Chronic wounds depict a silent epidemic challenging medical professionals worldwide. Regenerative medicine uses adipose-derived stem cells (ADSC) in promising new therapies. In this study, platelet lysate (PL) as a xenogen-free substitute for foetal bovine serum (FBS) in ADSC culture was used to create an ADSC secretome containing cytokines for optimal wound healing conditions. The ADSC secretome was tested on keratinocytes for migrational behaviour and viability. Therefore, human ADSC were characterized under FBS (10%) and PL (5% and 10%) substitution, regarding morphology, differentiation, viability, gene and protein expression. ADSC were then cultured in 5% PL and their secretome was used for stimulation of keratinocyte migration and viability. To enhance the effect, ADSC were treated with Epithelial Growth Factor  and hypoxia (1% O₂). In both PL and FBS groups, ADSC expressed typical stem cell markers. PL induced a significantly higher increase in cell viability compared to FBS substitution. ADSC secretome contained various beneficial proteins which enhance the wound healing capacity of keratinocytes. This could be optimized treating ADSC with hypoxia and EGF. In conclusion, the study shows that ADSC cultivated in 5% PL can effectively support wound healing conditions and can be considered as a promising new therapy for individual treatment of chronic wound disorders. Chronic wounds represent a common problem amongst patients, therapists, and the health care system. Individual factors, such as local tissue hypoxia, bacterial colonization and decreasing stress answer on cellular and systematic levels of ageing patients, result in a long-lasting non-healing tendency of anatomic and physiological skin layers . Thus, lThe process of tissue defect healing is generally divided into three major phases: inflammation, proliferation and remodelling, which involves fibroblasts, keratinocytes, endothelial cells and platelets directed by para- and autocrine mechanisms . MultipoPlatelets play a key role in wound healing, not only as active wound closers, but also as a source of growth factors, e.g., Platelet-derived Growth Factor (PDGF), Transforming Growth Factor Beta (TGF-β), Epidermal Growth Factor (EGF), FGF, VEGF und Connective Tissue Growth Factor (CTGF), and modulators of immune response. Platelet Rich Plasma (PRP), Platelet Rich Fibrinogen (PRF) or Human Platelet Lysate (PL) contain these factors and therefore could be beneficial for tissue engineering experiments . The comPhysiologically, wounds represent a low oxygen environment, hence a low oxygen incubation of ADSC cultures could lead to an accelerated amount of growth factors. Secretome of ADSC cultured in 1% and 5% oxygen was analysed, showing a higher amount of anti-apoptotic and angiogenic cytokines . In otheWithin this study, ADSC were cultivated in PL and effect on cell differentiation, viability, and expression of surface markers were analysed and compared to conventional cell culture conditions with FBS. Different types of ADSC secretome cultivated in PL under normoxia or hypoxia with or without EGF addition were produced and their influence on keratinocytes was analysed, thus mimicking physiological wound healing in vitro. The aim of the present study was to examine the efficacy of ADSC secretome from PL cultivated ADSC as possible therapeutic approach for chronic wound disorders, which would be easily accessible, storable, and cheap.From all donors, ADSC could successfully be isolated from abdominal fat tissue and cultivated at least over eight passages.The morphology of ADSC in different experimental groups was checked after 24 h and 72 h. Cell confluency was reached much faster in groups cultivated in PL. In all groups, ADSC showed the typical spindle-shaped, fibroblast-like morphology and grew homogenously distributed on the well plates a. No sigMKI67 was analysed by RT-PCR. It could be shown that MKI67 expression in 5% and 10% PL groups was 3.8 times and 5.3 times higher compared to FBS group  could be detected (<2%) .ADSC were differentiated into the adipogenic and chondrogenic cell lineage to analyse the effect of PL on their differentiation potential.Adipogenic differentiated ADSC were analysed by microscopy and Oil Red O staining after day 21. In all groups, ADSC could successfully be differentiated. ADSC in PL developed earlier signs of adipogenic differentiation such as intracellular lipid droplets around day 7 of induction. The amount and size of lipid droplets increased rapidly over time. However, due to the high proliferation of ADSC in PL groups, a time point of 21 days could not be analysed due to detachment of complete cell layers and only FBS groups were stained with Oil Red O a.Chondrogenic differentiation capability was proven after 4 weeks of chondrogenic differentiation. In all groups, alcian blue staining confirmed the expression of chondrogenic proteins, such as glycosaminoglycans without visible difference between the experimental groups b.n = 5). A range of cytokines important for wound healing  were highly secreted in PL groups compared to the FBS group  .Keratinocytes were then cultivated in ADSC secretome from ADSC cultivated in 5% PL in either normoxia or hypoxia with or without supplementation of EGF. The effect on cell viability and migration was analysed over 7 days, and 24 h, respectively . In all In normoxia groups, EGF significantly stimulated keratinocyte viability after 4 and 7 days b. LikewiPL is currently used in various studies in the field of regenerative medicine ,22,23,24Within this study, we could show that ADSC cultivated in cell culture medium supplemented with PL maintained stem cell characteristics such as typical stem cell pattern on protein level and multilineage differentiation capability. A systematic review by Guiotto et al.  demonstrMKI67, a known proliferation marker, was increased in PL-cultured ADSC, confirming a higher proliferation rate compared to the FBS cultures.Not only the cell viability, but also Overall, in all experimental groups, typical MSC surface markers were highly expressed, confirming no interference of PL in MSC characteristics. Negative markers, such as CD11b, CD19, CD31, CD34, CD45 and HLA-DR (Lin-cocktail), could not be detected, supporting the thesis of unaltered stem cell characteristics. A slight reduction in CD105 was noticed, although meeting the minimal criteria of MSC. This was described before by Riis et al. [In all groups, ADSC could be successfully differentiated into adipogenic and chondrogenic lineages. Interestingly, adipogenic differentiation induced an earlier development of lipid vacuoles in PL groups compared to the FBS groups, most probably because of stimulating growth factors in PL. Conversely, other authors postulate a PL-induced suppression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of MSC leading to a lower capability of adipogenic differentiation in PL-supplemented MSC compared to FBS-supplemented MSC, both meeting all common MSC criteria. This difference might be due to the fact that Lange et al.  used MSCBoth PL and FBS groups could be successfully differentiated into the chondrogenic lineage without visible differences. However, in previous studies, PL was described to enhance chondrogenic regeneration in vivo. In a clinical trial, autologous chondrocyte implantation was most successful under PL supplementation, showing faster regeneration