated liquid was centrifuged at 1,500 rpm for 10 min to obtain the precipitate. Then the precipitate was lysed in one mL sterile red blood cell (RBC) lysate for 3 min to dissolve the RBC. Harvested cells were washed with DPBS ≥ 3 times and used for 4 per well and treated with 0, 4, 20, 100 and 500 ng/mL PRL  for 24 h to validate the toxicity of PRL. The morphology of GCs was observed by inverted phase contrast microscope to evaluate the cytotoxicity with different concentrations of PRL .The GCs were seeded in 6-well-culture plates at a density of 6 × 103 per well and treated with 0, 4, 20, 100 and 500 ng/mL PRL for 24 h, respectively. Then a 10 µL CCK-8 was added into each well, and the cells were hatched in the dark for 1-4 h. A microplate reader was used to quantify the optical density of the cells at 450 nm. Cell viability values were calculated based on test data. Cell survival rate γ calculation common formula is γ = [(As-Ab)/(Ac-Ab)] × 100% .The direct cytotoxicity of materials was evaluated as percentage cell viability  using th4 per well and then trypsinized and collected in 1.5 ml centrifuge tubes. After incubation, cells were immediately centrifuged twice at 1000 × g for 10 min to remove the supernatant to obtain GCs, which were kept at −80 °C prior to analysis.GCs incubated with 0, 4, 20, 100 and 500 ng/mL PRL were cultured in a 6-well plate at a density of 6 × 10Superoxide dismutase (SOD) activity , malondialdehyde  and total antioxidant capacity (T-AOC)  were measured following the manufacturer’s instructions with respective analytical kits.3 per well and then incubated with fluorescent probe of ROS  for 20 min in dark at 37 °C. Then, incubated cells were washed twice with DMEM/F12 and images were captured under an inverted fluorescence microscope  using a green-fluorescence filter and images were analyzed using ImageJ 1.48v .GCs incubated with 0, 4, 20, 100 and 500 ng/mL PRL were cultured in a 96-well plate at a density of 5 × 10In the present study, we found that cells treated with 500 ng/ml PRL showed significantly lower cell viability and higher cytotoxicity and oxidative stress than other groups. By taking into account the results of prior investigations , the conGCs in 6-well plates from C and P groups were collected according to previously described methods .Samples were shredded with liquid nitrogen, lysed in 8 M urea lysis solution, and then stored on ice for 1 h, followed by sonication for 5 min. The lysate was centrifuged for one hour at 12,000 × g and 4 °C. The supernatant was transferred to a clean tube. The Bradford protein test was used to determine protein concentration. Each sample’s extracts were reduced with 5 mM DTT at 56 °C for 25 min before being alkylated by supplementation of 14 mM Iodoacetamide at room temperature for 30 min in the dark. The urea concentration from each sample, containing 2 M at less, was digested with Tyrisin at 1:100 enzyme-to-substrate ratio and digested at 37 °C for 16–18 h.Desalted peptides were labelled using TMT6/10-plex reagents  by the instructions. The labelling reagents were dissolved in acetonitrile. The differently labelled peptides were homogeneously mixed and incubated for 2 h and then desalted using a peptide desalting spin column .TMT-labelled peptide mix was fractionated using a C18 column  on a Rigol L3000 HPLC. The operation was as follows: mobile phases A  and B (100% acetonitrile) was used to develop a gradient elution. The solvent gradient was 3%–41% acetonitrile. And 20 components were collected after separation, which were concentrated into peptide powder by vacuum and stored at −20 °C.The present research employed EASY-nLC 1200 system  and Orbitrap Q Exactive HF-X mass spectrometer  to analyze the proteomics. The peptides were separated on a homemade analytical column with a linear gradient from 5% to 100% of eluent B (0.1% FA) using TMT-6 plex at a flow rate of 300 NL/min for eluent A (0.1% FA in water). The gradient of solvent was as follows: 3–5 percent of B, 5 s; 5–15 percent of B, 23 mins 55 s; 15–28 percent of B, 21 mins; 28–38 percent of B, 7 mins 30 s; 38-100 percent of B, 5 s; 100 percent of B, 12 mins 25 s.LC-MS/MS raw files were processed using Maxquant (v1.6.14) software for database search (Uniprot-sheep-proteome_UP000002356), building the database with the DDA method. Alkylation of cysteine was set as a fixed modification. Methionine oxidation and N-terminal acetylation of protein were set as a variable modification. The false discovery rate (FDR) of proteins and peptides was set at 0.01.P value <0.05 and FC >1.2 or FC <0.83 . The function of DEPs was determined by gene ontology (GO) analysis through GoPlot (v1.0.2) and GO enrichment analysis of DEPs was performed from biological process (BP), molecular function (MF) and cellular component (CC). Then, the molecular process pathways for differential protein enrichment were obtained through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.Different expression proteins (DEPs) were sifted by R23529; UBA52, 1:500, 16432; MT-ND1, 1:500, A8035; GAPDH, 1:5000, 200306-7E4) at 4 °C overnight, and incubated with secondary antibody  at room temperature for 1 h. ImageJ software was used for grayscale analysis.Total proteins from C and P groups were lysed for 30 min using phenylmethanesulfonyl fluoride (PMSF) and separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, membranes were subjected to a standard blocking with 5% non-fat milk, hybridized with primary antibody  used to be delivered to the GCs samples of C and P groups, and the samples were homogenized with the usage of a grinding mill after standing for 1 h. The extract was centrifuged at 3,000 × g for 15 min at 4 °C to obtain 400 µl supernatant, which was dried thoroughly. Next, the samples supplementation with 100 µl prechilled methanol-water , then the suspension was eddied  and centrifuged , to collect the supernatant for further analysis.LC-MS/MS analyses were conducted using Vanquish UHPLC system  with an Orbitrap Q Exactive HF-X mass spectrometer . Using a linear gradient, samples were injected into a ACQUITY UPLC HSS T3  column. The LC mobile phases A (5 mM ammonium formate), B (acetonitrile), C (0.1% formic acid) and D (0.1% formic acid acetonitrile) were used. The solvent gradient was set as follows: 2% B, 1 min; 2–50% B, 8 mins; 50–98% B, 3 mins; 98% B, 1.5 mins, 98–2% B, 1.5 mins and 2% B, 3 mins for the negative polarity mode. 2% D, 1 min; 2–50% D, 8 mins; 50–98% D, 3 mins; 98% D, 1.5 mins, 98–2% D, 1.5 mins and 2% D, 3 mins for the positive polarity mode. Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 2.5 kV, capillary temperature of 325 °C, auxiliary temperature of 300 °C, sheath gas flow rate of 30 arb and aux gas flow rate of 10 arb.Peak alignment, peak picking and quantitative analysis of raw data were performed by Compound Discoverer 3.0 . The main parameters were set as to the methods described previously .P value <0.05 and —FC—>1.5 . The DEMs of both groups their enriched KEGG pathway were analyzed.In LC-MS metabolomic analysis, the following criteria were employed for selecting the differentially expressed metabolites (DEMs) in C and P groups: variable influence on projection (VIP) value >1.0, post hoc test, was used to compare the cell activity and oxidative stress related indicators among multiple groups. Statistical comparisons between C and P groups were performed using the t-test. P < 0.05 indicated significance.Statistical analysis was performed using SPSS software . One-way ANOVA followed by Duncan’s Multivariate statistical analyses, principal component analysis (PCA), relative standard deviation (RSD), Pearson’s correlation coefficient, supervised partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted to evaluate data quality control. For the integrated analysis of the proteome and metabolome, the KEGG pathway database was used to identify relevant metabolic processes associated with DEPs and DMs in both groups. Additionally, information regarding their interaction network was also obtained.The morphology and viability of GCs are shown in P = 0.024), while 20 (P = 0.013), 100 (P < 0.001) and 500 ng/mL (P < 0.001) PRL obviously inhibited cell viability.The CCK-8 assay results  showed tP = 0.006), 4 (P = 0.004), 20 (P = 0.006) and 100 (P = 0.0255) ng/mL groups, while the content of MDA was higher in the 100 ng/mL group than in the 0 (P = 0.018), 4 (P = 0.006) and 20 (P = 0.020) ng/mL groups, and the content of MDA was elevated in the 0 (P < 0.001) and 20 (P < 0.001) ng/mL groups compared with the four ng/mL group. The activity of SOD was increased in the 0 (P = 0.004), 4 (P < 0.001), 20 (P = 0.004) and 100 (P = 0.044) ng/mL groups compared with the 500 ng/mL group, while the activity of SOD was lower in the 100 ng/mL group than in the 0 (P = 0.012), 4 (P < 0.001) and 20 (P = 0.011) ng/mL groups, and the activity of SOD was increased in the 0(P = 0.002) and 20 (P < 0.001) ng/mL groups compared with the four ng/mL group. Relative to the 500 ng/mL group, the T-AOC was decreased in the 0 (P = 0.003), 4 (P = 0.002), 20 (P = 0.015) and 100 (P = 0.017) ng/mL groups, while the T-AOC was lower in the 100 ng/mL group than in the 0 (P = 0.005) and 4 (P = 0.003) ng/mL groups. The level of ROS was shown in P < 0.001), 4 (P < 0.001), 20 (P < 0.001) and 100 (P = 0.007) ng/mL groups, while the ROS was higher in the 100 ng/mL group than in the 0 (P = 0.002), 4 (P = 0.001) and 20 (P = 0.002) ng/mL groups, and the level of ROS was increased in the 0 (P = 0.021) and 20 (P = 0.032) ng/mL groups compared with the 4 ng/mL group.The contents of MDA, SOD and T-AOC were shown in There were 821,838 LC-MS/MS spectra matched to the known spectra, whereas the number of available spectra was 120,505. Overall, 7,552 credible proteins and 85,761 peptides from all GCs samples were detected and quantified by 1% FDR. However, only 7,499 proteins could be quantified . PCA, RSP value <0.05 and FC >1.2 or FC <0.83, Relative to the C group, 93 proteins among all credible proteins were significantly downregulated or upregulated in P group. The volcano map and heatmap of the DEPs were shown in P0C276 (UBA52), Q9MZS7 (BCL2L1) and W5QEW6 (MAPK12) were associated with mitophagy. O78747 (MT-ND1) was contacted with reactive oxygen species (ROS). Q9MZS7 (BCL2L1) and W5QEW6 (MAPK12) enriched with functions correlated with apoptosis and NOD-like receptor signaling pathway. P0C276 (UBA52) and W5PQR5 (RPL18) were involved in ribosome. Q9MZS7 (BCL2L1) and W5QEW6 (MAPK12) were closely related to p53 signaling pathway, and 078747 (MT-ND1) in connection with oxidative phosphorylation  networks for DEPs. These interactions include both indirect functional links and direct physical connections.P0C276 and W5PQR5 proteins greatly influenced the regulation of ribosome, and the findings revealed that some proteins could not interact with one another directly. However, they still play a role through HPC.In The difference in specific metabolites is likely due to the different concentrations of PRL. The metabolites were identified following the treatments with 0 and 500 ng/mL PRL by LC-MS/ MS. A total of 1,118 metabolites were identified using the positive ion (POS) mode, and 809 were identified using the negative ion (NEG) mode (Table S1). The OPLS-DA  and 6B aWe compared the identified metabolites of ovine ovarian granulosa cells (GCs) from HPC and C groups to elucidate the underlying regulatory mechanism. The results were shown in P-value were presented in The pathway analysis utilised the KEGG database to identify the significantly enriched biochemical processes in DEMs. The results showed that the signaling pathways included 14 pathways at POS mode and 11 pathways at NEG mode. After pathway enrichment analysis, the regulated pathways ranked based on the An integrated analysis based on the aforesaid omics data was conducted to identify the related enriched pathways of the DEPs and DEMs. As a result, Therefore, the pathway information was captured by mapping these DEPs to the KEGG pathway database . OverallPRL has been reported to be associated with cell proliferation and apoptosis, with low concentration of PRL promoting cell proliferation and high concentration leading to apoptosis . In our via mitophagy, thereby enhancing oocyte developmental ability  group compared with the healthy group may be related to increased oxidative stress in the endometrium . High min cortex . Bcl-2 cn cortex . A studyn cortex . TherefoPRLR) exerts pleiotropic biological effects in mammals (PRLR regulates the pentose phosphate pathway in human by reducing the expression of two rate-limiting enzymes in pentose phosphate metabolism, G6PD and TKT. In this study, we screened the pentose phosphate pathway by both POS and NEG modes of the metabolomic. Researchers explored that HK2 attenuates cardiac hypertrophy by decreasing ROS accumulation via increased pentose phosphate pathway flux (PRL binding to the prolactin receptor ( mammals . Nie et way flux . Agarwalway flux . Data alway flux , which iway flux . Hence,  1.Low concentration of PRL inhibited cytotoxicity and oxidative stress while high PRL concentration induced cytotoxicity and oxidative stress. 2.High PRL concentration promoted oxidative stress in ovine ovarian GCs by the pentose phosphate pathway through regulating the related proteins MT-ND1 in the ROS pathway, and UBA52, MAPK12 and BCL2L1 in the mitophagy pathway.10.7717/peerj.15629/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15629/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15629/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15629/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15629/supp-5Supplemental Information 5Click here for additional data file.


