NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1  can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that  Lung cancer occurrence and mortality ranks top in China and around the world . About 8The Hippo signaling pathway controls growth and organ size, thus its dysregulation often leads to cell overgrowth and tumorigenesis . WW domaNDFIP1 promotes proliferation, invasion, epithelial mesenchymal transition (EMT), or glycolysis in several forms of cancer (Nedd4 family-interacting protein 1 (NDFIP1) is an adaptor of E3 ubiquitin ligases Nedd4 family, linking Nedd4 with target proteins to facilitate the ubiquitination . As prevf cancer . HoweverIn previous reports, NDFIP1 is identified to function in the loading of proteins into exosomes through the binding of WW domain in target proteins to PPxY motif in NDFIP1 . Given tNDFIP1 in NSCLC, the binding of NDFIP1 with TAZ and the recruitment of TAZ, but little YAP into NSCLC exosomes. Then combining evidences from clinical data, in vitro and in vivo experiments, we revealed that lower NDFIP1 led to lower TAZ packaged in exosomes and higher TAZ accumulation in cells, promoting cell proliferation and tumor growth eventually. Taken together, our data have disclosed NDFIP1 as a novel tumor suppressor and proposed a new exosome-related regulatory mechanism of TAZ, providing new insights for the development of biomarkers and treatment strategies of NSCLC.Here, we first verified the low expression of NDFIP1 mRNA expression and NDFIP1 DNA copy number in the different datasets of Oncomine. Decreased NDFIP1 expression levels were found in NSCLC in Hou Lung, Garber Lung, Selamat Lung, and Wachi Lung datasets  and LSQ  cell lines compared with the normal bronchial epithelial cell line, Beas-2B (NDFIP1 mRNA level in SPC-A1-BM cells (NDFIP1 level (top 50%) was associated with better overall survival  and progression-free survival    and 1O).Given that NDFIP1 contains PPxY motifs , and YAPg), cell debris , and extracellular vesicles   were isolated by ultracentrifugation method  and char,000 ×g) . Meanwhi,000 ×g)  levels w,000 ×g) . In addi,000 ×g) . Furtherexosomes  and 2M, exosomes  and 2K. per cell  and S6B,per cell  and S6D.pression , and S7BNDFIP1 in SPC-A1 cells and the knockdown of NDFIP1 in A549 cells resulted in the increase of TAZ protein level, rather than TAZ mRNA level   , and a mut cells  and 4F. ut cells  and S13A in vivo  and S13Bin vitro evidences have indicated NDFIP1 mediates TAZ packaging into exosomes and accumulation in cells, we next test this mechanism in vivo. Expectedly, the protein levels of TAZ were generally upregulated in the NDFIP1 knockout group and greatly decreased in tumors with NDFIP1 overexpression  and the plasma membrane . The forIn conclusion, NDFIP1 plays an essential role in the recruitment of TAZ into exosomes, therefore balancing the levels of intracellular and extracellular TAZ; NDFIP1, as a tumor suppressor, is downregulated in NSCLC, therefore releasing the brake of cell proliferation inhibition. Thus, the new function of NDFIP1 and the new regulatory mechanism of TAZ, uncovered in this study, have implications for the development of biomarkers and treatment strategies of NSCLC.Seventeen pairs of NSCLC tumor and paratumor tissue samples as well as 61 blood samples  were obtained from Shanghai Jiao Tong University-affiliated Shanghai Chest Hospital after surgical resection. The tissue samples were next subjected to IHC and PCR experiments. And blood samples were centrifuged at 3,000 rpm at 4°C for 10 min for serum collection, followed by exosomes isolation.This study was approved by the Ethical Committee of the School of Biomedical Engineering, Shanghai Jiao Tong University and carried out in accordance with the Declaration of Helsinki. All participants were provided written informed consents.2 incubator  for follow-up experiments. SPC-A1-BM is a highly bone metastatic cell line established from SPC-A1 by in vivo selection in BALB/c mouse models  while H520, H1581, HCC95, and H2170 cells were cultured in Roswell Park Memorial Institute  containing 10% fetal bovine serum  and 1% penicillin-streptomycin . Cells were incubated at 37°C in a 5% COe models .g for 16 h at 4°C. For exosomes isolation, cultures were incubated with exosome-free FBS for 24 h prior to collecting the cell culture medium.To deplete bovine exosomes from FBS, FBS was diluted to 20% by conditioned medium and then centrifuged at 120,000 ×g, 4°C, followed by 30 min, 10,000 ×g. Then, the supernatant was transferred into a 6-mL ultracentrifuge tube . The tube was then filled with PBS  and ultracentrifugation was done twice for 70 min at 100,000 ×g, 4°C in a Type 100 Ti swinging-bucket rotor (Beckman). As for conditioned media, it was first centrifuged for 5 min at 500 ×g, 4 ×g, followed by for 30 min at 2,000 ×g, and for 35 min at 10,000 ×g. Then, the supernatant was transferred into 38.5 mL ultracentrifuge tubes  and ultracentrifuged twice for 70 min at 100,000 ×g, 4°C, in an SW 32 Ti swinging-bucket rotor (Beckman). The deposits in each step are, respectively, referred to as intact cells in suspension during cell culture, cell debris, extracellular vesicles (EVs), and exosomes as shown in Exosomes from serum and cell conditioned media were both isolated by ultracentrifugation method. Briefly, the serum was first centrifuged 15 min at 3,000 ×The exosome pellet was resuspended in PBS for transmission electron microscopy . Briefly, 10 μL of exosomes was dripped onto a copper grid . After 1 min of sedimentation, the droplet was sucked out using the air-laid paper. And then, 10 μL of 2% uranyl acetate  solution was dripped onto the same copper grid for negative staining and sucked out again 1 min later.7 particles/mL). For each measurement, 3–5 mL of the diluted sample was injected into the instrument, and the concentration of this sample as well as the size distribution were measured by the machine software (ZetaView 8.03.04.01).The size distribution of exosomes was measured by ZetaView (Particle Metrix). In brief, the exosome pellet was resuspended in a proper volume of PBS to achieve the optimal detectable concentration  containing protease inhibitor cocktail , phosphatase inhibitor , and phenylmethyl sulfonyl fluoride . And then the protein concentration was measured using a BCA assay kit . As for exosomal TAZ level comparison, equal number of exosomes measured by NTA was used for Western blotting. Each sample with same amount of total proteins or exosomes was mixed with a loading buffer (5×) and heated at 95°C for 5 min. The denatured proteins were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel  and separated at constant 120 V for 80 min. Then, proteins were transferred to a nitrocellulose membrane  at constant 300 mA for 1.5 h. The membrane was blocked with 5% nonfat milk powder suspended in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature. The blots were probed with primary antibodies: TSG101 , HSP70 , ALIX , CD63 , CD9 , CD81 , GM130 , RAB27B , YAP/TAZ , TAZ , NDFIP1 , β-Tubulin , GAPDH , Lamin A/C , PCNA , and these primary antibodies were subsequently probed with appropriate horseradish peroxidase conjugated secondary antimouse or rabbit antibodies . Finally, the blots were visualized using the enhanced chemiluminescence  and chemiluminescence imaging system . The intensity of each band was analyzed by ImageJ software.Immunofluorescence (IF) was performed for co-localization. Generally, cells were seeded on coverslips in a 24-well plate and cultured, then fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, cells were rinsed three times with PBS followed by permeabilizing in 0.5% Triton X-100 for 10 min and blocking in 1% bovine serum albumin (BSA) in PBS for 1 h. And then cells were incubated with primary antibody  at 1:200 dilutions at 4°C overnight. Alternatively, cells were incubated with rabbit IgG conjugated with Alexa Fluor 488  and mouse IgG conjugated with Alexa Fluor 594  or mouse IgG conjugated with Alexa Fluor 488  and rabbit IgG conjugated with Alexa Fluor 594 . Cell nuclei were stained with DAPI  for 5 min at room temperature. Stained cells were photographed under an immunofluorescence microscope  or a confocal microscope  and qualified with ImageJ software.g centrifugation at 4°C for 10 min, the supernatant was divided into 200, 400, and 400 μL  was performed according to the manufacturer’s instruction . Briefly, cells grown in 10-cm dishes were washed once with PBS and lysed on ice for 15 min with 1-mL lysis buffer. Following 17,000 ×d 400 μL , and theRelative copy number and mRNA levels of NDFIP1 in different lung cancer datasets were downloaded from Oncomine database and analyzed using the Graphpad software. Kaplan-Meier overall survival (OS) and progression-free survival (PFS) curve of NSCLC patients with low or high expression of NDFIP1 was generated using Kaplan–Meier Plotter .Paraformaldehyde (4%)-fixed tissues were embedded in paraffin followed by sectioning (5 μm in thickness) with a microtome . After xylene dewaxing, PFA fixation, 0.3% Triton X-100 permeabilization, tissues were blocked with 10% goat serum for 1 h at room temperature and incubated with primary antibodies  at 4°C overnight. After three times washing, tissues were incubated with secondary antibodies  for 1 h at room temperature. Diaminobenzidine hydrogen peroxide (Sigma) was the chromogen, and the counterstaining was carried out with 0.5% hematoxylin. The intensity of immunohistochemistry (IHC) staining was qualified by an IHC Profiler in ImageJ software.2O was mixed with 5× gDNA eraser buffer and gDNA eraser at 42°C for 2 min to remove gDNA. Secondly, the sample was mixed with 5× PrimeScript Buffer 2, PrimeScript RT Enzyme Mix 1, and RT Primer Mix at 37°C for 15 min followed by 85°C for 5 s and 4°C forever. Finally, quantitative real-time PCR was performed on a Fast Real-Time PCR System  by using TB Green Premix Ex TaqTM II . Briefly, the synthesized cDNA was mixed with ROX Reference Dye II, TB green Premix Ex Taq II, and forward and reverse primer at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 34 s, and ending at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The sequence of each primer used was summarized as follows: GAPDH-F is 5ʹ-GGAGCGAGATCCCTCCAAAAT-3ʹ; GAPDH-R is 5ʹ-GGCTGTTGTCATACTTCTCATGG-3ʹ; NDFIP1-F is 5ʹ-CCAGCTGAGGATAGGAAACG-3ʹ; NDFIP1-R is 5ʹ-GGCATCTTCCGAACTTTTGC-3ʹ; TAZ-F is 5ʹ-CACCCGGCCTTGATGTTTAT-3ʹ; TAZ-R is 5ʹ-TGTGTGGTGTGCTATCAGGT-3ʹ.Total RNA was first extracted from cells with RNAiso Plus reagent . And then PrimerScript reverse transcriptase (RT) reagent kit with gDNA Eraser  was used to synthesize cDNA from total RNA. Briefly, total RNA diluted by RNase-free ddHNDFIP1 knockdown plasmid (shNDFIP1) and control plasmid (shNC), NDFIP1 overexpression plasmid (NDFIP1-OE) and negative control plasmid (PCDH) were purchased from OBIO . Human NDFIP1 knockout Crispr-cas9 plasmid (sgNDFIP1) and negative control plasmid (sgNC) were purchased from ZORINBIO . To generate the lentivirus containing plasmid, HEK 293T cells were co-transfected with VSVG, pDelta, and the indicated plasmid. And the concentrated lentivirus was obtained from conditioned medium after ultracentrifugation. For infection, the viral stock was added into SPC-A1 cells