and a lower inflammatory response compared to FBS supplemented groups . OsteogeELISA analysis of selected cytokines in PL-cultured ADSC secretome revealed that many markers were significantly increased in PL groups, e.g., VEGF, EGFR, FGF 4, MCSF, MSCFR and SCF. The VEGF family is a group of five factors, VEGF-A, -B, -C, -D and placenta growth factor (PIGF) known to regulate angiogenesis and lymphangiogenesis ,45,46, bAnother aspect to be investigated is the effect of PL-cultured ADSC secretome on the production of reactive oxygen species. There is not yet data highlighting this interplay, but there is little data regarding PRF. PRF reduced the ROS release provoked by lipopolysaccharide (LPS) in RAW 264.7 cells (macrophages) and  thereforEGF was used to optimize the effect of ADSC secretome for wound healing. In previous studies, EGF increased the VEGF and bFGF secretion in ADSC , positivInflammation and hypoxia are closely connected on molecular, cellular, and clinical levels. One of the key regulators in this pathway is the hypoxia-inducible-factor (HIF) . HypoxiaOn the other hand, anaerobic bacteria thrive under hypoxic conditions; half of all diabetic foot ulcers are infected. Since this is linked with worse outcome of such ulcers, often leading to amputations, antibiotic therapy is recommended . In clinOne limitation of the study is the small number of patients. Especially in the ADSC secretome analysis, a high patient-to-patient variance was observed. Additionally, practical working can be limited by the increased cell growth in PL groups, although PL showed to be an equally usable substitute in ADSC culture. This led to a cell detachment in differentiation assays, causing experimental delays. Another major question to solve which was not addressed within this study and should be focused on in further studies, is the exact composition of PL, e.g., using PL fraction, protein separation and MS-analysis . AnotherMoreover, FBS is used worldwide und manufactured under strict standards. PL is also commercially available, but lacks a consistency of protocols for PL preparation, of standardized terminology and therefore treatment regimens .PL was obtained from plateletpheresis donations from healthy volunteers at the Department of Transfusion Medicine and Haemostaseology, University Hospital of Erlangen, Germany. Platelet concentrates were stored at +22 °C ± 2 °C for 5 days under constant agitation until declared invalid for clinical transfusion. As all concentrates were intended to be transfused, all donors were healthy, met the minimal donor criteria and were aged 18 to 68 years. The gender was not recorded since all donors were anonymized in the process of possible transfusion. Only platelet concentrates containing 3 × 10¹¹ thrombocytes per 250 mL and less than 4 leukocytes per μL and less than 1000 erythrocytes per μL were used. One thousand (1000) mL of platelet lysate were obtained from 5 pooled thrombocyte concentrates each with a volume of 250 mL. Aliquots of platelet concentrates were frozen at −80 °C for a minimum of 48 h until further procession. Thawing was conducted using plasmatherm  at 24 °C. A system from MacoPharma  consisting of a freezing and centrifugation bag, a filtration, and a distribution kit were used for processing. The platelet concentrates then underwent two cycles of thawing at 37 °C and freezing at −80 °C for least 12 h and were then centrifuged for 30 min. A 0.65 μm filter was used for filtration by gravity. The bag containing filtered PL was recovered and connected to the 9-bag distribution kit. Aliquots of 25 mL were immediately stored at −80 °C until further usage. The entire procedure was performed under central temperature supervision systems and strictly closed systems.g for 10 min and the top fluid and fat layers were discarded. The cells were suspended in phosphate buffered saline (PBS) , filtered (70 µm) and centrifuged . Cell pellets were resuspended in minimal essential medium alpha (MEM α) (Biochrom GmbH) with supplements according to the following conditions: group 1: 5% PL, group 2: 10% PL, group 3: 10% foetal bovine serum ; 1 × 106 cells were seeded per T75 flask. One (1) % penicillin/streptomycin (Sigma-Aldrich) was added to the cell culture medium for the first two weeks. Cells were cultivated at 37 °C, 5% CO2. The medium was changed every 2–3 days and cells were split at about 90% confluence. Experiments were performed until passage 8.ADSC were isolated from human abdominal fat tissue of six female healthy patients aged 33 to 52 (mean 40.5 ± 7.4 years). Human tissue collection was approved by the Ethics Committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg (Germany) (Ethics number 264_13B) in accordance with the World Medical Association Declaration of Helsinki. Informed consent was obtained from all patients. About 20 mL fat tissue per patient was minced and digested with 0.1% collagenase type 1 . The homogenous fat solution was centrifuged at 400× Morphology of ADSC was checked on day 1 and 3 after isolation by microscopy . ADSC surface marker expression was analysed using flow cytometry (BD FACSVerse) . In total, 1 × 10⁶ cells per group were incubated simultaneously according to the manufacturer’s specifications with: APC mouse anti-human CD73 (clone AD2), FITC mouse Anti-Human CD90 (clone 5E10) and PerCP-Cy™5.5 mouse anti-Human CD105 (clone 266) as positive markers; APC-Cy™7 mouse Anti-Human CD31 (clone WM59), CD34 PE (clone 581), CD45 PE (clone HI30), CD11b PE (clone ICRF44), CD19 PE (clone HIB19) and HLA-DR PE (clone G46-6)  as negative control cocktail and mIgG1 PE (clone X40) and mIgG2a PE (clone GJ55-178) as a PE hMSC isotype negative control cocktail . All CD markers are listed under For comparing the cell viability of different experimental groups, a WST-8 assay was performed at time points 24 h, 48 h, and 72 h. In a 96-well plate, 2 × 10³ cells per well were seeded in triplicates in 100 µL cell culture medium according to the different experimental groups. After 24 h, 10 µL WST-8 solution  was added and absorbance was measured after 2 h of incubation (37 °C) at 450 nm and 600 nm (background) using a microplate reader . One 96-well plate was used for each measuring point. Relative values (24 h = 1) were analysed.® Green Supermix  and a Light Cycler (Bio-Rad CFX96). All experiments were performed according to the manufacturer’s protocols. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) served as housekeeping gene. Data analysis was performed using the 2−ΔΔCT method. Samples were tested as triplicates. Primer sequences are specified in RNA of the different experimental groups was extracted with the RNeasy Mini Kit with the corresponding QIAshredder Homogenizer . QuantiTect Reverse Transcription Kit (Qiagen) was used for reverse transcription with DNase I incubation. Quantitative real-time PCR was performed using SsoAdvanced Universal SYBRFor differentiation into the adipogenic and chondrogenic cell lineage, ADSC were cultivated in MEM α with supplements according to the experimental groups . When reaching 90% confluence, the medium was changed to a differentiation medium. MEM α with supplements according to the different experimental groups served as control. Experiments were performed in triplicates.For adipogenic differentiation, 2 × 10⁴ ADSC per well were seeded in 24-well plates and cultivated for 21 days in 500 µL MSC adipogenic differentiation medium I and II  , dexamethasone, rosiglitazone; ADMI II: basal medium, supplemented with 5% PL or 10% PL or 10% FBS, and insulin, glutamine, penicillin/streptomycin). ADSC were incubated in ADMI I for 3 days, followed by ADMI II for 24 h. This cycle was repeated 4 times. Differentiated ADSC were fixated in 4% buffered formaldehyde , rinsed in PBS and stained with Oil Red O (Sigma-Aldrich) for 15 min. Pictures were taken by microscopy (Olympus IX83) at day 0, 7, 21 and after staining.5 ADSC were transferred into a 15 mL tube in 0.5 mL chondrogenic differentiation medium (PeloBiotech GmbH) containing basal medium with ITS supplement , sodium pyruvate, proline, ascorbate, dexamethasone and TGF-β3. For creating cell pellets, tubes were centrifuged at 150× g for 5 min, and carefully placed into the incubator. The medium was changed 3 times a week. After 4 weeks, cell pellets were fixated overnight in 4% buffered formaldehyde. Cell pellets were embedded in paraffin. Five to ten slides per sample were cut and stained with alcian blue . Slides were analysed by microscopy (Olympus IX83).Chondrogenic differentiation was performed in a pellet culture system. In total, 2.5 × 10ADSC were cultivated in cell culture medium according to the experimental groups until 90% confluence. Cell layers were washed with PBS and incubated in MEM α without supplements for 24 h at 37 °C, 5% CO₂. Supernatant was collected and immediately stored at −80 °C. Samples were analysed by ELISA Quantibody  for Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor-Receptor 2 (VEGF-R2), Endocrine Gland Vascular Endothelial Growth Factor (EG-VEGF), Endothelial Growth Factor Receptor (EGF-R), Fibroblast Growth Factor 4 (FGF-4), Macrophage Colony Stimulating Factor (MCSF), Macrophage Colony Stimulating Factor Receptor (MCSF-R), Stem Cell Factor (SCF), Stem Cell Factor Receptor (SCF-R), Intercellular Adhesion Molecule 1 (ICAM-1), Monocyte Chemoattractant Protein 1 (MCP-1) and Chemokine Ligand 5 .g), filtrated (70 µm) and diluted in complete keratinocyte growth medium . Next, 1 × 10⁶ cells were seeded in collagen-coated  T75 flasks and cultivated at 37 °C, 5% CO2. A medium change was performed every 2–3 days and cells were split at about 70% confluence. Experiments were performed until passage number 2.Abdominal skin tissue of one female healthy patient aged 41 was used for isolation of keratinocytes. Human tissue collection was approved by the Ethics Committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany (Ethics number 264_13B), in accordance with the World Medical Association Declaration of Helsinki. Informed consent was obtained from the patient. Keratinocytes were isolated according to the protocol of the Epidermis Dissociation Kit, human . The skin was cut into 50 pieces with a diameter of about 4 mm. Ten (10) mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) and 250 µL of Enzyme G (Miltenyi Biotec) were added to the tissue pieces. Samples were incubated for 18 h at 4 °C under continuous shaking using a tube rotator. Subsequently, epidermis and dermis could be separated manually. Five (5) mL of RPMI 1640, 125 µL of Enzyme P and 25 µL of Enzyme A  were added to the epidermis samples and incubated at 37 °C for 60 min. The gentleMACS™ Octo Dissociator was used for sample shredding in gentleMACS™ C Tubes. Samples were centrifuged  antibody)  for 30 min, followed by 1 h incubation with the primary antibody  , which was diluted 1:100 with antibody diluent . This antibody reacts to cytokeratin 5, cytokeratin 6, cytokeratin 8, cytokeratin 17 and cytokeratin 19. A negative control was performed using isotype control mouse IgG1 (negative control code X0931) . Cells were washed three times in TBS-T  and counterstained with in 1:1000 distilled aqua diluted DAPI Staining Solution .For characterization, a cytokeratin immunofluorescence staining was performed. Cells were washed with PBS, fixed in 10% formaldehyde for 15 min. Specimens were blocked with 5% goat serum (Alexa Fluor2 in MEM α in 4 different conditions: group 1: normoxia (21% O2), group 2: hypoxia (1% O₂), group 3: normoxia (21% O2) with 100 ng/mL EGF, and group 4: hypoxia (1% O2) with 100 ng/mL EGF. As a control, MEM α was incubated without cells for 24 h at 37 °C, 5% CO2, 21% O2. Incubation was performed in technical triplicates. Supernatants from each group were pooled and 3-fold concentrated by centrifugation  4000× g, 30 min) and dilution with MEM α. ADSC secretome was immediately stored at −80 °C until further usage.For the preparation of ADSC secretome, ADSC were cultivated in T25 flasks in 3 mL MEM α supplemented with 5% PL until reaching 90% confluence. After washing with PBS, ADSC were incubated for 24 h at 37 °C, 5% COThe effect of ADSC secretome on viability of keratinocytes was measured with the WST-8 assay (PromoCell GmbH). First, 2 × 10³ keratinocytes per well were seeded into a 3 µg/cm² collagen-coated (Cell Guidance Systems Ltd.) 96-well plate in triplicates in 100 µL complete keratinocyte growth medium. After 4 h, keratinocytes were cultivated in different types of ADSC secretome according to the groups 1–4, which was diluted 50:50 with complete keratinocyte growth medium. Medium was changed after 2 d and 4 d. At time points 1 d, 4 d and 7 d, 10 µL WST-8 solution was added and absorbance was measured after 2 h of incubation (37 °C) at 450 nm and 600 nm (background) using a microplate reader (Thermo Fisher Scientific). One 96-well plate was used for each measuring point.2 collagen-coated  96-well plates were prepared with stoppers using the Oris™ Cell Migration Assembly Kit  to create a central cell-free zone. Per well, 6 × 104 keratinocytes were seeded in triplicates in 100 µL complete keratinocyte growth medium. After 4 h, stoppers were removed. Cell layers were washed twice with PBS and medium was added according to the ADSC secretome groups 1–4, diluted 50:50 with complete keratinocyte growth medium. Microscopic pictures were taken over a period of 24 h with an interval of 30 min (Olympus IX83).For the migration assay, 3 µg/cmp ≤ 0.05 and highly significant at p ≤ 0.01.All experimental data are displayed as the mean of all independent experiments ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism 8.4.3 . Comparisons between all groups were conducted using Kruskal-Wallis test, individual comparison between the groups was performed using Mann-Whitney U test. All experiments were performed in technical triplets using ADSC of 6 patients, except for ELISA quantibody, where due to technical issues only 5 patients were analysed. Differences were considered statistically significant at With this study, we could show that PL is a suitable substitute in ADSC culture. Stem cell characteristics are preserved including stem cell marker expression and ability to differentiate. PL significantly enhanced ADSC viability and proliferation compared to FBS. Secretome from PL-cultured ADSC contained increased amounts of proteins, such as proliferation, differentiation or pro-angiogenetic factors compared to FBS-cultured ADSC, which stimulate keratinocyte migration and viability. EGF substitution and most probably also hypoxia for ADSC secretome production support these effects and finally can lead to better wound healing. Lastly, the study illustrates that ADSC secretome can effectively support tissue regeneration and is a promising tool in individual treatment of wound healing.