To evaluate the utility of apparent diffusion coefficient (ADC) values for differentiating breast tumors.The medical records of 17 patients with phyllodes tumor , 74 patients with fibroadenomas , and 57 patients with breast cancers  confirmed by surgical pathology were retrospectively reviewed.−3 mm2/s, PTs from BCs was 1.100 × 10−3 mm2/s, and FAs from BCs was 0.925 × 10−3 mm2/s. There were significant differences between benign PTs, borderline PTs, and malignant PTs in ADCmean, ADCmax, and ADCmin values. The cutoff ADCmean for differentiating benign PTs from borderline PTs was 1.215 × 10−3 mm2/s, and borderline PTs from malignant PTs was 1.665 × 10−3 mm2/s.There were significant differences between PTs, FAs, and BCs in ADCmean, ADCmax, and ADCmin values. The cutoff ADCmean for differentiating PTs from FAs was 1.435 × 10DWI provides quantitative information that can help distinguish breast tumors. Phyllodes tumor (PT), first introduced by Muller in 1838, is a fibroepithelial neoplasm that is histologically similar to a fibroadenoma (FA). PTs are rare, accounting for 2% to 4.4% of all diagnosed FAs in one institution . PTs thain vivo. The motion of water molecules in tissues depends on tissue cellularity and the integrity of cell membranes. Differences in the motion of water molecules between tissues cause signal attenuation. To date, DWI for breast tumor applications has relied on the mono-exponential model with b-values of 0 and 800 s/mm2 , including a circular ROI (ROI-c), single-slice ROI (ROI-s), and whole-tumor ROI (ROI-w) (Diffusion-weighted imaging (DWI) has become clinically relevant , 13. DWI00 s/mm2 –16. Othes/mm2  –24. ADC The medical records of female patients diagnosed with breast tumors between 1 January 2017 and 5 April 2022 were retrospectively reviewed. This retrospective analysis of breast MRI data was approved by the Institutional Research Ethics Board of our institute . The requirement for informed consent was waived. Inclusion criteria were: 1) unilateral or bilateral solid breast tumor, 2) no history of surgery, 3) no history of other tumors or systemic diseases, and 4) surgical pathology provided a definitive diagnosis. All patients underwent MRI examination 3–7 days prior to surgery. Patients were divided into three groups based on pathological findings: Group A, PT; Group B, FA; and Group C, BC.2. DWI parameters: FOV (mm): RL × AP × FH, 340 × 196 × 150; voxel (mm): 3.04 × 1.87 × 3; REC voxel MPS (mm): 1.06 × 1.06 × 1.06; slice thickness (mm): 3; slice gap (mm): 0; matrix (slices): 112 × 105 × 50; REC matrix: 320; NSA: 2; scan percentage (%): 163.2; total scan duration (min): 3:07; SNR: 1.027; TR (ms): 12500; min.TR (ms): 11007; TE (ms): 82; EPI factor: 93; BW in EPI freq.dir (HZ): 2129.8; WFS (pix)/BW (hz): 24.817/17.5; fold-over suppression: oversampling; P (mm): 153; A (mm): 73; stacks: 1; type: parallel; slices: 50; slice gap: 0; slice orientation: transverse; fold-over direction: AP; fat shift direction: P; packages: 1; local torso SAR: <64%; whole body SAR/level: <1.7 W/kg/normal; SED: <0.3 kj/kg; coilpower: 51%; maxB1 + rms: 1.67 uT. ADC maps were processed using the post-processing software . Two radiologists placed an ROI-c (10–300 mm2) on a 2D single-slice of each breast tumor. The area of the ROI-cs (mm2), ADCmean, ADCmax, ADCmin, and standard deviation (SD) were calculated.Patients were scanned using a 3.0-T  superconducting MRI scanner. DWI sequences were obtained with b-values of 0 and 800 s/mmU test. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was used to assess the diagnostic performance of ADC parameters for breast tumors. p < 0.05 was considered statistically significant.Statistical analysis was conducted using SPSS v28.0.1. Descriptive statistics, including mean and standard deviation, were summarized for each ADC parameter. Normality of ADC values was evaluated with the single-sample Shapiro-Wilk test. Normally distributed data with homogeneity of variance were compared with ANOVA. Non-normally distributed data with heterogeneous variance were compared with the non-parametric Kruskal-Wallis H test. Pairwise comparison was made with the Mann-Whitney The medical records of 148 patients with breast tumors were retrospectively reviewed, including 17 patients with PTs [eight benign PTs Figure 1−3 mm2/s, 1.8112 (0.94–2.44) ± 0.28428 × 10−3 mm2/s, and 1.4113 (0.69–2.05) ± 0.28392 × 10−3 mm2/s, respectively, which were higher than the ADCmean, ADCmax, and ADCmin of FAs and BCs. Multiple group comparisons conducted with the Kruskal-Wallis H test and Mann-Whitney U test showed significant differences (p < 0.001)  ± 0.26015 × 10< 0.001) Table 1.−3 mm2/s, 1.3098 (0.83–1.68) ± 0.25017 × 10−3 mm2/s, and 1.7962 (1.45–2.16) ± 0.13255 × 10−3 mm2/s, respectively ; the cutoff ADCmin was 1.465 × 10−3 mm2/s  ± 0.14886 × 10−3 mm2/s Table 2.BI-RADS is a comprehensive guideline used by radiologists for breast tumor classification. Conventional MRI sequences are a complementary approach to assessing breast tumors. DWI technology is not included in the BI-RADS system, but the use of ADC values to evaluate breast tumors has become a research hotspot in recent years , 25, 26.Clinically, PTs are usually managed surgically. Benign and borderline PTs require wide excision. Malignant PTs >10 cm or PTs with rapid progression in a short period require whole mastectomy. PTs are likely to recur after surgery, but only malignant PTs develop distant metastases . PTs andIn this study, conventional MRI showed that the imaging characteristics of benign, borderline, and malignant PTs overlap, and benign PTs could not be precisely differentiated from other BCs. In previous reports, MRI findings for eight cases of benign PTs identified some characteristics of large benign PTs (>3 cm), but distinguishing small PTs from small FAs was difficult ; MRI of −3 mm2/s, 1.2711 (0.81–2.20) ± 0.31678 × 10−3 mm2/s, and 0.8496 (0.60–1.26) ± 0.14857 × 10−3mm2/s, respectively. The ADCmeans of PTs was significantly higher than those of FAs and BCs (p < 0.001). ADCmean had the best efficacy to discriminate between PTs, FAs, and BCs compared to ADCmax and ADCmin, and had the highest specificity. The specificity of ADCmean for differentiating between PTs and FAs or PTs and BCs was 81.90% and 94.2%, respectively. These findings suggest ADCmean has potential as a clinically useful technology. In 2020, Jelena et al.  ± 0.26015 × 10−3 mm2/s, 1.3098 (0.83–1.68) ± 0.25017 × 10−3 mm2/s, and 1.7962 (1.45–2.16) ± 0.13255 × 10−3 mm2/s, respectively, and were significantly different. ADC values of malignant PTs at b0/1000 s/mm2 have been reported as 1.37 ± 0.03 (10−3 mm2/s) (−3 mm2/s), and 1.45 ± 0.03 (10−3 mm2/s)  ± 0.14886 × 10t tumors .−3 mm2/s or malignant with internal liquefaction, necrosis, and cystic degeneration at ADCmean >1.665×10−3 mm2/s. ADCmin had clinical application for the differentiation of benign and malignant PTs, and PTs were considered malignant at ADCmin >1.465×10−3 mm2/s.The motion of water molecules in tissues depends on tissue cellularity and the integrity of cell membranes , 31. Con2; further research should include multi-b-value DWI. Fourth, distortion and deformation often occur at high b-values, which may disturb ADC parameters.This study was associated with several limitations. First, it was a retrospective study, and the clinical value of ADC values for discriminating between breast tumors should be verified in prospective studies. Second, the sample size was small, and there may have been interobserver variability with regard to ROI-c selection, which may have introduced bias. Third, DWI sequences included b-values of 0 and 800 s/mm2 images took 3 minutes. This enabled us to obtain satisfactory ADC values to evaluate the histological characteristics of a tumor. ADCmean differentiated PTs, FAs, and BCs, and benign PTs from borderline PTs and borderline PTs from malignant PTs. ADCmin helped differentiate benign PTs from malignant PTs. Overall, ADC values provided quantitative information that has potential to distinguish between PTs, FAs, and BCs, and classify PTs.Breast DWI acquiring b0 and 800 s/mmThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by People’s Hospital of Longhua. The patients/participants provided their written informed consent to participate in this study.Image evaluation, JF, JY, and RWL; Quality control, YZ and RWL; Literature retrieval, RFL; Data and statistics, LL and RWL; MR scanning, ZH, and LZ; Manuscript writing, RWL. All authors contributed to the article and approved the submitted version.