with 7 μg/mL polybrene. NDFIP1-OE and PCDH GFP+ cells can be subsequently selected by fluorescence-activated cell sorting  while sgNDFIP1 and sgNC cells need to be selected by 1 μg/mL of puromycin with a monoclonal cultivation by inoculating into a 96-well plate.Human Lentiviruses expressing shRNA were purchased from OBIO . The sequence of the shRAB27B was 5ʹ-CCCAAATTCATCACTACAGTA-3ʹ.g for 5 min. At this point, the supernatant is the cytoplasmic fraction. Then 50-μL nuclear protein extraction buffer was added into the pellet followed by an intermittent vortex for 30 min and centrifugation at 12,000 ×g for 10 min. The resultant supernatant is the nuclear fraction.Nuclear and cytoplasmic protein extraction were performed according to the manufacturer’s instruction . Briefly, cells grown in a 24-well plate were washed once with PBS and scraped for centrifugation to obtain cell pellet. Then 200 μL cytoplasmic protein extraction buffer A was added into cell pellet and vortexed for several seconds. After 15 min, 10-μL cytoplasmic protein extraction buffer A was added and vortexed for several seconds followed by centrifugation at 12,000 ×Cells were seeded in a 6-well plate in the absence or presence of drugs. For degradation rate assessment, 12.5 μg/mL of cycloheximide  was added for 1–6 h. For the inhibition of lysosome or proteasome-mediated degradation pathway, 5 μmol/L MG132  or 50 μmol/L chloroquine  was added for 24 h. To inhibit exosome secretion, 5 μmol/L GW4869  was added for 24 h.Cells were transfected with siRNAs using RNAFit Reagent  according to the manufacturer’s protocol. Briefly, the cells were incubated with the complex of siRNA and RNAFit reagent, and the gene knockdown efficiency was assessed after 48 h. siRNAs was synthesized by RiboBio. The siTAZ sequence was 5ʹ-GGTACTTCCTCAATCACAT-3ʹ.Cells were transfected with plasmids using Lipofectamine 3000  according to the manufacturer’s protocol. Briefly, the cells were incubated with the complex of plasmids, P3000 and Lipofectamine 3000 reagent, and the plasmid transfection efficiency was assessed after 48 h. The TAZ WT  and TAZ ΔWW (3XFlag pCMV5-TOPO TAZ (∂WW), 24811) plasmids were purchased from Addgene.n = 6). As for siTAZ interference, different stable expression cells were seeded in three 96-well plates at a density of 5,000 cells per well (n = 6) at 2 days after transfection. Every 24 h, 10 μL of CCK8 solution was added to each well in one plate and incubated for 90 min at 37°C. The OD value at 450 nm was measured by the microplate reader .For the proliferation assay, the cell viability at different times (every 24 h) was examined by CCK-8 assay kit . Briefly, different stable expression cells were seeded in five 96-well plates at a density of 2,000 cells per well (n = 6) were subcutaneously injected into the right flanks with a total of 5 × 106 stably transfected SPC-A1 cells in a volume of 50 µL. Tumor sizes and body weights were monitored every 3 days. All the mice were housed in the specific pathogen-free animal room of Shanghai Jiao Tong University. At day 21, mice were sacrificed and the blood samples were isolated by eyeball extirpating. Meanwhile, tumors were harvested and cut into three pieces for protein extraction, RNA extraction, and paraffin embedding.BALB/c nude mice (This study was approved by the Ethical Committee of the School of Biomedical Engineering, Shanghai Jiao Tong University, and all procedures were performed following the regulations and internal biosafety and bioethics guidelines of Med-X Research Institute, Shanghai Jiao Tong University.t-test or ANOVA were chosen to analyze the statistical significance. The probability value below 0.05 was considered significant.All statistical analyses were performed using the GraphPad Prism 6 software. Data are presented as mean ± SD, and the paired or unpaired pwac017_suppl_Supplementary_MaterialClick here for additional data file.


Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations—KrasG12D and Trp53R172H—present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in   Studies on the phenotypic transition and its role in pancreatic carcinogenesis revealed a considerable capacity of pancreatic acinar cells (PACs) to undergo morphogenetic changes and, as a result, acquire a duct cell-like phenotype. The consensus has been established that PC arises equally often from the acinar and ductal epithelia.y in PC6 .7 rarely alerts patients who may simply ignore symptoms of a “clinically silent” disease in the pancreas. However, the pathophysiological mechanisms that drive the transition of normal epithelial cells to the premalignant stages of pancreatic carcinogenesis are not well understood.The Kras-driven malignant transformation not only perpetuates pancreatic carcinogenesis but also induces clonal epithelial loss of function , thus re2+-rich fluid.2+ serve as a physiological signal for enzyme release from acinar epithelial cells. However, when these signals are dysregulated10 and escalate into global and sustained Ca2+ elevations, digestive enzymes become activated prematurely in situ in PACs,10 causing necrosis and the release of damage-associated molecular patterns (DAMPs) from injured and dead cells.11 Surprisingly, the role of pathophysiological Ca2+ signals is much less evident in the development of PC, compared to other pancreatic pathologies also characterized by the inflammatory background, such as acute pancreatitis (AP).In normal pancreatic epithelia, the secretion of digestive hydrolases by acinar cells occurs via exocytosis concurrent with the secretion of Ca2+ signals in PACs isolated from KPC  mice, a genetic animal model of pancreatic carcinogenesis.12 Owing to the presence of mutations in single alleles of the Kras and Trp53 genes under the control of the Pdx1-Cre construct, KPC mice recapitulate a multistep  model of PC development  that in normal PACs evoke cytosolic Ca2+ oscillation. In another set of experiments, we applied menadione (Men) that elicits a more irregular pattern of intracellular Ca2+ elevations in PACs: oscillations, calcium transients, formation of the cytosolic Ca2+ plateau, or a combination of all of the above. A detailed numerical analysis of the area under response curves, as well as a direct comparison of the proportions of different types of cytosolic Ca2+ signal patterns, shows that, on average, Ca2+ elevations in KPC cells were more prominent and prolonged compared to controls . The application of a Ca2+ extrusion protocol revealed a significantly slower Ca2+ efflux from the stores in KPC PACs compared to control-Cre cells, but the expression levels of the plasma membrane Ca2+ ATPase (PMCA) remained unchanged. Finally, the analysis of the cellular energy metabolism showed decreased mitochondrial respiration in KPC cells compared to normal PACs. Collectively, these findings suggest an impaired Ca2+ handling in premalignant secretory epithelium expressing KrasG12D and Trp53R172H, even when no morphological changes are yet evident in PACs.This has prompted us to study cytosolic Caelopment . Using Km HEPES, phenol red free, were purchased from Agilent, USA; horse anti-mouse IgG Antibody (H + L), peroxidase (#PI-2000), was purchased from Vector, USA; Matrigel, growth factor reduced, phenol red-free, was purchased from Corning, USA; NucView 488 & RedDot 2 Apoptosis and Necrosis Kit was purchased from Biotium, USA; TAE buffer was purchased from Bio-Rad, USA; and Thapsiargin was purchased from Cayman, USA.2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 3,12-Bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid (EGTA), 3-(morpholin-4-yl)propane-1-sulfonic acid (MOPS), acetylcholine, anti-vinculin mouse monoclonal antibody (#V9131), bicinchoninic acid (BCA), calcium chloride, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), collagenase type IV, dimethyl sulfoxide (DMSO), eosin with phloxine, ethanol, hematoxylin, Immobilon Western Chemiluminescent HRP Substrate, magnesium chloride, menadione, oligomycin, potassium chloride, radioimmunoprecipitation assay buffer (RIPA), rotenone with antimycin A, sodium chloride, Tris-buffered saline (TBS), and Tween 20 were purchased from Sigma-Aldrich/Merck, USA; Agarose, DNA Perfect Leader, and SimplySafe loading dye were purchased from EURx, Poland; Amersham Hybond 0.2 PVDF blotting membranes were purchased from Cytiva, UK; anti-calcium pump pan PMCA ATPase mouse monoclonal antibody (#ab2825) was purchased from Abcam, UK; CellTiter-Glo 3D Cell Viability Assay was purchased from Promega, USA; cOmplete mini, EDTA-free protease inhibitor cocktail, and KAPA mouse genotyping mix were purchased from Roche Diagnostics GmbH, Germany; distilled water, Genomic Mini isolation kit, and Tris buffer were purchased from A&A Biotechnology, Poland; Fluo-4 AM, Hoechst   33258, NuPAGE 4%-12% Bis-Tris Gels, and Nunc 96 Well White/Clear Bottom Plates, TC Surface were purchased from Thermo Fisher Scientific, USA; glucose, glutamine, pyruvate, Seahorse XF Cell Mito Stress Test Kit, and XF DMEM Medium pH 7.4, 5 m 5 mm tail tip samples were collected from young animals after weaning. DNA was extracted using a Genomic Mini isolation kit, eluted in Tris buffer, and stored for a long term at −20°C.The animal genotype was confirmed by polymerase chain reaction (PCR) using DNA extracted from the tail tips and specific primers under the conditions described in Kras and Pdx1-Cre; 3%: Trp53, respectively), 1% or 3% agarose gels were made by dissolving agarose in TAE buffer. SimplySafe loading dye was then added to allow DNA detection in agarose gels. Casted gels were positioned in a Sub-Cell GT Horizontal Electrophoresis System , covered with TAE buffer, loaded with 10 μL of DNA or 3 μL of DNA Perfect Leader, and the samples were run at 90 V for 30-60 min. The samples were visualized using a ChemiDoc XRS + Imaging System , and the genotypes were determined based on product sizes given in For the separation of PCR products 12 mouse breeding pair (mixed background) was transferred from Cardiff University to the Jagiellonian University institutional animal units in 2018. The animals were kept in a 12 h light/dark regimen, in individually ventilated cages (up to 5 mice) with aspen wood bedding material and environmental enrichment, and free access to food (standard rodent chow diet) and water. All procedures involving animals were performed in accordance with the ARRIVA guidelines,13 and ultrasonographic tumor detection/palpation was carried out according to license No. 113/2020, issued by the II Local Ethics Committee for Animal Experimentation . Up to 9-month-old tumor-free KPC mice , or age/sex-matched control animals , were humanely killed by cervical dislocation14 (for cytosolic Ca2+ measurements) or CO2 inhalation (other experiments). The pancreatic tissue was removed for further experimental procedures.The KPC (LSL-Kras16In our mouse model of pancreatic carcinogenesis (KPC mice), the presence of pancreatic abnormalities  was examined by abdominal palpation on a weekly basis. In certain cases, KPC mice were also monitored by ultrasound imaging (USG) using a Vevo 2100 high-frequency, high-resolution digital imaging platform , according to the previously described protocols.18 inspected under a light microscope DMi8 , photographed, and