Ventilator-induced lung injury (VILI) is caused by stretch stimulation and other factors related to mechanical ventilation (MV). NOD-like receptor protein 3 (NLRP3), an important innate immune component, is strongly associated with VILI. This study aimed to investigate the effect and mechanisms of aerobic exercise (EX) on VILI.To test the effects of the PKC inhibitor bisindolylmaleimide I on PKC and NLRP3, male C57BL/6 mice  were randomly divided into four groups: control group(C), bisindolylmaleimide I-pretreated group(B), MV group, and bisindolylmaleimide I-pretreated + MV (B + MV) group. The mice were pretreated with bisindolylmaleimide I through intraperitoneal injection (0.02 mg/kg) 1 h before MV. MV was performed at a high tidal volume (30 ml/kg). To explore the ameliorative effect of EX on VILI, the mice were randomly divided into C group, MV group, EX group and EX + MV group and subjected to either MV or 5 weeks of EX training. After ventilation, haematoxylin-eosin (HE) staining and wet/dry weight ratio was used to assess lung pathophysiological changes. PKCɑ, P-PKCɑ, ASC, procaspase-1, caspase-1, pro-IL-1β, IL-1β, NLRP3 and occludin (tight junction protein) expression in lung tissues was determined by Western blotting. The level of IL-6 in alveolar lavage fluid was determined by ELISA.NLRP3, P-PKCɑ, and PKCɑ levels were inceased in MV group, but bisindolylmaleimide I treatment reversed these changes. Inhibition of PKC production prevented NLRP3 activation. Moreover, MV increased ASC, procaspase-1, caspase-1, pro-IL-1β, and IL1β levels and decreased occludin levels, but EX alleviated these changes. HE staining and lung injury scoring confirmed an absence of obvious lung injury in C group and EX group. Lung injury was most severe in MV group but was improved in EX + MV group. Overall, these findings suggest that MV activates the NLRP3 inflammasome by activating PKCɑ and inducing occludin degradation, while Exercise attenuates NLRP3 inflammasome and PKCɑ activation. Besides, exercise improves cyclic stretch-induced degradation of occludin.PKC activation can increase the level of NLRP3, which can lead to lung injury. Exercise can reduce lung injury by inhibiting PKCɑ and NLRP3 activation. Exercise maybe a potential measure for clinical prevention of VILI.The online version contains supplementary material available at 10.1186/s12871-022-01874-4. Acute lung injury (ALI) is a common clinical respiratory illness caused by a variety of noncardiac factors. The main manifestations are injury to pulmonary capillary endothelial cells and alveolar epithelial cells and increased vascular permeability, which can develop into acute respiratory distress syndrome (ARDS). The characteristics of ARDS are acute and progressive dyspnoea, refractory hypoxemia and pulmonary edema . MechaniNOD-like receptor protein 3 (NLRP3) is an important component of innate immunity and is strongly associated with the occurrence of VILI. Previous studies have shown that cyclic stretch activates the NLRP3 inflammasome by activating NIMA-related kinase 7 (NEK7) . By inhiIn this study, we established a mouse model of aerobic exercise in VILI to explore the protective role of aerobic exercise and the mechanisms of NLRP3 in VILI. We observed that PKCɑ upregulated NLRP3 activation, whereas 5 weeks of aerobic exercise inhibited the activation of NLRP3, thereby reducing lung injury. All our findings suggest that exercise may be a new direction for the prevention of VILI.Seven-week-old male C57BL/6 N mice weighing 19–23 g were supplied by the Experimental Animal Center of Shandong University and fed for 1 week to allow them to adapt to the environment. The mice were kept in a constant temperature of 25 °C under a 12-hour light/dark cycle and were fed a standard diet of pellets and water.To determine the relationship between PKC activation and NLRP3, the mice were randomly divided into the following four groups (n = 6 in each group): control (C) group, bisindolylmaleimide I-pretreated (0.02 mg/kg)(B) group, mechanical ventilation (MV) group and bisindolylmaleimide I-pretreated (1 h) and mechanically ventilated (B + MV) group. Mechanical ventilation was not conducted in C and B groups. The other two groups were mechanically ventilated for 4 h using an ALC-V8 animal ventilator. The ventilation parameters were set as follows: tidal volume 30 ml/kg, respiratory rate 60 times/min, I/E ratio of 1:2, no positive end-expiratory pressure, fraction of inspired oxygen 21% and room temperature 25 °C.For another study, the mice were randomly divided into the following four groups (n = 6 in each group): control (C) group, mechanical ventilation (MV) group, exercise (EX) group (which only underwent 5 weeks of aerobic exercise training), and exercise + mechanical ventilation (EX + MV) group . The ventilation parameters were set as described above . All exp, 5]5]]. Mice in C group and MV group were raised in standard cages for 5 weeks without intervention.Mice were subjected to mechanical ventilation or 5 weeks of aerobic exercise training. The specific aerobic exercise regimen was as follows: mice were acclimated to a treadmill for 3 days . The maximum aerobic capacity of the mice was determined, and the treadmill speed was increased by 0.1 km/h every 2.5 min until exhaustion . The maximum speed each mouse achieved during treadmill training was recorded as the maximum aerobic capacity (100%). Mice in EX group and EX + MV group received aerobic exercise training at a moderate aerobic exercise capacity (50%) for 60 min a day, 5 days a week for 5 weeksShanghai Alcott Biotech Co.  provided the ALC-V8 animal ventilator. The animal experiments platform ZH-PT/5S was purchased from Guizhou Zhenghua Biological Instrument Co., Ltd. . Enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β and IL-6 were purchased from Elabscience Biotechnology Co., Ltd. . Rabbit anti-mouse NLRP3, GAPDH and ɑ-tubulin antibodies were purchased from Cell Signaling Technology, Inc. . Antibodies against ASC, procaspase-1, caspase-1, and P-PKC α were purchased from Santa Cruz, Inc. . Antibodies against IL-1β, pro-IL-1β, PKCα, and occludin were purchased from Abcam, Inc. . A bicinchoninic acid kit, radio immunoprecipitation assay lysis buffer and protease inhibitor were purchased from Shanghai Biyuntian Biological Company . A haematoxylin-eosin (HE) staining kit was purchased from Beijing Zhongshan Biotechnology Co., Ltd. .Mice in C, B and EX groups were sacrificed after tracheotomy, while the other four groups were sacrificed after ventilation (4 h). Mice were given 150 mg/kg sodium pentobarbital to keep them under deep anaesthesia and then sacrificed by exsanguination through the abdominal aorta. Arterial blood was quickly harvested from the four groups. Western blotting was performed on the superior lobe of the right lung, and the remaining right lung tissue was fixed for HE staining. The other lung was used to calculate the lung wet/dry (W/D) weight ratio .After mechanical ventilation, lung tissues were collected and washed rapidly, then the wet lung weight was determined. After