Furthermore, our investigations into cell apoptosis and reactive oxygen species (ROS) production revealed that compound 5i significantly induced apoptosis and triggered ROS generation. Additionally, Western blot analysis revealed the pronounced elevation of LC3 expression in H460 cells and γ-H2AX expression in H1299 cells subsequent to treatment with compound 5i. These molecular responses potentially contribute to the observed cell death phenomenon. These findings highlight the potential of compound 5i as a promising candidate for further development as an anticancer agent especially lung cancer.In pursuit of discovering novel anticancer agents, we designed and synthesized a series of novel 1,2,3-triazole hybrids based on cabotegravir analogues. These compounds were subjected to initial biological evaluations to assess their anticancer activities against non-small-cell lung cancer (NSCLC). Our findings indicated that some of these compounds exhibited promising antitumor abilities against H460 cells, while demonstrated less efficacy against H1299 cells. Notably, compound  Lung cancer is the leading cause of cancer-related deaths worldwide and advanced non-small-cell lung cancer (NSCLC) is the most common type. Surgical intervention is the preferred treatment for patients with stage I-II non-small-cell lung cancer (NSCLC), while chemotherapy combined with radiotherapy is recommended for stage III NSCLC patients . ApproxiE. coli with MIC of 0.0063 μmol/mL   is the lwith HIV .In recent years, efforts have been continuously directed towards the development of safer and less side-effect anticancer drugs. Repurposing mature drugs that are already on the market and evaluating their potential anticancer effects is an important approach in anticancer drugs development. This ensures both drug safety and clinical applicability. Integrating 1,2,3-triazole hybrids with known drugs has become a popular strategy for anticancer drug discovery. Preliminary researches have revealed promising anticancer effects by combining the 1,2,3-triazole hybrids with natural compounds such as betulin, myrrhanone B, coumarin, hydnocarpic acid, curcumin and others . While c1H NMR and 13C NMR spectra were performed in DMSO-d6 solution. Chemical shifts d) were reported in parts per million with tetramethylsilane as internal reference, and coupling constants were expressed in hertz using the following abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet).Elemental analyses were conducted using an Elementar Analysensystem Vario Macro cube in CHNSO mode. NMR spectra analyses were recorded on a Bruker400 spectrometer. 1)  was combined with anhydrous formic acid . The reaction mixture was heated to 65°C and allowed to react for 3 h under an argon atmosphere. The progress of the reaction was monitored using TLC (V(Petroleum ether):V(ethyl acetate) = 3:1, RF = 0.3–0.6). Afterward, the reaction mixture was concentrated under vacuum at 45°C to eliminate the formic acid, resulting in the formation of a crude oil compound. To remove any residual formic acid, acetonitrile (300 mL) was added, and the solvent was subsequently evaporated under vacuum. This process led to the isolation of compound 2 in the form of an oil.This step is a classic reaction of the demethylation of dimethyl aldehyde under acidic conditions. 1--1,4-dihydro-3-methoxy-4-oxo-2,5-pyridinedicarboxylic acid-2-methyl ester , and then S-2-aminopropanol  was added to the solution. The reaction mixture was heated to 80°C–82°C and stirred for 2 h. The progress of the reaction was monitored using TLC (V(Petroleum ether):V(ethyl acetate) = 3:1, RF = 0.3–0.6). The mixture was then concentrated under 45°C, and dichloromethane (2000 mL) was added. After complete mixing, water  was added, followed by the addition of 2 N hydrochloric acid to adjust the PH to the range of 1–2. The lower organic phase was separated and the upper aqueous phase was extracted three times with methylene chloride (500 mL). Combined with the organic phase, washed these using saturated salt water (400 mL) for three times. The resulting crude product, compound 3 (165 g), was further purified using methyl alcohol. 1H NMR  δ 15.46 , 8.75 , 5.43 , 4.87 , 4.41 , 4.14 , 3.89 , 3.72 , 3.14 , 1.33 .Compound 3 (30 g), HATU (38 g), DIPEA (25 g), aminophenylacetylene (23 g), and N,N-dimethylformamide (1500 mL) was mixed at room temperature, and reacted for 12 h. Water (500 mL) was added, and the mixture was stirred, filtered, and dried overnight to obtain compound 4 (34 g), yield 86.5%. 1H NMR  δ 12.53 , 8.69 , 7.95 , 7.58 , 7.38 , 7.22 , 5.42 , 4.87 , 4.40 , 4.14 , 3.80 , 3.13 , 2.81 , 1.29 .This step is a classic reaction involving the condensation of a carboxyl group and an amino group. Compound 4 (0.5 g), TBA (10 mL), water (10 mL), tetrahydrofuran (10 mL), chalcanthite (0.25 g), and sodium ascorbate (0.5 g) were mixed and reacted at 70°C. After reacted completely, dichloromethane (20 mL) was added, and the reaction was then filtered. The aqueous phase was extracted twice with dichloromethane, and the organic phase was combined, dried using anhydrous magnesium sulfate, followed by evaporated. The obtained residues was subsequently recrystallized using methanol, resulting in the final isolation of the target compounds 5a-5s, 6a-6i.This step is the classic of a click compound reaction, which has been previously reported in articles . Azide cFor biological experiments, Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 Medium, Fetal bovine serum (FBS) and penicillin/streptomycin were bought from Gibco . Dimethylsulfoxide (DMSO) was got from Sigma-Aldrich . Enhanced Cell Counting Kit-8, Reactive Oxygen Species (ROS) Assay Kit, Calcein/PI Live/Dead Viability Assay Kit and Giemsa dye were obtained from Beyotime Biotechnology . Annexin V-FITC/Propidium iodide (PI) staining kit was bought form BD Biosciences .2.Human lung cancer cell line H460 and H1299, human liver normal cell line LO2 were bought from ATCC. DMEM or RPMI 1640 was used as the cell culture medium with 10% FBS and 1% penicillin/streptomycin. Cells were maintained in an incubator with 37°C humidified atmosphere and 5% CO50 values were determined using the CCK8 assay kit. Cells were seeded at a density of 5 × 103 cells per well in a 96-well plate. After overnight adhesion, 25 μM of compounds or vehicle control DMSO which all dissolved in the culture medium were added to cells and cultured for 48 h or 72 h. CCK8 reagent, dissolved in the medium, was then added to cells and incubated for 1 h. The absorbance at 450 nm was measured using a microplate spectrophotometer (Thermo). Cell viability was calculated, with the untreated group taken as 100%. For IC50 values determination, different concentrations  of compounds were added to cells for 72 h. Subsequent CCK8 measurement allowed the calculation of inhibition percentage based on cell viability. The IC50 values were computed by the prism statistical software. The CCK-8 assay was conducted three times and the repetitions in each time were at least three. All the values were expressed as Mean ± SD, p < 0.05 was considered statistically significant.Cell viability and ICp < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.Cells with the density of 500 cells per well were seeded in a 6-well plate. Different concentrations  of compounds were added to cells and maintained for a 72- hour duration. And cells were cultured until the monoclonal appeared. Afterward, cells were fixed with 4% paraformaldehyde and stained by Giemsa