analyzed.Pancreatic tissues were fixed in formalin , embedded in paraffin, and cut into 4 μm sections. The sections were then stained using our standard hematoxylin and eosin (HE) staining protocol,m) NaCl, 140; KCl, 4.7; HEPES, 10.0; MgCl2, 1.0, and glucose, 10.0 was supplemented with 1.0 m m Ca2+19 (in Ca2+ extrusion protocol: 10.0 m m Ca2+14). PACs were isolated as described previously14 from the pancreata of control-Cre mice, or tumor-free pancreatic tissues of KPC mice, and loaded with a Ca2+-sensitive fluorescent indicator Fluo-4 AM . After loading, PACs were transferred to a flow chamber, and the experiments were performed in continuous perfusion with NaHEPES-based solutions. The cells were visualized using an LSM-880 AiryScan AxioVert confocal microscope equipped with an EC Plan-Neofluar 40×/1.30 Oil DIC M27 objective . Excitation was set to 488 nm laser light and emission to 593-630 nm. A series of images were recorded at 256 × 256 pixel resolution; two consecutive frames were averaged, and the time resolution was 1 image per 2 s. Fluorescence signals were plotted as F/F0, where F0 was an averaged signal from the first 10 baseline images, normalized as previously described.20Unless otherwise stated, extracellular buffer NaHEPES containing  for 10 min in the absence of extracellular Ca2+. Next, again in Ca2+-free extracellular buffer, 10 μm Tg was applied for 200 s, and then the extracellular Ca2+ concentration was increased to 10 m m, which induced Ca2+ influx to the cytosol. Once a cytosolic plateau was reached, extracellular Ca2+ was reduced to 0 in the presence of 2 m m EGTA. This abrupt removal of extracellular Ca2+ unmasked Ca2+ extrusion across the plasma membrane. This phase of the response was further analyzed and compared between PACs isolated from control-Cre and KPC mice. For every recorded Ca2+ trace, the normalized fluorescence that corresponds to half the decrease between the maximum and minimum F/F0 values was calculated as follows: F1/2 = Fmin + (Fmax − Fmin)/2, according to the previously described methods.21 The time values corresponding to Fmax (tmax), and F1/2 (t1/2) were calculated from the linear fit to the extrusion phase. Finally, t1/2 was calculated as the difference between t(F1/2) and tmax.For the CaKPC and control-Cre mouse pancreatic tissue samples were homogenized using a TissueLyser , in RIPA buffer supplemented with a protein inhibitor cocktail. The protein concentration was assessed by the BCA assay. 15 μg protein samples were loaded into the NuPAGE 4%-12% Bis-Tris precast gels and resolved  using SDS/PAGE electrophoresis in MOPS buffer. The overnight transfer (25 V) in a Mini Trans-Blot Cell  was performed onto the 0.2 μm PVDF blotting membranes. Then the membranes were blocked in 5% non-fat dry milk in TBS supplemented with 0.05% Tween-20 (TBS-T). The membranes were incubated  with primary antibodies: anti-calcium pump pan PMCA ATPase mouse monoclonal antibody (1:1000) or anti-vinculin mouse monoclonal antibody (1:5000), and then were washed with TBS-T . Next, the membranes were incubated  with the secondary antibody: horse anti-mouse IgG Antibody (H + L), peroxidase (1:5000), and then were washed with TBS-T . Protein detection was performed in a ChemiDoc Imaging System  using Immobilon Western Chemiluminescent HRP Substrate. The ImageLab software was used for the densitometric analysis.m Men  in extracellular NaHEPES buffer, containing 1 m m Ca2+ or Ca2+-free. One hour before the incubation end, the cells were stained with the NucView 488 & RedDot 2 Apoptosis and Necrosis Kit, which reports caspase-3/7 activity with a green fluorescence signal and the presence of dead cells with far-red fluorescence signal, according to the manufacturer’s instructions. PACs were transferred to a glass-bottom chamber and imaged using a DMI8 fluorescence microscope equipped with an HC PL APO 40x/1.30 OIL objective and a DFC7000GT camera . The following parameters were applied for NucView 488 imaging: excitation 490 nm, 5% illumination power, an FITC emission filter; and for RedDot 2 imaging: excitation 660 nm, 9% illumination power, and a CY5 emission filter. A total of 15 random images were collected, and live, apoptotic, and necrotic cells were counted, and their numbers were averaged and presented as percentages of the total ± SEM. (2) For the ATP-based cell viability measurements, a CellTiter-Glo 3D Cell Viability Assay was used to quantify ATP levels in acinar cells freshly isolated from KPC and control-Cre mice, following the manufacturer’s protocol. Briefly, equal volumes of extracellular NaHEPES buffer containing PACs and CellTiter-Glo 3D Reagent were transferred to a 96 Well White/Clear Bottom Plate, shaken for 5 min, and left for 25 min at RT to stabilize the samples. The luminescence signals were recorded using an Infinite M200 Plate Reader , and the results were normalized post-assay to the protein levels assessed by the BCA assay run in a reference plate, using the same cell aliquot as for the assay.(1) For cell death assessments, PACs were stimulated for 2 h at RT with 60 μ23 with some modifications. Briefly, 8-well cell culture plates were thin-coated with Matrigel 12 h before the measurements. PACs were isolated on ice, transferred to the plates, and allowed to attach. Next, extracellular NaHEPES buffer was replaced with XF DMEM pH 7.4 medium, supplemented with 10 m m glucose, 1 m m pyruvate, and 2 m m glutamine. The cells were incubated for 15-30 min at 37°C in a CO2-free I5110 dry oven , and then a mitochondrial stress test was performed using a Seahorse Bioscience XF HS Mini Analyzer . The cells were injected with sequential fluxes of:23 1 µg/mL oligomycin applied to inhibit ATP synthase and cause a rapid hyperpolarization of the mitochondrial membranes; 0.3 µm FCCP, an uncoupling agent of oxidative phosphorylation, which restores proton transport across the mitochondrial inner membrane; and 2 µg/mL mix of antimycin A and rotenone, an inhibitor of complexes I and III, and thus a blocker of mitochondrial phosphorylation. The results were normalized post-assay to the cell numbers calculated by counting cell nuclei stained with Hoechst 33258 and analyzed using the QuPath software.24 Basal respiration, ATP production, maximal respiration, proton leak, spare respiratory capacity, non-mitochondrial oxygen consumption, and coupling efficiency were calculated with normalized oxygen consumption rate (OCR) changes.Metabolic functions of acinar cells isolated from KPC and control mice were assessed using a Seahorse XF Cell Mito Stress Test Kit, according to the previously described protocols,2+ traces), box and whisker plots  showing individual data points together with a median and/or a mean, 10 × 10 dot plots (response abundancies), or bar charts (cell death assay) showing a mean ± SEM. For statistical analysis, appropriate parametric or nonparametric tests were applied using the GraphPad Prism 8.0.1 software (detailed description in the figure legends), and the significance threshold was set at P < .05.Quantitative results were presented as averaged/representative linear plots (CaKras (G12D) and Trp53 (R172H), under the control of the Pdx1-Cre system, which promotes the development of PC. Control cells were obtained from Cre mice expressing the Pdx1-Cre construct, but in these animals, both alleles of Kras and Trp53 genes were free from cancer-inducing point mutations. KPC and control-Cre PACs were isolated by collagenase digestion of the mouse pancreata, and they were essentially identical in terms of their morphological features. In order to characterize the capacity of KPC or control-Cre PACs to maintain physiological Ca2+ signals evoked by ACh, we first applied low 50 n m concentrations of the stimulant for 600 s and recorded Ca2+ responses induced in control-Cre  in the oscillatory responses recorded in KPC PACs compared to control-Cre acinar cells (m Ca2+) for 200 s, PACs were treated with a supramaximal concentration of 10 μm ACh in order to trigger large responses. Global Ca2+ releases recorded in PACs isolated from control-Cre mice represented a typical single Ca2+ response characterized by a rapid Ca2+-release phase followed by a steady decline in cytosolic Ca2+ levels (P < .001) areas under the responses recorded in KPC vs control-Cre PACs  that carry driver mutations in trol-Cre  and KPC trol-Cre . While cmulation , in KPC esponses . As evidmplitude  and B. Decreased , but theecreased  and B. Aar cells , with ex+ levels . Similar+ levels , but in Cre PACs , with me2+ signals in KPC and Cre acinar cells. Historically used as a cost-effective substitute for vitamin K in fortified foods, Men evokes cytoplasmic Ca2+ elevations that take the form of oscillatory responses, Ca2+ transients, or Ca2+ plateaus . Owing to the presence of double bonds in its naphthoquinone ring, Men can initiate the production of reactive oxygen species in PACs. Cell stimulation with 60 μm Men elicited cytosolic Ca2+ responses in control-Cre , a single spike followed by the plateau (13%), and the plateau alone (25%). The corresponding values in control-Cre cells were much lower: 22% of the overall plateau-type responses, that is, 10% of transients on top of the plateau, 4% of a single spike followed by the plateau, and the plateau alone in 8% of total responses recorded in control-Cre cells 21 (2+-free extracellular buffer supplemented with thapsigargin (Tg), a potent inhibitor of the endomembrane Ca2+ pump, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), to empty intracellular Ca2+ stores. The extracellular Ca2+ concentration was then changed to 10 m m, which caused an influx of Ca2+ into the cytosol —this was evidenced by the increase in cytosolic Ca2+ until a prolonged plateau was formed in both control-Cre and KPC cells  sodium-calcium exchanger (NCX) activity,25 almost the entire Ca2+ extrusion is dependent on the plasma membrane Ca2+ ATPase (PMCA) in these cells.21 When extracellular Ca2+ was removed, KPC cells not only maintained a Ca2+ plateau longer than control-Cre cells but were also characterized by slower rates of Ca2+ extrusion than control PACs. The analysis of cytosolic Ca2+ clearance revealed a statistically significant difference (****P < .0001) in the PMCA-driven extrusion between KPC and control-Cre cells, here described as the time required for Ca2+ concentration to decrease by half the maximum amplitude (t1/2). The t1/2 values were much higher in KPC cells compared to control-Cre PACs: 47.91 vs 33.91 [s], respectively (2+ extrusion across the plasma membrane was slower than in control-Cre cells and this effect was not due to decreased expression of PMCA (In order to compare the speed of Cations)21 . We firsPC cells . Abrupt PC cells . The conectively . Importaectively  and D. T of PMCA .2+ extrusion rates found in KPC acinar cells compared to control-Cre PACs, we investigated mitochondrial respiration in these cells (P < .05) in KPC cells compared to normal PACs (6 cells]), spare respiratory capacity (30598.66 vs 28917.73 [pmol/min/106 cells]), non-mitochondrial oxygen consumption (6009.10 vs 4867.59 [pmol/min/106 cells]), proton leak (10 845.15 vs 11 641.88 [pmol/min/106 cells]), and coupling efficiency (66 vs 57 [%]) were not markedly altered in KPC cells  