that, the lung tissue was heated to measure dry weight (70 °C for 72 h). Then, we calculated the pulmonary W/D ratio to evaluate the degree of pulmonary edema.Mice were tracheotomized and cannulated with a 20-gauge catheter. The lungs were lavaged three times with 1.0 ml of ice-cold D-PBS to collect BALF. Then, the BALF was centrifuged at 1500 rpm at -4 °C for 5 min, and the supernatant was collected and stored at -20 °C for ELISA detection.The levels of IL-6 were determined according to the ELISA kit instructions .In brief, the lung tissue was fixed with formalin, embedded in paraffin, sectioned and observed under a 400× light microscope. Previous studies have introduced criteria for evaluating pulmonary pathological changes. The criteria required at least three visual fields to be observed for each slice. Pulmonary structure destruction, pulmonary edema, alveolar septum thickening, hyaline membrane formation, alveolar haemorrhaging and inflammatory cell infiltration were evaluated. The severity of lung injury was described on a scale of 0–4, where 0 indicates no injury, 1 indicates mild injury, 2 indicates moderate injury, 3 indicates severe injury, and 4 indicates serious injury. The cumulative total score was the final lung injury score .The tissue fragments were lysed in radioimmunoprecipitation assay buffer and then centrifuged for 5 min at 12,000 × g. The lysate was collected, and the protein concentration was measured using a test kit. Equal amounts of protein were denatured and separated on 10% SDS‒PAGE gels and then transferred to polyvinylidene difluoride membranes. When we performed WB, the blots were cut prior to hybridization with antibodies, and we developed strips of different proteins on the same PVDF film separately. The membranes were blocked in 5% nonfat milk for 2 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted the following proteins: NLRP3 (1:500), ɑ- tubulin (1:1000), GAPDH (1:3000) , ASC (1:200), procaspase-1, caspase-1 (1:500), p-PKC α (1:500) , IL-1β, pro-IL-1β (1:1000), PKC α (1:1000), and occludin (1:2000) . Then, the membranes were incubated with secondary antibodies (1:5000) for 2 h at room temperature, and ECL Super Signal reagent  was used to detect protein bands. Image J software was used to measure the relative band density of different proteins on the scanned membrane .Statistical analysis was performed using SPSS 22 and Prism 8. All the results are expressed as the mean ± SD. The unpaired T test or one-way analysis of variance (ANOVA) was used for Gaussian data, and the Mann‒Whitney U test or Wilcoxon rank sum test was used for nonparametric data. P ≤ 0.05 was considered to indicate statistical significance.p < 0.01)  . However, the degradation of occludin was alleviated in EX + MV group (5 weeks excise before ventilation for 4 h) (p < 0.01)  influence of aerobic exercise on NLRP3 or PKCα has been reported previously , but theTo determine the effects and mechanisms by which mechanical ventilation and aerobic exercise on NLRP3, mice were divided into four groups, and the expression of P-PKCɑ, ASC, procaspase-1, caspase-1, pro IL-1β and IL-1β were examined. We found that the expression of ASC, procaspase-1, and caspase-1 were obviously increased in the MV group; in contrast, these changes were alleviated by aerobic exercise pretreatment. These findings indicate that mechanical ventilation and aerobic exercise may play opposing roles by influencing NLRP3 inflammasome formulation . The expAdherens proteins and tight junction proteins play vital roles in maintaining normal alveolar permeability , 25. MecInterleukins play important roles in the maturation, activation, proliferation and immune regulation of immune cells . A previNoncardiogenic pulmonary edema is the most visible manifestation of VILI. Its fundamental causes are destruction of alveolar membrane integrity and increased pulmonary capillary permeability , 29. In Mechanical ventilation is a double-edged sword; it is often used as a life support to improve oxygen supply in critically ill patients, but its long term use or inappropriate parameter settings may inhibit pulmonary circulation and induce VILI , 31. HowTaken together, our findings demonstrate that aerobic exercise for 5 consecutive weeks can reduce the induction of lung injury by mechanical ventilation. In terms of the mechanisms, we found that activation of PKC and NLRP3 can lead to ALI. Specific and highly efficient PKC inhibition and aerobic exercise can reduce lung injury by inhibiting the activation of PKCɑ and NLRP3, suggesting a potential measure for the clinical prevention and treatment of VILI.Below is the link to the electronic supplementary material.Supplementary Material 1


The health risk stemming from drinking alcohol is serious, sometimes even life-threatening. Alcoholic steatohepatitis (ASH) is a critical stage leading to cirrhosis and end-stage liver disease. However, its pathogenesis is still far from clearly understood, and a treatment that is widely recognised as effective has not been discovered. Interestingly, PDPK1,3-phosphoinositide-dependent protein kinase 1, also known as PDK1, was observed to be obviously increased in the ASH model by our researchers. We also investigated the protective role of autophagy in ASH. Here, we studied the function of PDPK1 and found an efficient treatment to alleviate symptoms by targeting PDPK1 in ASH. In our study, PDPK1 affected hepatocyte self-healing by inhibiting autophagy. Both inhibiting PDPK1 and the phosphorylation of PDPK1 (ser241) could protect hepatocytes from suffering heavy alcoholic hepatitis. A rising death toll of alcoholic liver disease (ALD) reveals such the phenomenon that liver injury has become a major threat to human health . ALD refAutophagy is an important cellular component degradation and energy-recycling process in the body , 8. It iPDPK1,3-phosphoinositide-dependent protein kinase 1, also known as PDK1, is a phosphorylation-regulated kinase that is expressed in various organs of eukaryotes and plays a central role in activating a variety of cell signalling pathways –16. StudThen, we found a drug targeting PDPK1 by SPR to be a promising drug for ASH treatment. Surprisingly, phillyrin was demonstrated to target PDPK1 and inhibit the phosphorylation of PDPK1 (ser241). Phillyrin is a representative extract from Forsythia suspensa, which is a traditional Chinese herbaceous plant and certain functional foods. The effect of phillyrin has been tested on different kinds of diseases. Zhong et al. showed the anti-inflammatory action of phillyrin in lung injury . Jiang eAccording to many convincing articles , 23, theSince our former experiments showed that further promoting autophagy in ethanol-treated mice could alleviate ASH, we next detected whether autophagy was blocked in the process of activating self-protection. Interestingly, we found a higher expression of PDPK1 in ASH mouse livers than in normal livers from the results of immunohistochemical staining and western blotting Fig. . AdditioThen, we constructed a PDPK1-overexpressing cell stable transfection line by