dye. The plate clone numbers were photographed and counted using a microscope. Values were expressed as Mean ± SEM, 3 cells per well were seeded in a 96-well plate. After overnight incubation, cells were treated with different concentrations  of compounds for 24 h. The LIVE/DEAD Assay Kit was used to dye cells, and cells were observed and photographed under a fluorescent microscope. Live and Dead cell numbers were counted and the ratio of dead to living cells were calculated. Values were expressed as Mean ± SEM, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.Cells with a density of 5 × 105 cells per well were seeded in 6-well plates. After overnight adhesion, cells were treated with different concentrations of compounds for 72 h. Cells were harvested and stained with Annexin V-FITC and propidium iodide. Apoptotic ratio was quantified using the flow cytometry (BD Biosciences) and analyzed using the FlowJo software v10. Values were expressed as Mean ± SD, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.Cells with a density of 3 × 103 cells/well were cultured in a 96-well plate. The cells were treated with different concentrations  of compounds for 24 h. Then the medium was replaced with 10 μM DCFH-DA dissolved in culture medium and incubated for 30min at 37°C in dark. The fluorescent cells were observed and photographed using a fluorescent microscope.Cells with a density of 5 × 10p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.Western blot was used to analyze protein expressions. Cells were cultured in 12-well plates and treated with different concentrations  of compounds for 72 h. Cell proteins were extracted using RIPA buffer with protease/phosphatase inhibitor cocktail (CST). 10%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate proteins and nitrocellulose membranes (Millipore) was used to collected proteins. Antibodies used in western bolt were LC3 , caspase 3 , cyclin D , cyclin E , β-Catenin , γH2AX , PARP , and β-actin . Values were expressed as Mean ± SEM, 1) using formic acid, resulting in the formation of 1-(formylmethyl)-5-methoxy-6-(methoxycarbonyl)-4-oxopyridine-3-carboxylic acid (compound 2). Subsequently, the formic acid was removed by direct vacuum concentration without any additional treatments. Compound 2 was then subjected to a reflux reaction with acetonitrile and (S)-2-aminopropanol, leading to the formation of -6-methoxy-3-methyl-5,7-dioxo-3,5,11,11a-tetrahydro-2H-oxazolopyridopyrazine-8-carboxylic acid (compound 3). Using HATU and DIPEA as coupling agents, compound 3 reacted with 3-aminophenylacetylene to yield terminal alkynes compound 4. Subsequently, compound 4 was subjected to reaction with azides containing various substituents, resulting in the synthesis of 28 novel target compounds, namely, 5a-5s and 6a-6i. The structures of these newly synthesized compounds are presented in 1H NMR, 13C NMR, elemental analyses, and MS  of selected compounds against H460 cells, the results of which were summarized in 5i exhibited the most potent cytotoxicity with an IC50 value of 6.06 μM, followed by compounds 5c and 5s with IC50 values of 9.65 μM, and 13.38 μM, respectively.In order to assess the potential cytotoxicity of the synthesized compounds, we selected two types of human non-small-cell lung cancer cell line, H460 and H1299. Cell viability and cytotoxicity were evaluated using the CCK8 assay . The cel5i, live/dead cell staining was performed using Calcein Acetoxymethyl Ester  and Propidium Iodide (PI) dyes  dyes . Calcein60 cells . In the 60 cells . Similarad cells . In addiad cells  to furth60 cells . Similarells , and visualized using a fluorescent microscope. DCFH-DA has no fluorescence and it can pass through cell membrane freely. After entry the cell, it could hydrolyze by intracellular esterase to DCHF and intracellular ROS can further oxidize DCFH to produce green fluorescent DCF. Results showed a significant augmentation in ROS generation within H460 cells treated with compound 5i (5i could increase ROS levels in H460 cells which may lead to cancer cell apoptosis and cell death.Reactive oxygen species (ROS) levels were determined quantitatively using the probe 2′-7′-dichlorodihydrofluorescin diacetate (H2DCFDA) . Under npound 5i . It is ipound 5i . Previoupound 5i . Our fin5i, and whole cell proteins were collected for analysis. Results demonstrated a significant increase in the expression of ubiquitin like molecule light chain 3 (LC3) , γ-H2AX  , an impoLC3) (5i . This su, γ-H2AX , and Pol, γ-H2AX , after c60 cells . In H12960 cells . Additioells (5i . Notablyells (5i . Treatmeaffected . These raffected . A numbeaffected . In our 5i emerged as the most potent cytotoxic agent, with an IC50 value of 6.06 μM. Besides, compounds 5c and 5s showed IC50 values of 9.65 μM and 13.38 μM, exhibited potential antitumor activities. Further studies found that compound 5i significantly induced cell apoptosis and stimulated ROS generation, which may finally lead to cell death. Western blot analysis revealed the dysregulation of proteins associated with autophagy and DNA damage in either H460 or H1299 cells. These findings suggested that the novel derivatives especially compound 5i holds promise as a potential therapeutic candidate for the treatment of lung cancer, warranting further investigations and explorations. 1,2,3-triazole derivatives have demonstrated potential antitumor activities against various types of tumors, which could be further investigated in our future work. Previous studies have also highlighted diverse activities of 1,2,3-triazoles, including antioxidant, antiviral, and acaricidal properties. In line with our findings, this novel compound induced ROS generation, indicating a potential antioxidant effect. Moving forward, our ongoing investigations will explore additional activities of these novel compounds.This study successfully synthesized a series of novel compounds by incorporating 1,2,3-triazole units into the cabotegravir backbone. The biological evaluations demonstrated that several of these molecular hybrids exhibited potent antitumor activities. Notably, the synthesized compounds displayed stronger cytotoxic effects against H460 cells than H1299 cells, with some compounds achieving cell viability rates below 50% in H460 cells but below 70% in H1299 cells. The effects became more pronounced with an extended duration of treatment. Compounds 5c, 5d, 5i, 5s, 6b, and 6 h showed a cell proliferation inhibition rate exceed 50% against H460 cell lines after 72-h treatment. Among them, compound