area under the response curve to 50 n m ACh, but a significantly lower (***P < .001) area under the transient-type responses to supramaximal 10 μm doses of ACh in PACs isolated from KPC mice compared to control-Cre cells , wherein the mutant KRAS allele was deleted owing to the homologous recombination.26 Colorectal cancer cells were found to have SOCE repressed, and that protected these mutant cells from the apoptotic stimuli and cell death.26 Active removal of Ca2+ from the cytosol to the extracellular environment across the biological membranes is essential for maintaining low resting cytosolic Ca2+ levels.27 Processes of Ca2+ extrusion by Ca2+ pumps require constitutive hydrolysis of ATP, but this is the metabolic “price to pay” in order to prevent accumulation (overload) of Ca2+ in the cytosol and cell death.28 Notably, sustained elevations in cytosolic Ca2+ reduce ATP synthesis.29 Since KPC acinar cells were characterized by significantly lower rates of Ca2+ extrusion (****P < .0001) than control-Cre PACs , and so was the production of ATP (*P < .05) in PACs isolated from KPC mice  in mitochondrial respiration (G12D. This, in turn, decreases their capacity for fine-tuning the compartmental Ca2+ concentration, resulting in a less regular pattern of the cytosolic Ca2+ signals than in control-Cre acinar cells (Secretory acinar epithelia may undergo a phenotypic transformation into duct-like cellular structures, which, under the condition of cell stress and the presence of driver mutations in genes that encode Kras and Trp53 proteins, contribute toward pancreatic carcinogenesis . Here we signals -4, the c signals , and mit signals . First, al cells  and B. Sre cells . Althougre cells , the prore cells  and B. Wre cells  and B, tsolution . James eCre PACs , and PMCCre PACs  and D, wCre PACs . ProductCre PACs  and founKPC mice . Of notecapacity , the spacapacity , were noxtrusion  and B. Axtrusion  and C, axtrusion , it is ppiration , but alsar cells  and 4.


Radioresistance is a major cause of radiotherapy failure among patients with cervical cancer (CC), the fourth most common cause of cancer mortality in women worldwide. Traditional CC cell lines lose intra-tumoral heterogeneity, posing a challenge for radioresistance research. Meanwhile, conditional reprogramming (CR) maintains intra-tumoral heterogeneity and complexity, as well as the genomic and clinical characteristics of original cells and tissues. Three radioresistant and two radiosensitive primary CC cell lines were developed under CR conditions from patient specimens, and their characteristics were verified via immunofluorescence, growth kinetics, clone forming assay, xenografting, and immunohistochemistry. The CR cell lines had homogenous characteristics with original tumor tissues and maintained radiosensitivity in vitro and in vivo, while also maintaining intra-tumoral heterogeneity according to single-cell RNA sequencing analysis. Upon further investigation, 20.83% of cells in radioresistant CR cell lines aggregated in the G2/M cell cycle phase, which is sensitive to radiation, compared to 38.1% of cells in radiosensitive CR cell lines. This study established three radioresistant and two radiosensitive CC cell lines through CR, which will benefit further research investigating radiosensitivity in CC. Our present study may provide an ideal model for research on development of radioresistance and potential therapeutic targets in CC.The online version contains supplementary material available at 10.1007/s13577-023-00882-x. Cervical cancer (CC) is the fourth most common malignancy among women, accounting for approximately 7% of cancer cases , 2. AlthMost CC researches are based on traditional, established cell lines, such as SiHa, C33A, and ME180 etc. which are homologous in both morphology and phenotype and proliferate easily in vitro. However, loss of intra-tumoral heterogeneity is the main obstacle in developing drugs, including radiosensitizers, and research investigating radioresistance . A compaConditional reprogramming (CR) methods have been used to culture tumors from patients with CC with the support of feeder cells and conditional medium, enabling infinite proliferation in vitro . Whole-eThe development of high-throughput approaches, such as single-cell RNA sequencing (scRNA-seq), has enabled analysis of the molecular characteristics of individual cells in tumors. Nevertheless, the transcriptional spectrum of radioresistant and radiosensitive CC cells has not yet been investigated. The goal of current study was to establish radioresistant and radiosensitive CR cell lines from CC specimens and verified their characteristics in vitro and in vivo. For the first time, this study proclaims the molecular characteristics and biological changes of radioresistant and radiosensitive CC cells via scRNA-seq, providing a greater understanding of radiosensitivity that will benefit predictions of prognosis and radiotherapy response.Three radioresistant CC specimens were collected from local recurrent lesions of patients who received radiotherapy within 12 months and without re-irradiation or other treatments. Two radiosensitive CC specimens were collected from patients at diagnosis who achieved clinical remission after radiotherapy and remained disease-free survival for at least 12 months.2 incubator. All experiments were performed using mycoplasma-free cells. After reaching 80% confluence, the primary cells were passaged with 0.02% EDTA  and 0.05% trypsin/EDTA  consequently.CC tumor tissues were excised and cell suspensions were prepared following the protocol described by Liu et al. . BrieflyComplete F medium was prepared using DMEM , 10% fetal bovine serum , 1% 100 × Pen-Strep , 1% 100 × L-glutamine , 5 µg/mL insulin , 250 ng/mL amphotericin B , 10 µg/mL gentamicin , 0.1 nM cholera toxin , 0.125 ng/mL EGF , 25 ng/mL hydrocortisone  and 10 µM ROCK inhibitor Y-27632 .4 cells were seeded onto a 6-well plate in triplicate, and cell numbers were counted at 1, 3, 5, and 7 days. The growth rate and population doubling time were calculated from the growth curves.Cellular morphology was examined and photographed using a phase-contrast microscope . The population doubling times were calculated for cells in early passages. Briefly, 1 × 10Equal numbers of cells were plated in triplicate onto 6-well plates for 24 h, then received radiotherapy of 0, 2, 4, 6, 8 Gy. The plates were incubated at 37 °C for 2–3 weeks. Then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 0.05% crystal violet. Colonies with > 50 cells were counted and photographed.Genomic DNA from primary cell lines and corresponding peripheral blood samples of patients was isolated using the Tissue DNA Kit . Matching comparison of DNA fingerprinting including a total of 21 STR loci  was conducted between peripheral blood samples and CR cell lines, as well as between the CR cell lines and data for traditional cell lines in the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Japanese Collection of Research Bioresources (JCRB) and RIKEN databases.6 cell suspensions which were prepared in 100 μL DMEM were conducted on left flank of the hindleg of anesthetized female mice. Injected mice were observed and the tumors were examined every three days. IHC straining was performed following the general procedure using the IHC kit .The animal research was performed under the NIH Guidelines for the Care and Use of Laboratory Animals and supervised by the Animal Care Committee of Tongji Medical College. In vivo primary cell lines were injected into 4–6 weeks old BALB/c nude mice . Briefly, subcutaneous injections of 5 × 104 cells were seeded on a chamber slide  in a 24-well plate for 24 h, followed by washing 3 times with PBS before being fixed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 for 15 min, the cells were incubated with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then cells were covered with primary antibodies against CK5 , Ki67 , p63 , and p16 , respectively, overnight at 4 °C. After 3 times washes, the secondary antibody  was added on the slides for 1 h. Nuclei were stained with DAPI . Chamber slides were analyzed and imaged by fluorescence microscope .A total of 5 × 10Raw disembarkation data quality statistics, cell ranger, cell refiltration, dimensionality reduction and clustering, differential genes and functional enrichment analysis, pseudo-time trajectory analysis, gene set variation analysis, CNV evaluation and transcriptional factor prediction were described in the Supplementary Methods .p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Results were represented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Data shown were representatives of at least three independent experiments, which showed similar results.Statistical significance was determined by the Student’s t-test or two-way ANOVA using GraphPad Prism 7.0 software , and presented as *The primary radioresistant CR cell lines  4, UCR7, and UCR8) and radiosensitive CR cell lines  14, and UCS19) formed cancer nests when cultured with irradiated feeder cells . However, the STR loci in the CR cell lines differed from those of traditional cell lines reported in the ATCC, DSMZ, JCRB, and RIKEN databases.Next, xenografts of CR cell lines were established in mice and their tumorigenicity was verified. The xenografts kept the radiosensitivity of PDTs after irradiation Fig. a, b, exhTo investigate the crucial oncogenes or pathways associated with radiosensitivity in CC, CR cell lines UCR4 and UCS19 were selected to conduct scRNA-seq using 10 × Genomics Chromium platform. High-dimensional data were visualized into low dimensional space by t-distributed stochastic neighbor embedding (t-SNE). Cells with similar characteristics were classified into a cluster according to the algorithm of t-SNE, and the identified clusters were displayed. A mixture of human CR cell lines and murine feeder cells were captured Fig. a, c. A tIn general, cells in different cell cycle phases exhibit different radiosensitivity. Previous studies have shown that cells were most sensitive to irradiation during mitosis and in the G2 phase, less sensitive in the G1 phase and least sensitive in the latter S phase . The CR Next, the scRNA-seq results for UCR4 and UCS19 were combined to better understand the radiosensitivity of CC cells, yielding 13 cell clusters using the graph-based clustering method  were addressed in state 1, whereas the majority of radiosensitive cell clusters were placed in state 4. Branched expression analysis modeling (BEAM) indicated that cell cluster 8 was at one end of the pseudotemporal trajectory, while cell cluster 9 was located at the other end , these cell lines may serve as a promising platform for cancer research, including studies exploring cancer biology, high-throughput drug screening, personalized treatment, and biobanking repositories. Radioresistant cells are in G1 and S phases and increase the FoxO signaling and oxidative stress pathway to support the cells after irradiation. This study infers that USP7 can be the potential radiosensitization targets after careful scientific researches in the future.Supplementary file1 (PDF 1094 KB)Supplementary file3 (XLSX 32 KB)Below is the link to the electronic supplementary material.