transfection with HBLV-m-PDPK1-3xflag-mcherry-PURO. The transfection efficiency was detected by RT‒qPCR and immunoblotting Fig. . Both imKd = 3.026 × 10−3), phillyrin truly had a binding affinity to PDPK1 in a concentration-dependent manner. Additionally, a cellular thermal shift assay (CETSA) was performed to verify the interaction between phillyrin and PDPK1. According to the interaction principle, proteins tend to withstand high temperatures, triggering degradation when they bind with drugs  to different concentrations as follows: 1000, 333.3, 111.1, 37, 12.3, 4.1, 1.37, 0.46, 0.15 and 0.05 μM. A random concentration was double used. Samples were injected into the channels at a flow rate of 30 μL/min and then, washed with HBS buffer. The binding RU (Response Unit) values of phillyrin to PDPK1 were recorded directly by the Biacore T200 instrument and analysed by Biacore T200 Evaluation Software.7 cells/ml with PBS. The cell suspension was divided into 10 parts, which were heated for 3 min at different temperatures . The heated cells were kept at −80 °C for 12 h and then at room temperature for 5 min. This freezing and thawing process was repeated three times. After that, cell lysates were extracted by centrifugation at 20,000×g for 20 min. The levels of PDPK1 were assessed by western blotting.AML-12 cells were incubated with or without phillyrin (200 μg/ml) for 24 h. Then, the incubated cells were collected and resuspended to 1 × 10t-tests . Experimental data in this study were analysed by using Prism 8.0 GraphPad Software (USA). P < 0 .05 was considered statistically significant.Differences in multiple groups were determined by one-way ANOVA analysis. Tukey’s post hoc test was performed after a one-way ANOVA analysis. However, differences between the two groups were determined by Student’s A reproducibility checklistSupplementary figure legendsSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Original Data File


The Fe3O4@COF(TAPT–DHTA) was prepared by a simple template precipitation polymerization method, using Fe3O4 as magnetic core, and 1,3,5-tris-(4-aminophenyl) triazine (TAPT) and 2,5-dihydroxy terephthalaldehyde (DHTA) as two building units. Fe3O4@COF(TAPT–DHTA) could effectively capture the targeted mycotoxins by virtue of its abundant hydroxyl groups and aromatic rings. Several key parameters affecting the performance of the MSPE method were studied, including the adsorption solution, adsorption time, elution solvent, volume and time, and the amount of Fe3O4@COF(TAPT–DHTA) nanomaterial. Under optimized MSPE conditions, followed by analysis with UHPLC–MS/MS, a wide linear range (0.05–200 μg kg−1), low limits of detection (0.01–0.5 μg kg−1) and satisfactory recovery (74.25–111.75%) were achieved for the nine targeted mycotoxins. The established method was further successfully validated in different kinds of fruit samples.In this study, a simple and efficient magnetic solid-phase extraction (MSPE) strategy was developed to simultaneously purify and enrich nine mycotoxins in fruits, with the magnetic covalent organic framework nanomaterial Fe Alternaria, Aspergillus, Fusarium, etc. [1(AFB1), ochratoxin A (OTA), zearalenone (ZEN) and Alternaria toxins, mainly including tentoxin (TEN), altenuene (ALT), altenusin (ALS), alternariol monomethyl ether (AME), alternariol (AOH) and tenuazonic acid (TeA), are the most frequently found in various fruits [1, OTA and ZEN in different types of food have been set in the European Union, USA, Canada, China and other countries [Mycotoxins are toxic secondary metabolites produced by toxigenic fungi under suitable environmental conditions. Fruits increasingly favored by consumers, owing to their high moisture content, rich nutrition and improper harvest or storage conditions, are highly susceptible to various fungi such as um, etc. ,2. Amongs fruits ,4. Thoses fruits ,6. Owings fruits . Maximumountries ,9. Givenountries ,11.Conventional methods for the analysis of mycotoxins in foodstuffs commonly rely on high-performance liquid chromatography (HPLC) coupled with ultraviolet, fluorescence or mass spectrum detectors ,13. Neve3O4@COF(TpDA) material to enrich plant growth regulators from fruits and vegetables through π-π and hydrogen bonding interactions [Covalent organic frameworks (COFs) are a new class of crystalline material, which can be constructed with organic building units by covalent bonds of elements  . COFs, wractions . Neverthractions .3O4 as magnetic core, and 1,3,5-tris-(4-aminophenyl) triazine (TAPT) and 2,5-dihydroxy terephthalaldehyde (DHTA) as two building units, Fe3O4@COF(TAPT–DHTA) was firstly designed and applied as an MSPE adsorbent to extract nine mycotoxins (3O4@COF(TAPT–DHTA) adsorbents were expected to effectively enrich mycotoxins by virtue of their abundant hydroxyl groups and aromatic rings. The features of Fe3O4@COF(TAPT–DHTA) were characterized and several key factors affecting MSPE were optimized. In addition, the possible adsorption mechanism was also discussed. The proposed method was further validated and applied to the analysis of mycotoxins in fruits including watermelon, hawthorn, melon, tomato and strawberry. The schematic of the fabrication and application of Fe3O4@COF(TAPT–DHTA) is shown in In this study, using Fecotoxins  from frucotoxins . The pre3O4 has a regular spherical structure with a diameter of approximately 200 nm. However, Fe3O4@COF(TAPT–DHTA) exhibited a dense surface with a significantly larger particle size of nearly 850 nm, proving that COFs were successfully grafted onto Fe3O4 nanoparticles.The morphology of the prepared nanomaterial was characterized by SEM A,B. It c3O4@COF(TAPT–DHTA) was verified by FT-IR spectroscopy , the peaks at 1670 cm−1 and 3329 cm−1 were assigned to the C=O stretching vibration of DHTA and N–H stretching of TAPT, respectively [−1 and 1501 cm−1 owing to the presence of the triazine ring [−1 might be ascribed to the formation of imine bonds, proving the successful condensation of formyl linker and amine node [The formation of Fetroscopy C. For Feectively . The addine node ,29.3O4 and Fe3O4@COF(TAPT–DHTA), respectively. Compared with Fe3O4, Fe3O4@COF(TAPT–DHTA) showed an absorption peak at 345 nm, which was attributed to the existence of the conjugated double bond in COF. The strong conjugated double bond made Fe3O4@COF(TAPT–DHTA) nanocomposites ideal adsorbents for the extraction of benzene ring-containing mycotoxins. In fact, after capturing nine mycotoxins (3O4@COF(TAPT–DHTA) at 345 nm was red-shifted to 351–358 nm, indicating the strong π-π-stacking interaction between the nanocomposites and targeted mycotoxins [cotoxins , the abscotoxins .3O4@COF(TAPT–DHTA)  line-scanning method was conducted to analyze the elements contained in FePT–DHTA) E. The re3O4 and Fe3O4@COF(TAPT–DHTA) were also investigated, and the magnetization curves are shown in 3O4 and Fe3O4@COF(TAPT–DHTA) were 46.542 and 18.457 emu g−1, respectively. Although the magnetization values of Fe3O4@COF(TAPT–DHTA) decreased compared with pure particles, it could still provide sufficient magnetism for MSPE. As shown in