Exercising on regular basis provides countless health benefits. To ensure the health, well-being and performance of athletes, optimal indoor air quality, regular maintenance and ventilation in sport facilities are essential.10, PM2.5, and particle number concentration in size range of 20–1,000 nm, i.e., – PNC20-1000 nm), gaseous pollutants  and comfort parameters  in different functional spaces of health clubs (n = 8), under specific occupancy and ventilation restrictions.This study assessed the levels of particulate, down to the ultrafine range (PM10: 1.9–988.5 μg/m3 PM2.5: 1.6–479.3 μg/m3) and number (PNC 1.23 × 103 – 9.14 × 104 #/cm3). Coarse and fine PM indicated a common origin (rs = 0.888–0.909), while PNC showed low–moderate associations with particle mass (rs = 0.264–0.629). In addition, up to twice-higher PM and PNC were detected in cardiofitness & bodybuilding (C&B) areas as these spaces were the most frequented, reinforcing the impacts of occupational activities. In all HCs, TVOCs (0.01–39.67 mg/m3) highly exceeded the existent protection thresholds (1.6–8.9 times) due to the frequent use of cleaning products and disinfectants (2–28 times higher than in previous works). On contrary to PM and PNC, TVOCs were higher (1.1–4.2 times) in studios than in C&B areas, due to the limited ventilations combined with the smaller room areas/volumes. The occupancy restrictions also led to reduced CO2  than previously observed, with the lowest values in HCs with natural airing. Finally, the specific recommendations for RH and T in sport facilities were largely unmet thus emphasizing the need of proper ventilation procedures in these spaces.In all HCs human occupancy resulted in elevated particles (up to 2–3 times than those previously reported), considering mass concentrations (PM All clubs were located in the urban-background and urban-traffic areas of Oporto Metropolitan Area  where vehicle traffic and local industry are the main emission sources in the respective ambient air , 32. ThrIn general, all HCs had a similar organization, comprising main area cardiofitness and bodybuilding (C&B) for cardiovascular fitness  and bodybuilding training . Additionally, they featured studios for group classes , and dressing rooms with bathroom with associated functions. Typically, all HCs also included a bar area furnished with vending machines, dedicated spaces for physical and nutritional assessment offices, and administrative and support spaces . In addition to these standard features, HC2 and HC5 encompassed functional areas for spa and beauty/healthcare services; HC7 included an indoor studio with a hairdresser. Three out of the eight clubs  included indoor swimming pools, along with the necessary supplementary facilities. More detailed descriptions of all HCs are summarized in The indoor air quality (IAQ) monitoring was conducted in October 2020 – November 2021, which coincided with the post-lockdown period in Portugal. During this period, stringent sanitary recommendations were enforced for sport facilities to mitigate the transmission of infections. These recommendations encompassed various aspects including hygiene practices, ventilation strategies, and occupancy limitations , 34. Spe2.23, and CO2) and comfort parameters  were sampled by a multi-parametric probe GrayWolf Sensing Solutions . Particulate matter was continuously monitored by Lighthouse Handheld particle counter , which allowed concurrent monitoring of six different size fractions (N300 nm –10μm). Particle number concentration, in a diameter range between 20 and 1,000 nm (PNC20-1000 nm) was monitored with TSI P-Trak™ condensation particle counter sampler, model UP 8,525 . All pollutants were registered with logging interval of 60 s, resulting in a large set of measurements . Further details on study protocol are presented in Supplementary Text S1.IAQ monitoring was conducted continuously (during 24 h) for a period of 12 days in each club, considering different indoor functional spaces. Gaseous pollutants  and Kolmogorov–Smirnov (n > 30) tests. One sample t test was used for comparison with the current protective thresholds; independent samples t test was used to verify the influence of ventilations. The relationships between the pollutants were analyse by Spearman’s correlation coefficients. Finally linear regression was employed to model and assess the variables potentially affecting indoor air quality. All analysis were conducted using the level of statistical significance p < 0.05.All data were analyzed using descriptive and inferential statistical analysis using Microsoft Excel (Microsoft 365 MSO) and Statistical Package for the Social Sciences . Samples of the study population were independent and the normality of the data was verified using the Shapiro–Wilk  are presented in 10 and PM2.5 in different functional areas across the eight HCs (when occupied). The overall results showed large variations of PM levels between the HCs and the respective functional spaces. Notably, across the eight clubs, PM10 medians (of each HC) ranged between 8.6–48.8 μg/m3 .PM10  and PM2.5  were identified in HC3, being followed by HC7 .When occupied , the hig10 and PM2.5, respectively, when occupied  were observed in C&B than in studios (3 and 25 μg/m3 for PM10 and PM2.5) (expressed as 8 h means) ; PM2.5: (27.4–37.2 μg/m3)]. These results indicated the possibly health risks for the respective occupants. It is noteworthy that in studios the threshold was exceeded only in HC3 (median: 60.7 μg/m3), most likely due to the overall high PM levels  , 37 or O studios ,B. Thoug studios , and as  studios ], the in studios  (with thh means) . The resM levels ,B. Conce10 maxima reached high values, up to 989 μg/m3 whereas it was up to 479 μg/m3 for PM2.5. In agreement with the previous studies (10 concentrations between 6.2–312.4 μg/m3 and PM2.5 between 4.6–196.2 μg/m3.As demonstrated in  studies  26), th, th10 ma studies , 40. Occ studies . For the10 and PM2.5 were positively and significantly correlated in all functional indoor spaces, with high Spearman correlation coefficients (rs) 0.830–0.961 (median of 0.909) and 0.461–0.916 (median 0.888) respectively for C&B and studios  and 2.15 × 104 #/cm3 (HC3), whereas it was 3.39 × 103 #/cm3 (HC5) and 1.78 × 104 #/cm3 (HC3) for the group class studios. These results showed that similar concentrations range in all HCs (103), with the exception of HC3 (C&B: 2.15 × 104 #/cm3 and studios: 1.78 × 104 #/cm3) and HC8 (C&B: 1.01 × 104 #/cm3 and studios: 1.31 × 104 #/cm3) where higher values were found (104). Overall, the highest PNC  were obtained in both functional spaces of HC3. The club unusual layout and direct connection with restaurant areas and the possible emissions (p = 0.948). In addition, it shall be noted that higher PNC were observed in C&B areas than in studios in 62.5% of HCs  in view of these spaces being predominately frequented.Particle number concentrations in all spaces of the eight HCs  showed hmissions  resultedmissions , 45–47. missions , 48. Nev3 #/cm3), (20-1000) when spaces were unoccupied , due to2.5 rs = 0.264, PM10 rs = 0.305; studios: PM2.5 rs = 0.496, PM10r = 0.629), which is understood given the different characteristics and behaviors of both particle modes . Thus, it would be important to identify the individual VOCs in these spaces to better determine the respective associations.In all functional spaces, PNC showed low to moderate associations with both PM .Overall, the results in 3 in HC2 and 2.86 mg/m3 in HC5, while in group class studios the observed concentration varied between 0.27 mg/m3 in (HC1 and HC2) and 5.34 mg/m3 (in HC3). These results showed that on contrary to PM, in 88% of HCs TVOCs concentrations were 1.1–4.2 times higher in group studios that in C&B areas. This was most likely due to the specific ventilation conditions, imposed during the evaluated periods. Furthermore, the group studios volumes  varied between 0.24 mg/m volumes  were muc3) exceeded the protective threshold of 600 μg/m3  and with mechanical ventilation (0.003–12.4 mg/m3). In this study, the lowest TVOCs were observed in HCs with direct connections to the outside  and / or in clubs with natural ventilations (HC6 and HC8): HC2 < HC1 < HC8 < HC6. These results are in agreement with Canha et al.  – 2,040 mg/m3 (HC7). In all HCs, CO2 levels were significantly higher (up to 1.5 times) when occupied; these differences were similar in clubs with natural ventilations and mechanical ventilation (21.7–42.1% in HC6 and HC8 vs. 11.5–50.5% in other HCs). Similar to TVOCs, the lowest CO2 concentrations (HC6) were observed in naturally ventilated spaces ] of 2,250 mg/m3. The stricter recommendation of the American Society of Heating, Refrigerating, and Air-Conditioning Engineers [ASHRAE; 1,800 mg/m3; (2 levels (1.1–1.4 times) were observed in group class studios , with medians of 1,043 mg/m3 in HC1 and 2,538 mg/m3 in HC7, than in C&B areas . Overall, the results observed in this study were lower than those previously reported for health clubs with mechanical ventilation (3) and 3.4 times  higher values. Similarly, Ramos et al. (3), whereas Slezakova et al. (3) and natural ventilation . During the period in this study, the sports facilities were subject to specific rules, also in terms of occupancy  to 79.2 μg/m3 (in HC3) . Regarding the functional spaces, the average ozone levels varied between 34.3 μg/m3 (in HC5) - 68.0 μg/m3 (in HC6) in the C&B areas and 25.5 μg/m3 (in HC6) - 92.4 μg/m3 (in HC3) in the group class studios. In indoor air, ozone results either from infiltrations of ambient air emissions  (p = 0.113). Concerning the occupancy impact, when people were exercising  the observed concentrations were 0.9–1.0 times higher for HC with natural ventilations and 1.0–1.7 times higher for mechanical one. It is necessary to enhance that restricted use of mechanical ventilation system might lead to considerable implications for IAQ  . Similar1 μg/m3)  estimate for IAQ . While t for IAQ .3.4When exercising, breathing and perspiration generate substantial amount of water vapor, which may impact the measured RH in these spaces . The resSimilar to TVOCs, in studios (62.5%) the observed RH were higher than in C&B, ranging from 53.5% (in HC3) - 78.7% (in HC6). In C&B areas with natural ventilation systems (HC6 and HC8), the RH was 1.1–1.2 times lower during the occupancy period than when vacant . For all HCs with mechanical systems, the opposite trend was observed with the RH being 1.0–1.2 times higher when occupied. Hence these results emphasize the importance of ventilations and its maintenance in sport facilities when equipped with mechanical systems in order to assure optimal indoor conditions for its occupants. Furthermore, the prevention of humidity accumulation is especially relevant to have a suitable indoor microenvironment.Regarding the temperature - T, the IFA suggests 18.3–20°C (65–68°F) for spaces where aerobics, cardio, bodybuilding and Pilates activities are carried out and 26.7°C 80°F) for yoga activities, regardless of the season. When occupied , T varie°F for yo410 and PM2.5 originated from the same emission sources (rs of 0.888–0.909). In addition, higher levels (up 2 times for both PM and PNC) were observed in the C&B as these spaces were predominantly frequented, reinforcing the impacts of occupational activity. On the contrary, TVOCs levels were higher when HCs were unoccupied (1.1–2.0 times) emphasizing the importance of ventilation procedures to accumulation of pollutants. In addition, it needs to be emphasized that when occupied TVOCs highly exceeded the protection thresholds (1.6–8.9 times) due to the specific hygiene requirements that implied frequent use of cleaning products and disinfectants in HCs. In order to better characterize the respective health risks, identification and quantification of individual VOCs would be required. The specific rules for occupancy and human distancing in sport facilities led to positive CO2 impacts, with decreased levels than those previously reported for these indoor environments  unfulfilled in all HC. These results emphasized the importance for ventilation procedures in sport facilities when equipped with mechanical systems in order to assure optimal indoor conditions for its occupants.This study assessed IAQ in different functional spaces of eight fitness centers under specific occupancy and ventilation restrictions, showing potential impacts for all pollutants and comfort parameters. Specifically, the obtained results showed that the human occupancy resulted in increased indoor particulate levels in all HCs, considering both particle number (1.2–2.0 times) and mass concentrations (1.1–4.7 times); PMronments , 38, 65;The original contributions presented in the study are included in the article/CP: Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. MC: Funding acquisition, Supervision, Writing – review & editing. SM: Funding acquisition, Project administration, Supervision, Writing – review & editing. KS: Conceptualization, Methodology, Supervision, Writing – original draft, Writing – review & editing, Formal analysis.


Porcine sapelovirus (PSV) is a ubiquitous virus in farmed pigs that is associated with SMEDI syndrome, polioencephalomyelitis, and diarrhea. However, there are few reports on the prevalence and molecular characterization of PSV in Fujian Province, Southern China. In this study, the prevalence of PSV and a poetical combinative strain PSV2020 were characterized using real-time PCR, sequencing, and bioinformatics analysis. As a result, an overall sample prevalence of 30.8% was detected in 260 fecal samples, and a farm prevalence of 76.7% was observed in 30 Fujian pig farms, from 2020 to 2022. Noteably, a high rate of PSV was found in sucking pigs. Bioinformatics analysis showed that the full-length genome of PSV2020 was 7550 bp, and the genetic evolution of its ORF region was closest to the G1 subgroup, which was isolated from Asia and America; the similarity of nucleotides and amino acids to other PSVs was 59.5~88.7% and 51.7~97.0%, respectively. However, VP1 genetic evolution analysis showed a distinct phylogenetic topology from the ORF region; PSV2020 VP1 was closer to the DIAPD5469-10 strain isolated from Italy than strains isolated from Asia and America, which comprise the G1 subgroup based on the ORF region. Amino acid discrepancy analysis illustrated that the PSV2020 VP1 gene inserted twelve additional nucleotides, corresponding to four additional amino acids (STAE) at positions 898–902 AAs. Moreover, a potential recombination signal was observed in the 2A coding region, near the 3′ end of VP1, owing to recombination analysis. Additionally, 3D genetic evolutionary analysis showed that all reference strains demonstrated, to some degree, regional conservation. These results suggested that PSV was highly prevalent in Fujian pig farms, and PSV2020, a PSV-1 genotype strain, showed gene diversity and recombination in evolutionary progress. This study also laid a scientific foundation for the investigation of PSV epidemiology, molecular genetic characteristics, and vaccine development. Porcine sapelovirus (PSV), taxonomically designated sapelovirus A, belongs to the genus sapelovirus within the family Picornaviridae [aviridae . PSV is aviridae . The aveaviridae . Althougaviridae ,4,5,6,7.aviridae . Traditiaviridae . Howeveraviridae ; followiaviridae .PSV infections can cause either asymptomatic co-infection with other enteric pathogens or a symptomatic result in multisystemic wasting syndromes, such as encephalomyelitis, diarrhea, respiratory tract symptoms, and reproductive disorders of sows . ExperimPSV was first identified in England in 1960 and then reported in Japan, America, Republic of Korea, Italy, and other countries ,16,17,18® (Code No: RR02MQ), and pMD 18-T Vector (Code No: 6011) were purchased from TaKaRa Bio Inc. .For the epidemiological survey of PSV, a total of 260 clinical samples, including