IFIT2, IRF1, and XAF1. Therefore, this novel approach using a global gene expression analysis may shed light on the pleiotropic effects of palmitic acid on astrocytes, and provide a basis for future studies addressed to elucidate these responses in neurodegenerative conditions, which is highly valuable for the design of therapeutic strategies.Chronic intake of a high-fat diet increases saturated fatty acids in the brain causing the progression of neurodegenerative diseases. Palmitic acid is a free fatty acid abundant in the diet that at high concentrations may penetrate the blood–brain barrier and stimulate the production of pro-inflammatory cytokines, leading to inflammation in astrocytes. The use of the synthetic neurosteroid tibolone in protection against fatty acid toxicity is emerging, but its transcriptional effects on palmitic acid–induced lipotoxicity remain unclear. Herein, we performed a transcriptome profiling of normal human astrocytes to investigate the molecular mechanisms by which palmitic acid causes cellular damage to astrocytes, and whether tibolone could reverse its detrimental effects. Astrocytes undergo a profound transcriptional change at 2 mM palmitic acid, affecting the expression of 739 genes, 366 upregulated and 373 downregulated. However, tibolone at 10 nM does not entirely reverse palmitic acid effects. Additionally, the protein–protein interaction reveals two novel gene clustering modules. The first module involves astrocyte defense responses by upregulation of pathways associated with antiviral innate immunity, and the second is linked to lipid metabolism. Our data suggest that activation of viral response signaling pathways might be so far, the initial molecular mechanism of astrocytes in response to a lipotoxic insult by palmitic acid, triggered particularly upon increased expression levels of The online version contains supplementary material available at 10.1007/s12035-023-03366-z. Astrocytes are located in the entire central nervous system (CNS), playing different roles such as regulation of homeostasis and synapses; they participate in inflammatory processes and contribute to the maintenance of the blood–brain barrier (BBB), among others. Due to these important functions, astrocytes have been related to the development of several diseases such as neurodegenerative and neuropsychiatric disorders (ND) . AstrocyIn order to know the possible role of astrocytes in the development of ND, a large number of studies have analyzed the effects of several molecules in in vitro and in vivo models –9. NowadOn the other hand, previous studies have found that tibolone (TIB), a synthetic steroid with estrogenic, progestogenic, and androgenic actions, protects astrocytes against the detrimental effects induced by PA. For instance, TIB attenuates inflammatory responses induced by PA ; it reduConsidering the relevance of PA to elucidate the role of astrocytes in relation to ND, this study analyzes the transcriptome profile of normal human astrocytes (NHA) to investigate the molecular mechanisms of lipotoxic-triggering stimuli by PA, and whether TIB might reverse its detrimental effects, according to conditions previously standardized by . Interes2. The cells were seeded at 5000 cells/cm2 and kept for 12 days until they reached 80% of confluence using the astrocyte basal medium (ABM)  supplemented with SingleQuots supplements .NHA cells were obtained from Lonza . Three different batches of NHA cells: (i) 0000514417 , (ii) 00005656712 , and (iii) 0000612736  were grown at 37 °C with a humidified atmosphere and 5% COThe experimental model employed in the current work was previously described by . BrieflyThe controls for these experimental conditions were the following: (i) untreated cells (DMEM) were used as a control for TIB treatment; and (ii) a vehicle (VH) containing 1.35% bovine serum albumin (BSA) , and 2 mM carnitine  was considered as a control for PA treatment, and the combination of TIB plus PA.The total RNA was isolated from 30 samples of cultured astrocytes, namely from the 3 treatments , alongside the 2 controls , which were carried out in 3 biological replicates per duplicate, using RNeasy mini kit  according to the manufacturer’s recommendations Table . To remoWe analyzed the RNA-seq data from astrocytes with the “rnsaseq” (v3.6) pipeline implemented in nf-core . An initM-values (TMM) normalization ) , using tns [MF])  pathway ns [MF]) , with th(v1.0.2) . GO term(v1.0.2) , and the(v1.0.2) .k-core = 2, and max.depth = 100. The hub genes were screened out by cytoHubba plug-in (v0.1) [The up- and downregulated DEGs in GO terms were employed to establish the protein–protein interaction (PPI) network. PPI network was constructed from the STRING database (v11.5)  using Cyn (v0.1) , which iGAPDH) gene was used as housekeeping gene for normalization. Statistical analysis was carried out in the GraphPad Prism (v8.0.1) software . An unpaired t-test was used to compare the treatments and a p-value < 0.05 was considered significant.For this analysis, the three batches of NHA cells were treated with PA and VH as described above, and RNA was isolated from three technical replicates for each treatment. RNA was isolated using the RNeasy mini kit  according to the manufacturer’s recommendations. These samples were quantified through a NanoDrop 2000 , and treated with DNase I . Next, cDNA was generated using the OneScript® Plus cDNA Synthesis Kit , employing 13 μL of RNA at 20 ng/μL, 0.5 μM Oligo(dT), 0.5 mM dNTP, 1 × RT buffer, and 1 μL of reverse transcriptase following the manufacturer’s recommendations. Before running the quantitative PCR (RT-qPCR), a dilution of 1:2 of the cDNA was done. RT-qPCR was performed with the SensiFAST™ SYBR No-ROX Kit , which was mixed with 1 µL of cDNA, 400 nM of each primer (forward and reverse), and water was added for a total volume of 10 µL. The PCR program consisted of a cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 60 s, followed by melting analysis. The annealing temperature and the primer sequence for each gene analyzed are described in Table The viability of NHA is altered by 2 mM PA for 24 h Fig. ; at the DUSP1), which perhaps might be induced by TIB.Although TIBPA with PA and TIB with DMEM did not show DEGs, the comparison between TIBPA and VH showed a higher number of DEGs than PA. Taking into account these results, we implemented the strategy of cross-referencing DEGs in order to explore whether TIB could be modulating the expression of specific genes. Therefore, further comparisons between TIB with VH, and DMEM with VH were performed. This resulted in 32 DEGs, where 15 were up-  comprised the cytokine family such as C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 9 (CXCL9), and C–C motif chemokine ligand 8 (CCL8). In contrast, downregulated DEGs ranged in three more enriched terms, as follows: (i) actin filament organization (GO:0007015), (ii) regulation of actin filament-based process (GO:0032970), and (iii) positive regulation of cytoskeleton organization (GO:0051495)  included Leiomodin 1 (LMOD1), Actin Alpha Cardiac Muscle 1 (ACTC1), and Refilin A (RFLNA)  Fig. , whose DA) Table .Fig. 3CoCXCL10 and pro-inflammatory genes such as interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), and radical S-adenosyl methionine domain containing 2 (RSAD2), considered the most significant DEGs (log2FC = 5.66 to 4.61). Regarding the second most enriched pathway, lipid metabolism, we detected steroid and cholesterol biosynthesis as well as the peroxisome proliferator–activated receptor (PPAR) signaling pathway. In addition, DEGs involved in fat body induction (log2FC = 4.62 to 3.42), such as angiopoietin Like 4 (ANGPTL4), matrix metallopeptidase 1 (MMP1), and fatty acid binding protein 3 (FABP3) were highlighted. Collectively, the enrichment analyses suggest that PA may induce pro-inflammatory cytokine expression by upregulating pathways of antiviral innate immunity as well as genes related to lipid metabolism in human astrocytes.KEGG and Reactome were used to elucidate the most enriched pathways in the dysregulated astrocytes. Even though diverse pathways were uncovered in the databases for upregulated DEGs, most of them hinted at two main processes: (i) antiviral innate immunity pathways; and (ii) lipid metabolism Fig. . Among tMX2) as the seed protein  as seed protein , and subsequently removed redundant proteins, retrieving 380 of them. The PPI network from STRING was composed of 778 edges and 417 nodes, with an average local clustering coefficient of 0.36 Fig. A. Additiein Fig. B. In addein Fig. . Conversein Fig. C. Based ein Fig. . InteresIFIT2, IRF1, XAF1, MX1, IFI35, STAT1, IFI6, and IFI27  with CytoHubba. Upon intersection of the UpSet diagram, 8 common hub genes were found: IFIT2, IRF1, and XAF1 were increased by PA in comparison to VH    by means of Toll-like receptors (TLRs) and retinoic acid-inducible gene – I (RIG-I) . InteresAPOE protein [β- and γ-secretase, a mechanism associated with AD [SREBP2 expression [SREBP2 might explain the link