the inset of 3O4@COF(TAPT–DHTA) was well-dispersed in the solvent, and could be collected within 30 s by an external magnetic field.The magnetic properties of Fe3O4@COF(TAPT–DHTA) nanomaterial, with rich hydroxyl groups and aromatic rings, was utilized as competent adsorbent in MSPE for the selective enrichment of multiple mycotoxins. To achieve excellent MSPE procedure, several important parameters were studied by using spiked tomato samples (20 μg kg−1), including adsorption solution, pH, ionic strength, adsorption time, elution solvent, elution volume and time, and Fe3O4@COF(TAPT–DHTA) amount.Fev/v) were examined. As shown in 1 were obtained (83–104%). For AFB1, there was no significant difference in recovery between 1% acetonitrile (79%) and 2% acetonitrile (81%)  composite for all the targeted analytes, 1% acetonitrile in water was used as the adsorption solution.The contents of organic solvent in the adsorption solution played a crucial role in the adsorption process. Therefore, the effect of different contents of acetonitrile , as well as on the stability and existing forms of the targeted mycotoxins. p > 0.05, 3O4@COF (TAPT–DHTA) under alkaline conditions. Therefore, the optimal pH was set to 4.0.The pH value of the sample solution had a great influence on the charge property of the surface of the Fe−1) were evaluated  were much higher than those obtained when the adsorption solution contained sodium chloride  [To explore the effect of ionic strength on the extraction efficiency of mycotoxins, different sample solutions with various amounts of sodium chloride  . Hence, p > 0.05, The adsorption time could also affect the extraction efficiency. A series of experiments was conducted to optimize the adsorption times . It was observed that the recoveries of the nine mycotoxins gradually increased in the range of 2–8 min D. When 83O4@COF(TAPT–DHTA) nanomaterial on the recoveries of the nine mycotoxins were compared (3O4@COF(TAPT–DHTA) was used, the adsorption and desorption of the nine mycotoxins achieved equilibrium, and the highest recoveries were achieved within the acceptable range (75–97%) except for AFB1. Although the recovery of AFB1 was a little lower than that in the case of 30 mg, there was no significant difference between them  could not provide adequate adsorption sites for these mycotoxins. Conversely, when the amounts of adsorbent were excessive, such as 25 and 30 mg, it might not have been easy to elute these mycotoxins from Fe3O4@COF(TAPT–DHTA). Thus, 20 mg of Fe3O4@COF(TAPT–DHTA) was selected for study.The amount of adsorbent is a critical factor in the MSPE process. The effects of various amounts  of Fecompared E. When 23O4@COF(TAPT–DHTA) nanomaterial, the π-π, hydrogen bonding and hydrophobic interactions between adsorbent and analytes could be destroyed by organic solvents. Therefore, elution solvent with different polarities was optimized. As indicated in 1 and ZEN were much lower compared to methanol  nanomaterial. Consequently, 3.0 mL of pure methanol for 4 min were the optimal elution conditions.The volume of the elution solvent is crucial in the MSPE procedure. Different volumes  of elution solvent were investigated . The res3O4@COF(TAPT–DHTA) as magnetic adsorbent, extraction time of 8 min using 3 mL water containing 1% acetonitrile as adsorption solution (pH 4.0), and 3 mL methanol as elution solvent with desorption time of 4 min.Therefore, based on the optimization of these key parameters, the optimal MSPE conditions were determined as follows: 20 mg Fe3O4@COF(TAPT–DHTA), the purification efficiency of the synthesized nanomaterials was investigated in five kinds of fruits including watermelon, hawthorn, melon, tomato and strawberry. The sample solutions treated with Fe3O4@COF(TAPT–DHTA) were almost colorless and transparent (3O4@COF(TAPT–DHTA) could eliminate the interferences. The matrix effects of the targeted mycotoxins in the five kinds of fruits were in the range of 80.04–106.87% except for TeA (127.19–144.88%), ALS (46.31–102.74%) and ZEN (55.01–83.33%), as shown in For further characterization of the MSPE performance of Fensparent A in comp2) > 0.99 over the concentration range of 0.05–200 μg kg−1 in the matrices for all five kinds of fruits , 55% (3.4–54.8 μg kg−1), 25% (29.5–56.3 μg kg−1), 50% (43.4–123.7 μg kg−1) and 45% (38.8–190.4 μg kg−1) in the tomato, strawberry, watermelon, melon and hawthorn samples, respectively. TeA was detected in the tomato and strawberry samples, with incidences (concentration ranges) of 50% (3.8–6.5 μg kg−1) and 45% (1.9–5.6 μg kg−1), respectively. The incidence (concentration ranges) of AOH in the tomato, strawberry and hawthorn samples was 10% (3.05–4.0 μg kg−1), 15% (4.9–20.0 μg kg−1) and 25% (3.7–14.2 μg kg−1), respectively. These results are similar to previous reports, with pollution levels at the same level as other regions [−1, 0.6–18.2 μg kg−1 and 1.3–8.1 μg kg−1, respectively. OTA, AFB1 and ZEN were not detected in the five kinds of fruit samples. Therefore, the survey results demonstrated that the fruits were mainly susceptible to contamination by Alternaria mycotoxins, which might impose health risks to the consumer.A validated approach was employed to investigate mycotoxin contamination of a total of 100 samples of the five kinds of fruits, i.e., tomato, strawberry, watermelon, melon and hawthorn. As shown in  regions ,32. In ap > 0.05, To demonstrate the accuracy of the method, a comparison between the current established approach and the reference method  was perf3O4@COF(TAPT–DHTA) was synthesized and utilized as an MSPE adsorbent for the first time to enrich and determine trace levels of nine mycotoxins in fruits by coupling with UHPLC–MS/MS. Owing to the presence of abundant aromatic rings and the carbonyl group in the structure of the adsorbent, the effective enrichment of the targeted mycotoxins was achieved by virtue of a strong π-π interaction and hydrogen bonding between the mycotoxins and Fe3O4@COF(TAPT–DHTA). The established method showed a wide linear range, high sensitivity, satisfactory recoveries and good precision, and was successfully employed for the analysis of mycotoxins in real fruit samples. The fruits were found to be easily contaminated with different mycotoxins, i.e., TeA, AOH, ALT, etc. Therefore, continuous monitoring of the occurrence of multiple mycotoxins is essential to ensure of the safe consumption of fruits.In summary, Fe3·6H2O (≥99.5%) and FeCl2·4H2O (≥99%) were provided by Sinopharm Chemical Reagent Co. .1,2-dichlorobenzene, 1-Butanol and 1,4-Dioxane were supplied by Macklin Co. . DHTA and TAPT were obtained from Yuanye Bio-Technology Co. . Nylon filters (0.22 μm) were obtained from Navigator Lab Instrument Co. Ltd. . Deionized water was prepared by a Milli-Q water purification system .All the organic solvents, acids, alkalis and salts were HPLC or analytical grade. Acetonitrile and methanol were purchased from Merck . Ammonium acetate, formic acid, aqueous ammonia, FeCl1, OTA and ZEN were obtained from Sigma-Aldrich . Standard stock solutions of the nine mycotoxins (10 μg mL−1) were