feces, fecal swabs, and intestine samples, were collected between 2020 and 2022 from sows and suckling, post-weaning, and fattening pigs that were asymptomatic or with clinical diarrhea  from 30 farms in Fujian Province. A description of the studied pig population and the number of collected samples for each farm is shown in Since the 5′ UTR gene region of PSV is highly conserved, it was selected as the molecular target for the real-time PCR. The real-time primer sequences are shown in g for 5 min, and the PSV total RNA (0.2 mL of the supernatant of samples) was isolated using the RNA extraction kit. Then, reverse transcription from RNA to cDNA was performed using the RT kit, stored at −80 °C. Utilizing the cDNA as template and PSV-F and PSV-R as specific primers, amplification was carried out according to the 25 µL reaction system, including Taq Plus DNA Polymerase (5 U/µL), 0.5 µL; 10× PCR Buffer, 2 µL; PSV-F, 0.5 µL; PSV-R, 0.5 µL; dNTP (10 mM), 0.5 µL; MgCl2 (25 mM), 0.5 µL; cDNA, 2 µL; and ddH2O, 17 µL. Reaction conditions of 95 °C for 3 min; 95 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s, 35 cycles, and 72 °C for 8 min were used for full-length elongation. PCR amplificons were analyzed using 1% agarose gel electrophoresis. Target fragments were recovered using a SanPrep kit, then ligated into pMD® 18-T Vector, and recombinant plasmids were transformed into DH-5α competent cells. The ampicillin plate was used for screening the positive recombinant plasmid, and a single colony was selected for the next culture. The positive recombinant plasmid (PSV-T) was obtained by using the SanPrep column plasmid DNA mini extraction kit and was identified via PCR and Sanger sequencing. The standard plasmid was quantified using a NanoDrop2000 spectrophotometer , and copy numbers of PSV-T were calculated following the method described by Lee et al. [7 copies/µL) and stored at −80 °C.Aliquots of 0.5 g of fecal material were re-suspended in 1 mL of ice-cold phosphate-buffered saline (PBS), vortexed for 1 min, and clarified using centrifugation at 12,000× e et al.  then dil2O, 7.8 µL; and standard plasmid (PSV-T), 1 µL. The reaction conditions were 95 °C for 3 min; 95 °C for 10 s, 57 °C for 15 s, and 72 °C for 30 s. The optimized real-time PCR detection method was used to amplify the recombinant plasmid with a concentration of 4.67 × 101~107 copies/µL, repeated three times in succession to obtain the amplification curve and standard curve. The standard logarithm (standard curve) was derived with the common logarithm (lgC) of the standard starting copy number as the abscissa and the cycle threshold  as the ordinate.To obtain the lowest threshold cycle (Ct) and maximum fluorescence intensity, the concentration of the primers and probe and the annealing temperature were optimized. The best parameters were selected after all the reactions had been conducted in triplicate. The optimized real-time PCR system included. 2 × TaqMan Fast qPCR master mix, 10 µL; PSV-F, 0.4 µL; PSV-R, 0.4 µL; PSV-Taq, 0.4 µL; ddH7 copies/µL; each dilution was amplified via real-time PCR with three replicates and independent experiments carried out three times. The repeatability test was carried out with 4.67 × 102, 4.67 × 104, and 4.67 × 106 copies/µL plasmids, repeated three times. The intra- and inter-assay of the method were determined by calculating the coefficient of variation (CV).The DNA or cDNA of CSFV, PRV, PRRSV, PEDV, TGEV, PoRV, PDCoV, and PTV were used as templates for evaluating the specificity of the established real-time PCR. When the curve was above the threshold before 35 cycles had passed, we judged the result to be positive. The detection limit of the established real-time PCR was carried out using tenfold dilution plasmids with a concentration of 4.67~4.67 × 102O, 6.3 µL; cDNA, 1 µL; and Taq polymerase (5 U/µL), 0.2 µL. The reaction conditions were 95 °C for 3 min; 94 °C for 10 s, 55 °C for 15 s, 72 °C for 30 s, 35 cycles, and 72 °C for 7 min. The 3′ RACE experiments included two steps. Firstly, reverse transcription of the RNA into cDNA with 3′ adaptor primer was carried out according to the manufacturer’s instructions, and then nested PCR was performed using 3′ RACE-F1 and 5-3′ outer, or 3′ RACE-F2 and 5-3′ inner, as primers (http://ibs.biocuckoo.org/ (accessed on 26 April 2023).Reverse transcription (RT) was performed using a PrimeScriptTM RT Master Mix reverse transcription kit according to the manufacturer’s recommendations and a primer 3′ adapter. The viral RNA covering the internal genome was amplified via RT-PCR with primers based on the nucleotide sequences of the V13 strain (GenBank ID: NC_003978.1) , accordi primers . 5′ RACE primers . Then, a primers  accordinBioinformatics analyses were performed with 61 strains obtained from the GenBank database . The nuc7 copies/µL  but only showed a significantly amplified signal of PSV , indicat4 to 5.89 × 109 genomic copies per mL of supernatant. Notably, high PSV viral loads  were also identified in sows.PSV was detected in 260 samples across all 30 farms. Globally, 23 out of 30 (76.67%) farms were positive, and the rate of positive samples was 30.8% (80/260). However, different age distribution patterns were observed regarding the positive rates. Remarkably, a 60.0% farm prevalence and 41.18% sample prevalence were observed in suckling pigs, which were higher than those in sows, post-weaning pigs, and fattening pigs . It is nThe PCR amplicons were analyzed using 1% agarose gel electrophoresis, showing that we amplified the 10 target fragments , coverinA phylogenetic tree was constructed based on the alignment of complete ORF nucleotide sequences using data on PSV2020 and other PSVs retrieved from GenBank . PSVs weHomology comparison of the complete ORF of PSV2020 with those of other PSV strains retrieved from GenBank  revealedp = 9.950 × 10−10)  C, indica× 10−10) A. These porcine sapelovirus is a ubiquitous virus in farmed pigs and wild pigs, infections have been associated with a wide spectrum of symptoms. In China, the prevalence of PSV has been shown to range from 20.4% to 46.39% in different provinces, and an outbreak of gastroenteritis associated with respiratory disease and polioencephalomyelitis was reported in Shanghai [Although Shanghai . In the Shanghai . In the Shanghai . PSV infShanghai ,23,24,25Shanghai ,19,24,25Shanghai ,27, withThe PSV genome contains only one open reading frame (ORF), which encodes a polyprotein precursor, which is further cleaved into 12 mature functional proteins by self-encoded proteases ,22. ThroPhylogenetic analysis based on gene sequencing is widely used to determine the taxonomy of picornaviruses ,31,32. PIn this study, similarity plot analysis showed that the structural proteins VP2, VP3, and VP1) of the PSV2020 P1 region were highly similar to the DIAPD5469-10 strain, which was representative of the G2 European subpopulation A, and th, and VP1In conclusion, we have established strong specificity, high sensitivity, and good repeatability from TaqMan-based real-time PCR, indicating that an overall sample prevalence of 30.8% was detected in 260 fecal samples, and a farm prevalence of 76.7% was observed in 30 Fujian pig farms, collected from 2020 to 2022. Meanwhile, a PSV-1 genotype strain, PSV2020, showed gene diversity and recombination events while undergoing genetic evolution. These results also lay a scientific foundation for the investigation of PSV epidemiology, molecular genetic characteristics, and vaccine development.