between saturated fatty acid and ND. Moreover, our results highlight that the TLR4 receptor is key for the induction of PA effects since several genes are associated with pathways that need its activation.The second module in the PPI was associated with lipid metabolism, mainly cholesterol biosynthesis. Cholesterol is part of membranes and is a precursor of signaling molecules, impacting processes such as neurotransmission, and synaptic formation. In the brain, cholesterol should be synthesized in situ mainly by astrocytes, and its dysregulation has been related to ND, including the altered transport by  protein , 55. Bes with AD . Anotherpression . ConsideMX2 and SREBP2. Seed proteins facilitate the understanding of pathogenic mechanisms that trigger the onset and progression of diseases [MX2 is important for an antiviral response, which is induced by interferons [MX2 is also increased by an inflammatory stimulus [MX2 was upregulated [MX2 protein was detected in most cases compared to controls [SREBP2, a gene involved in cholesterol biosynthesis, was upregulated by PA in NHA cells. This finding was similar by another study on mouse neuroblastoma cells containing the double mutant AβPP69 [SREBP1 and SREBP2 genes. Thus, our results are relevant because these genes confirm the effects observed in previous studies. Nevertheless, further analysis will be important to elucidate the role of these seed proteins in inflammatory processes or activation in astrocytes.In the PPI analysis, two seed proteins were identified, diseases . Based oerferons . Interesstimulus . Moreoveegulated . Likewiscontrols . These dt AβPP69 , where PDUSP1 gene. This gene is involved in mechanisms of dephosphorylation of MAP kinase MAPK1/ERK2 [DUSP1 is a common gene induced by astrocytes under different stressful stimuli [Finally, we observed that TIB could modulate the expression of the PK1/ERK2 . Previou stimuli . Therefo stimuli , our fin stimuli . These d stimuli , 66, or  stimuli . Moreove stimuli , which i stimuli .In this work, we performed the first transcriptome-wide investigation of the impact of saturated fatty acids and to what extent they respond to protective functions against inflammation in NHA. We demonstrate that at 2 mM PA astrocytes suffer an increased transcriptional alteration, which initially activates a defensive response against lipotoxicity to viral-like mechanisms. It also underscores the widespread immune-metabolic dysregulation induced by PA on astrocytes, whereby TIB was not able to fully reverse the lipotoxic impact of this saturated fatty acid. These findings may shed light on the pleiotropic effects of PA on astrocytes, in which high concentrations of this fatty acid are a risk factor for the development of different ND. Further studies are needed to elucidate the complete role of saturated fatty acids in ND, which will be relevant for testing and designing new therapeutic strategies.Supplementary Figures (DOCX 2.62 MB)Supplementary Table 1 (DOCX 12 KB)Supplementary Table 2 (DOCX 13 KB)Supplementary Table 3 (DOCX 15 KB)Supplementary Table 4 (XLSX 168 KB)Supplementary Table 5 (XLSX 50 KB)Supplementary Table 6 (XLSX 78 KB)Below is the link to the electronic supplementary material.


In Masaaki Kusunose et al. (2015),Figure 1(g) has been replaced by the correct image shown below.Figure 2(f) has been replaced by the correct image shown below.Figure S2(B) has been replaced by the correct image shown below.The label of “G4A Δ tail” in Figure S2(e), S2(f), S2(g), and S2(h) should be corrected to “G4A tail only”. Figure legend of S2 is correct and remained same.The authors apologize for the errors. Masaaki Kusunose, Naozumi Hashimoto, Motohiro Kimura, Daisuke Aoyama, Koji Sakamoto, Shinichi Miyazaki, Akira Ando, Norihito Omote, Kazuyoshi Imaizumi, Tsutomu Kawabe, Yoshinori Hasegawa agree with this notice. Ryo Ogata disagrees with this notice.This correction does not affect the results or conclusions drawn from the data in any way.


Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the  As a key component of the innate immune system, neutrophils are generally considered rapid responders in the first line of defense against pathogen invasion. They are the predominant population among leukocytes and can clear pathogens by a number of mechanisms, including phagocytosis, production of reactive oxygen species (ROSs) and other antimicrobial products, as well as neutrophil extracellular traps (NETs) ,2. The rThe human microbiota is the aggregate of more than 100 trillion symbiotic microorganisms that live on and within the body, including bacteria, archaea, eukaryotic viruses and bacteriophages (phages) ,6. It isPseudomonas aeruginosa phage PAK_P1, which has been thoroughly investigated by our team as an efficient candidate for treating experimental pulmonary infection in mice [Pakpunavirus genus that includes, so far, only phages infecting P. aeruginosa [Moreover, to address the rise of multidrug-resistant infections, phage therapy, the use of phages as antibacterials, is supported by an increasing number of successful case reports in Europe and USA ,16,17. T in mice ,20,21. Truginosa . The synruginosa .P. aeruginosa phage PAK_P1 with human peripheral blood neutrophils, resting and ex-vivo activated, and measured a large panel of neutrophil responses. Using increasing neutrophil:phage ratios up to 1:10,000, we found no evidence supporting the fact that phage PAK_P1 affects phagocytosis, oxidative burst, or NETosis. The only significant signal that was detected was a moderate increase of IL-8 at the highest neutrophil:phage ratio.Here, we co-incubated the P. aeruginosa strain PAK [g for 30 min; (2) tangential-flow ultrafiltration, diafiltration in TN buffer  and concentration; (3) cesium chloride step density gradient ultracentrifugation followed by 24 h PBS dialysis; (4) endotoxin removal by 3 passages through an Endotrap Spin Column , as per the manufacturers’ protocol; and (5) 0.22 µm syringe filter sterilization followed by 4 °C until use [−1 of phage stocks) with the EndoZyme II Recombinant Factor C kit , according to the manufacturer’s protocol. Purified phage stocks (4 × 1011 plaque-forming unit mL−1) and dilutions in Hank’s balanced saline solution  were titrated on lawns of strain PAK.rain PAK  was cultntil use . The end6 mL−1 in HBSS, and purity >98% and viability >99% was confirmed by flow cytometry, as previously reported [Neutrophils were separated from human peripheral blood collected from healthy donors on EDTA by negative magnetic sorting , according to the manufacturer’s instructions. Neutrophils were washed once in HBSS and resuspended at 3 × 10reported .6 mL−1, which was chosen according to standard conditions previously reported [−1 Zymosan A (Dectin-1 agonist), 5 µg mL−1 Staphylococcus aureus peptidoglycan , or 100 µg mL−1 ovalbumin/anti-ovalbumin immune complex at a 1:5 ratio in PBS (FcγRs agonist), all added at the same time as phages, respectively. All stimuli were from Sigma-Aldrich .Equal volumes (50 µL) of phage solution or buffer control and neutrophils were mixed to give a final neutrophil concentration of 1.5 × 10reported ,26. Inflreported ,27: eith2, H20), cells were incubated with fluorochrome-conjugated monoclonal antibodies at 4 °C for 30 min in darkness using standard procedures [After incubation at 37 °C in a standard cell culture incubator  conjugates at 5–50 µg mLreported . Phagocy6 mL−1) were suspended in HBSS in the presence of luminol (100 μM) for 10 min at 37 °C. Cells were then stimulated with phages , with or without Zymosan A (5 µg mL−1) or PMA (25 nM). Chemiluminescence was evaluated with a luminometer , where light emission was recorded in relative luminescence units (RLUs) during 40 min at 37 °C. The area under the curve (AUC) was calculated for each sample tested in triplicate.The production of reactive oxygen species (ROSs) was evaluated by chemiluminescence, as previously described . Briefly6 mL−1 neutrophils were co-incubated in a cell-culture incubator at 37 °C, 5% CO2, H20, with HBSS (negative control), phage PAK_P1 alone , or PAK_P1 with either PMA, A23187, PAF, or IC for 3 h in the presence of 5 µM Sytox Green (Thermo Fischer Scientific). Samples were analyzed by a TristarTM LB941 microplate reader (Berthold Technologies). Fluorescence was monitored every 15 min, and the fluorescence of the unstimulated sample was subtracted as background from each sample tested in triplicate.Extracellular DNA release was measured by fluorescence, as described previously . Briefly2, for 18 h. Cell-free supernatant was collected, and IL-8 levels were measured by sandwich ELISA , according to the manufacturers’ instructions, with a Multiskan EX spectrophotometer (Thermo Fischer Scientific).Neutrophils were co-incubated with HBSS (negative control), phage PAK_P1 alone , or PAK_P1 with either PAF or PGN at 37 °C, 5% CO6.mL−1) were co-incubated with phages at multiple cell:phage ratios  at 37 °C, 5% CO2 for 3 h or 18 h and 5 µL of FITC Annexin V , according to the manufacturer’s instructions. Staining with Annexin V was used in conjunction with the vital dye 7-amino-actinomycin (7-AAD) to show membrane permeability by flow cytometric analysis. In addition, cell viability was evaluated using Trypan blue staining (Sigma-Aldrich).To determine the percentage of cells actively undergoing apoptosis (apoptotic rate), neutrophils . Results are shown as the mean ± SEM, and p < 0.01; We first established the potential effects on resting neutrophils of purified phage PAK_P1 compared to the HBSS diluent control. Phage PAK_P1 was found to neither induce nor delay neutrophil apoptosis after either short (3 h) or long (18 h) co-incubations a,b. In aBeyond characterizing phage effects on resting blood neutrophils from healthy controls, we also questioned whether phages could potentially upregulate activation to other agonists, resulting in exacerbated inflammation. Indeed, it was suggested that certain phages induce downregulation of inflammatory responses . To evalS. aureus PGN activation of neutrophils was not modulated during phage co-exposure compared to unexposed controls  c,d. Lastcontrols e.Together, phage PAK_P1 did not significantly modulate the main functions of activated neutrophils with either weak or strong physiological or artificial agonists.P. aeruginosa phage PAK_P1 triggered low IL-8 (CXCL8) production in freshly isolated human blood neutrophils. However, phage exposure did not further affect resting neutrophil apoptosis or induce necrosis, oxidative burst, and NET release. Activation-induced neutrophil effector responses were also unaffected by phage PAK_P1 co-exposure. Our findings contrast with those by Przerwa et al., who found that neutrophil exposure to the Escherichia coli phage T4 elicited weak oxidative burst, a critical antimicrobial mechanism of neutrophils [E. coli cells or E. coli lipopolysaccharide (LPS) dampened neutrophil ROS production [S. aureus phage A3/r did not increase granule marker expression in neutrophils [9 PFU of PAK_P1 did not elicit inflammatory cytokine responses [In this study, we show that a high concentration of purified trophils . Furtheroduction . Howevertrophils . Previouesponses . Howeveresponses . It appe–Barr virus (EBV), have been shown to also stimulate IL-8 production. However, a major distinction is that other neutrophil effector responses are elicited, such as apoptosis and oxidative burst [Phage PAK_P1 at a high cell:phage ratio of 1:10,000 induced a significant twofold increase in pro-inflammatory chemokine IL-8 release . IL-8 plve burst ,36. Indeve burst ,38,39. E−1 endotoxin. For comparison, most in vitro IL-8 secretion studies of co-incubated isolated human neutrophils had between 10 ng mL−1 and 1 µg mL−1 of endotoxin to induce expected levels of effector responses [However, a major challenge to deciphering anti-phage immune responses is the separation of phages from bacterial cell debris, such as endotoxin , peptidoglycan, exotoxins, flagella, nucleic acids and other compounds. If not adequately removed, these gross impurities could trigger inflammatory responses. Importantly, neutrophils express an abundance of bacteria-specific receptors, including the LPS-sensing TLR 4 . Phage Pesponses ,41. In aesponses , but we esponses .Although neutrophils are the first and predominant immune cell population recruited to an infected site, it remains unclear if this occurs when triggered by phage signals. Importantly, our results suggest that phages are unlikely to stimulate neutrophils into inadvertently releasing their intracellular toxic contents and causing collateral tissues damage. As a major component of the human microbiota , phages P. aeruginosa phage PAK_P1 does not influence the inflammatory functions of human neutrophils. Therefore, adverse reactions to this phage used as an antibacterial agent should be limited. However, in the context of some diseases, such as cystic fibrosis, one must keep in mind that other inflammatory factors may modulate this phage-induced IL-8 release. While most phages may display similar safety characteristics, it is likely that given the diversity of phages, some could elicit stronger responses, which justifies innate immune response monitoring during phage treatments. The interaction between human immune cells and phages, which either make up part of the human microbiota or are administered as human therapeutics, remains underappreciated in relation to the vast genomic diversity of phages ,46. Our


The OPTRELL (Biosense Webster) multielectrode array mapping catheter includes 48 electrodes symmetrically distributed on 6 splines. A unique feature is an additional unipolar reference electrode  located in the distal end of the irrigation lumen, serving as an internal close unipolar reference electrode to reduce far-field unipolar signals. The catheter provides visualization of real-time conduction vectors for better understanding of beat-by-beat wave propagation over the catheter surface, showing the direction of most significant change in local activation times and highlighting areas of slow conduction and scarring. An enhanced bipolar mapping module enables selection of the measurement either along or across the splines, whichever best conforms with wave propagation. Studies in a swine model demonstrated the ability of this catheter to provide high-density and high-resolution mapping in complex substrates , 2. We aThe OPTIMUM study  was a prospective, a multicenter, a single-arm, and an open-label study. Participants with scar-related atrial tachycardia (AT), paroxysmal or persistent atrial fibrillation (PAF or PsAF), ventricular tachycardia (VT), or premature ventricular complexes (PVCs) underwent preablation mapping with the study catheter Fig. , followeBetween August 25 and October 26, 2021, 31 participants were enrolled and mapped . Mean ± standard deviation (SD) age was 59.8 ± 14.4 years and 71% were male. Mean ± SD duration of preablation mapping was 29.9 ± 17.8, 20.0 ± 20.0, and 26.7 ± 8.8 min for AT, PsAF, and PAF, respectively, and 122.2 ± 43.9 and 51.3 ± 28.3 min for VT and PVC, respectively; mean total procedural time was 101.2 ± 32.5, 170.0, 102.5 ± 24.9, 223.7 ± 53.1, and 185.0 ± 25.5 min, respectively. Fast anatomical mapping was performed for all participants. Voltage mapping was performed in all redo-PAF, PsAF, and VT procedures and most AT and PVC procedures. Local activation-time mapping was most often performed for AT, VT, and PVC procedures.No SAEs were related to the study catheter. Three participants reported 4 SAEs unrelated to the study catheter . All participants recovered or resolved at follow-up. There were no occurrences of cardiac perforation, thromboembolic events, catheter entanglement with cardiac structure, or device malfunction. Primary effectiveness endpoint was achieved in 100% of participants.Almost all operators rated highly (≥ 5) on noise reduction in bipolar maps (30/31 operators) and short learning curve (27/31 operators). Operators rated the study catheter highly on: arrhythmogenicity versus PENTARAY (31/31 operators) and versus other multielectrode catheters (25/25 operators); design and coverage for confirming PVI versus PENTARAY (18/21 operators) and versus other multielectrode catheters (17/21 operators); and ability to characterize tissue versus PENTARAY (31/31 operators) and versus other multielectrode catheters (25/25 operators).This first-in-human investigation of the novel multielectrode array mapping catheter provided clinical support for the results observed in preclinical studies. In a swine model, average voltage amplitudes between 2 sampling areas were comparable when using either standard or direction-aware bipolar mapping, suggesting minimal impact of bipolar map directionality. Minimizing directionality has the potential benefit of improving ability to recognize surviving myocardial bundles in the scar [In patients with complex cardiac arrhythmias, identification of the mechanisms maintaining or sustaining arrhythmia is a critical aspect of ablation procedures, and successful identification of these targets facilitates therapy . The emeOther grid-type catheters have recently been investigated in larger patient populations. Hsu et al. evaluated the EnSite Precision cardiac mapping system, which can be used with a variety of electrophysiology catheters; in that study, 101 of a total 925 procedures were performed with the Advisor HD Grid catheter . The EnSLimitations of this study include the small sample size of 31 participants split across several arrhythmia subgroups, specifically chosen to demonstrate the performance of the catheter in the atria and ventricles. The purpose of the study was to show the safety and feasibility of the novel multielectrode array catheter in both atrial and ventricular arrhythmias as a first step before comparing it with other catheters. Subsequent studies including larger populations, comparison with other mapping catheters, and long-term outcomes of ablation will determine the performance of this catheter and its abilities in standard clinical use.


Streptococcus suis is a bacterium that can cause infections in pigs and humans. Although oxidative stress is common occurrence during bacterial growth and infection, the regulation networks of S. suis under oxidative stress remain poorly understood. To address this, we utilized RNA-Seq to reveal the transcriptional landscape of S. suis in response to H2O2 stress. We identified novel genes responsible for S. suis resistance to oxidative stress, including those involved in DNA repair or protection, and essential for the biosynthesis of amino acids and nucleic acids. In addition, we found that a novel aquaporin, Aagp, belonging to atypical aquaglyceroporins and widely distributed in diverse S. suis serotypes, plays a crucial role during H2O2 stress. By performing oxidative stress assays and measuring the intracellular H2O2 concentrations of the wild-type strain and Aagp mutants during H2O2 stress, we found that Aagp facilitated H2O2 efflux. Additionally, we found that Aagp might be involved in glycerol transport, as shown by the growth inhibition and H2O2 production in the presence of glycerol. Mice infection experiments indicated that Aagp contributed to S. suis virulence. This study contributes to understanding the mechanism of S. suis oxidative stress response, S. suis pathogenesis, and the function of aquaporins in prokaryotes. Streptococcus suis can cause systemic diseases such as septicaemia and meningitis in pigs. It is also considered as a zoonotic pathogen for humans in close contact with infected pigs or contaminated by-products [S. suis may encounter oxidative stress. Reactive oxygen species (ROS) like hydroxyl radicals (HO•), hydrogen peroxide (H2O2), and superoxide anion (O2−) are generated during many cellular activities [2− and H2O2 result from one-electron and two-electron reduction of O2, respectively [2O2 can oxidize Fe2+ in ferritin, leading to inactivation of the enzymes and releasing Fe3+, and then Fe3+ is reduced to Fe2+ in the intracellular environment [2+ reacts with H2O2, Fenton chemistry is occurred and generates highly-reactive HO• [2−, H2O2, and HO• can cause cell damage by oxidizing amino acids, DNA, and lipids [S. suis has developed various mechanisms that are regulated by transcriptional regulators, including PerR, Rex, SpxA1, FlpS, and SrtR, which target genes encoding superoxide dismutase, catalase, thioredoxin, glutathione reductase, iron uptake (Fur), and ferritin [products . During tivities . O2− andectively ,6. H2O2 ironment . When Fetive HO• . O2−, H2d lipids ,8. When d lipids . In addid lipids . In ordeferritin . HoweverS. suis. Our findings revealed a comprehensive defence network in response to H2O2 stress, and we identified a novel aquaporin, Aagp, that plays a crucial role in facilitating S. suis H2O2 efflux and contributes to virulence.In this study, we performed RNA-sequencing (RNA-Seq) analysis to explore the global oxidative stress response of S. suis strains and plasmids. S. suis serotype 9 virulent strain GZ0565 was isolated from a meningitis pig [S. suis strains were cultured in Todd-Hewitt broth  and plated on an agar (THA) medium containing 6% (vol/vol) sheep blood at 37 °C with 5% CO2. Escherichia coli strains were grown in Luria-Bertani  broth at 37 °C. Antibiotics were added when in need as follows: spectinomycin , 50 μg/mL for E. coli, and 100 μg/mL for S. suis, 10% (w/v) sucrose  for S. suis.Supplementary Table S1 lists itis pig . S. suisS. suis strains were grown in THB to mid-exponential phase (OD600 = 0.6), washed twice in PBS, and then transferred (with a dilution of 1:100) to a 50 mL flask with 10 mL of 1/3 diluted THB or 1/3 diluted THB containing glycerol with a final concentration of 10 mM or 100 mM. The growth rates were measured at OD600 at 37 °C in a shaking incubator.For studying glycerol utilization, S. suis strain GZ0565 was grown in THB to reach the mid-exponential phase. For H2O2 treatment group, when the bacteria had reached the mid-exponential phase, a final concentration of 25 mM H2O2 was added to THB medium and incubated for 25 min. After incubation, the cultures were centrifuged at 5000×g for 10 min at 4 °C, and the bacterial pellets were collected. The total RNA was extracted using the FastRNA Pro Blue Kit  according to our previous report [p < 0.01 and the absolute value of Fold change ≥ 2 were used to identify the differentially expressed genes.To serve as a control group, s report . The exts report . Sequencs report . The gens report . The thrAagp (BFP66_RS01250) in strain GZ0565 background using S. suis-E. coli shuttle plasmid pSET4s was described in our previous study [mut) by inserting single “G” base into its coding sequence (CDS), causing a frameshift that rendered Aagp non-functional. This mutation strain was constructed through a two-step natural transformation [Aagp with SacB-Spc cassette and resulted in sucrose sensitive and spectinomycin resistant; step II was the process of cassette replacement for single base insertion via the negative selection on sucrose THA plate . We also employed the same methodology to construct deletions of exodeoxyribonuclease III , AguB (BFP66_RS06520), metQ (BFP66_RS08225) in strain GZ0565 background, as well as Aagp in serotype 2 strain P1/7 background (AagpP1/7). Supplementary Table S2 contains a list of primers used for constructing all these strains.The detailed protocol for deleting us study . In addiormation . Step I S. suis strains were challenged with H2O2. Wild-type strain GZ0565 (WT), ΔAagp, and Aagpmut were cultured to the mid-exponential phase. Then the final concentration of 25 mM H2O2 was added to THB. After incubation at 37 °C for 25 min, the number of bacteria was determined by spreading serial dilutions on THA plates. The survival rate at each time point was calculated as CFU at time point 25 min/CFU at time point 0. The functions of exo III, AguB, metQ and PAagp1/7 in response to oxidative stress were evaluated using the same methodology. Experiments were performed with three biological replicates. The statistical analysis was performed with a two-tailed unpaired t test.To assess the role of the Aagp in oxidative stress response, Aagp, and Aagpmut to H2O2, the Oxford cup supplemented with 10 µL 1 M H2O2 was conducted on the THA plates. After incubation at 37 °C with 5% CO2 for 24 h, a transparent circle formed, indicating an inhibition zone.To further assess the susceptibility of WT, Δ2O2 for 25 min, one mL bacterial cultures were centrifuged at 5000 × g at 4 °C for 10 min and subsequently washed once with PBS and resuspended in 1 mL PBS. According to the manufacturer’s protocol, the concentration of H2O2 in cells was measured using the Pierce Quantitative Peroxide Assay Kits . Experiments were performed with two biological replicates and repeated two times. A two-tailed unpaired t test was used for statistical analysis.After treated with 25 mM HAagp was assessed in mice according to our previous report [8 CFU of the WT or ΔAagp. All mice were euthanized at 24 h post-infection. Blood samples were collected from the heart, and liver, kidney, and brain samples were taken, weighed, suspended in PBS, and homogenized. Plating serial dilutions on THA determined the number of viable bacteria in organs and blood. Experimental results were shown as mean ± standard error of the mean (SEM). The statistical analysis was performed with a two-tailed unpaired t test.The virulence of WT and Δs report . Mice in2O2 treatment and RNA extraction were performed as described above for RNA-Seq analysis. The detailed protocol for RT-qPCR was described previously [BFP66_RS5620 (ParC) was used as the internal control. Three biological replicates were used. The 2−ΔΔCT method was used to calculate the relative fold change. Data were shown as mean ±SEM.Heviously . SupplemS. suis in response to H2O2. Compared with the control condition in the THB medium, 371 differently expressed genes (DEGs) were observed in the H2O2 treatment condition, consisting of 112 upregulated and 259 downregulated genes (Supplementary Table 3). The putative functions of these genes were classified into different functional categories according to the gene ontology (GO) . The DEGs are involved in various biological processes, such as biosynthesis of amid acids, carbohydrate transport and metabolism, metal ion transport, and transcriptional regulation.The RNA-Seq was used to explore the transcriptional landscape of 2O2 stress, including BFP66_RS05940 encoding an exodeoxyribonuclease III (exo III) and BFP66_RS06520 encoding an N-carbamoylputrescine amidase (AguB). The gene expression of enzymes encoded by BFP66_RS01580, BFP66_RS05715, BFP66_RS06935, BFP66_RS07745, BFP66_RS04160 (LDH) was upregulated under H2O2 condition, and these enzymes contribute to producing oxidized dinucleotide cofactors  which are essential for the biosynthesis of amino acids and nucleic acids. As shown in 2O2 condition, contributing to producing more ATP. In contrast, several genes related to energy-consuming pathways were downregulated, including six genes related to sugar ABC transporters, 13 related to the PTS system, and seven related to glycerol metabolism. RT-qPCR was performed to verify the transcriptional data. Eight upregulated and four downregulated genes were selected, and their expressions were consistent with those obtained from the RNA-seq .As shown in S. suis to H2O2 at the transcriptional level, we selected four DEGs, namely exo III, AguB, metQ (encoding a methionine transporter) and Aagp (encoding an aquaporin), to examine their function under oxidative stress conditions. After subjecting the deletion mutations to H2O2 treatment, the survival rates of mutants ΔAagp, Δexo III, ΔAguB, and ΔmetQ were 0.7075%, 0.0063%, 0.69%, and 9.1867%, respectively, which were significantly lower than that of wild-type (WT) strain (59.3133%) (S. suis in response to H2O2.To further investigate the response of 9.3133%) . These fAagp showed the highest fold change R as the motif and water-transporting aquaporins with F(H/I)XR as the motif  was 8.4067 pmol per 106 cells, which was dramatically lower than 430.731 pmol per 106 cells in ΔAagp   . Despiteus study . Similaran in WT . AdditioT strain . These rAagp and WT were performed in diluted THB medium (1/3 THB as a poor medium condition) containing 10 mM glycerol or 100 mM glycerol under aerobic condition. As shown in Aagp was not affected in the absence or presence of glycerol. As shown in S. suis strain GZ0565 possesses genes involved in the phosphorylation pathway for glycerol metabolism, which is an energy-consuming pathway. Under the aerobic condition, glycerol may be phosphorylated by glycerol kinase (GlpK) and then oxidized by glycerol-3-phosphate oxidase (GlpO), which is accompanied by H2O2 production that can be toxic to bacteria [2O2 in WT and ΔAagp were measured in 1/3 THB containing 10 mM glycerol or 100 mM glycerol. The concentration of H2O2 in WT cultured in 1/3 THB was 0.0178 pmol per 106 cells, but in the presence of additional glycerol, the concentration of H2O2 increased and reached to 0.1007 pmol per 106 cells when cultured in 1/3 THB containing 100 mM glycerol. Conversely, in ΔAagp, the concentration of H2O2 was similar in the absence or presence of glycerol  (p < 0.01), RT-qPCR analysis revealed a 10.65-fold upregulation . Moreover, compared to the WT, ΔmetQ was more sensitive to H2O2, confirming the role of metQ under H2O2 conditions  was upregulated upon H2O2 stress. The AguAB pathway converts agmatine to putrescine, protecting E. coli from DNA damage via ROS scavenging [2O2 resistance in S. suis  and PerR (encoded by BFP66_RS01545), accompanied by a significant upregulation of Aagp during H2O2 stress in S. suis. These findings suggest that MntR and PerR in S. suis may play similar roles in regulating Aagp under H2O2 stress conditions. Additionally, we propose that Aagp may possess dual functions of both H2O2 efflux and glycerol transport. In Lactobacillus plantarum, GlpF2, GlpF3, and GlpF4, sharing 30.37%, 36.63%, and 32.73% amino acid identity with Aagp, respectively, have been shown to be permeable to both H2O2 and glycerol [2O2 efflux by oxidative stress assays and measurement of intracellular H2O2 level. However, further investigation is required to determine the glycerol transport ability of Aagp.The aquaporins are divided into three subfamilies: supergene channel superaquaporins, water-transporting aquaporin, and glycerol-transporting aquaglyceroporins . Recent e stress . Howevert damage . Both S.ous H2O2 . In contand PerR ,41. In oglycerol . In our Streptococcus, GlpF, sharing 81% amino acid identity with Aagp, enhances the invasion capacity of Group B Streptococcus into eukaryotic cells [S. pneumococci, Pn-AqpC contributes to pneumococcal pathogenicity by modulating H2O2 production and pneumolysin release [L. monocytogenes, the expression of GlpF, sharing 37% amino acid identity with Aagp, is upregulated during intracellular growth [2O2. Neutrophils, monocytes, and macrophages, abundant in blood and tissues, play crucial roles in producing ROS to eliminate bacteria. The deletion of Aagp would impair the defence capability of S. suis survival in blood and tissues. Finally, we assume that Aagp contributes to S. suis oxidative stress resistance and promotes its survival in the host by facilitating H2O2 efflux.Through in-depth analysis, we have identified homologs of Aagp that play a role in virulence in other pathogenic bacteria. In Group B ic cells . In S. p release . In L. mr growth . When baS. suis defence network response to H2O2 stress and identified a novel aquaporin, Aagp, that alleviated S. suis oxidative stress via facilitating H2O2 efflux, which contributes to virulence. In addition, Aagp might be involved in glycerol transport.In summary, we depicted Click here for additional data file.