prepared in acetonitrile and stored at −20 °C in the dark. Their chemical structures and physicochemical parameters are shown in High purity (≥98%) standards of TeA, AOH, AME, TEN, ALT, ALS, AFBA total of 100 random fruit samples were provided by local markets and supermarkets in Shanghai. The samples were ground into powder or pulp and stored at −20 °C. −1 was performed with an FTIR spectrometer . Ultraviolet-visible (UV–VIS) absorption spectra were obtained using a JENA2010 spectrophotometer . A Super-X Spectrometer  used for elemental analysis and energy spectrum analysis. The magnetic property was studied with a LakeShore7404 vibrating sample magnetometer .Scanning electron microscopy (SEM) images were achieved on a ZEISS Gemini SEM 300 electron microscope  operated at 3.0 kV. Fourier transform infrared (FTIR) spectroscopy with a recording range of 500–4000 cm3O4@COF(TAPT–DHTA) are shown in 3O4 magnetic nanoparticles (MNPs) were fabricated according to the previously reported Massart method with minor changes [3·6H2O and 1.59 g FeCl2 4H2O were dissolved in 12.5 mL water containing 0.43 mL of 30% HCl under nitrogen atmosphere. Afterwards, the obtained mixture was added dropwise into 125 mL 1.5 M NaOH aqueous solution under vigorous stirring for 40 min. The synthesized Fe3O4 product was collected using a strong magnet and washed several times with water, then dried at 65 °C for further use. Secondly, Fe3O4@COF(TAPT–DHTA) was fabricated using a simple synthesis method [3O4 MNPs (200 mg), DHTA (105 mg) and TAPT (85 mg) were mixed in the 40 mL 1,4-dioxane/butanol  solvent system, and then sonicated for 5 min. This was followed by adding 0.5 mL of 36% acetic acid, and the mixture was then stirred for 2 h at 25 °C. Then, 4.5 mL acetic acid/deionized water  was dropped into the mixture and refluxed at 75 °C for 48 h. Finally, the obtained Fe3O4@COF(TAPT–DHTA) was separated by magnet, washed several times with methanol and dried for further use.The preparation procedures of Fe changes ,35. Bries method . The prev/v) for 1 min, and then ultrasonicated for 60 min at room temperature (25 °C). After centrifugation at 8000 rpm for 5 min, a 3 mL aliquot of the supernatant was evaporated to dryness under nitrogen flow and dissolved to 3 mL with an aqueous solution containing 1% acetonitrile (pH 4.0). This was followed by the addition of 20 mg of Fe3O4@COF(TAPT–DHTA), the mixture was vortexed for 8 min to fully adsorb the mycotoxins onto the nanocomposites. Afterwards, the supernatant was discarded by a magnet and 3 mL methanol was added to elute the targeted mycotoxins for 4 min under ultrasonic conditions. The elution was collected and dried with nitrogen at 50 °C. Finally, the residues were re-dissolved with 1 mL acetonitrile/water containing 5 mmol L−1 ammonium acetate , passed through a 0.22 μm nylon filter before analysis. The schematic illustration of MSPE is shown in 3O4@COF(TAPT–DHTA) nanocomposites were used as disposable purification adsorbents.A fruit sample of 2.0 g was vortexed in 10 mL acetonitrile/formic acid  with an analytical column EC-C18   maintained at 40 °C. The binary gradient mixture consisted of (A) methanol and (B) water containing 5 mmol L+) and negative (ESI−) mode. The capillary voltages were set at 2.5 kV (ESI+) and 1.5 kV (ESI−). The source and desolvation temperatures were 150 °C and 500 °C, respectively. The desolvation gas flow and cone gas flow were 800 L h−1 and 150 L h−1, respectively. Multiple reaction monitoring (MRM) mode was established and is shown in Waters XEVO TQ-S mass spectrometer system  was performed to detect separated mycotoxins both in positive (ESI−1) of mixed standard solutions of the nine mycotoxins were freshly prepared both in blank matrix solution and standard solutions , respectively. The calibration curves were obtained by plotting the responses (y) versus analyte concentrations (x). The limit of detection (LOD) and limit of quantification (LOQ) were applied to evaluate the sensitivity of the method, which were defined by the signal-to-noise ratio (S/N) of 3 and 10, respectively. The recoveries and intra- and inter-day precision were measured using blank samples spiked with three different concentrations of mycotoxins . The relative standard deviations (RSDs) in a single day and five consecutive days were devoted to estimation of the intra-day precision and inter-day precision, respectively. The matrix effect (ME) (%) was calculated according to the following equation:The performance of the established method was carefully validated according to the recommendations of European Commission Decision 2002/657/EC by determination of linearity, matrix effect, sensitivity, accuracy (recovery), and precision (%RSD) . DiffereData were analyzed using SPSS Statistics 18  and presented as mean ± SD. Significant tests were conducted by the analysis of variance (ANOVA) followed by Duncan’s least significant test. The Shapiro–Wilk test was used to check the normality of the data before using ANOVA.


C19orf12 gene. C19orf12 has been implicated in playing a role in lipid metabolism, mitochondrial function, and autophagy, however, the precise functions remain unknown. To identify new robust cellular targets for small compound treatments, we evaluated reported mitochondrial function alterations, cellular signaling, and autophagy in a large cohort of MPAN patients and control fibroblasts. We found no consistent alteration of mitochondrial functions or cellular signaling messengers in MPAN fibroblasts. In contrast, we found that autophagy initiation is consistently impaired in MPAN fibroblasts and show that C19orf12 expression correlates with the amount of LC3 puncta, an autophagy marker. Finally, we screened 14 different autophagy modulators to test which can restore this autophagy defect. Amongst these compounds, carbamazepine, ABT-737, LY294002, oridonin, and paroxetine could restore LC3 puncta in the MPAN fibroblasts, identifying them as novel potential therapeutic compounds to treat MPAN. In summary, our study confirms a role for C19orf12 in autophagy, proposes LC3 puncta as a functionally robust and consistent readout for testing compounds, and pinpoints potential therapeutic compounds for MPAN.Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a relentlessly progressive neurodegenerative disorder caused by mutations in the  C19orf12 (*614297) [C19orf12 orthologs in fruit flies leads to neurodegeneration comparable to humans [C19orf12 negatively affects neuronal and muscular development [Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a rare form of neurodegeneration with brain iron accumulation (NBIA) . Patient*614297) ,4,5,6. Telopment .C19orf12 is ubiquitously expressed, with highest expression levels in adipose tissue, brain, and muscle.In humans, the gene encodes a membrane-associated protein that localizes to mitochondria, endoplasmic reticulum (ER), and endoplasmic reticulum–mitochondria contact sites ,9,10. AcC19orf12 expression in whole blood is co-regulated with genes involved in fatty acid biogenesis and valine, leucine, and isoleucine degradation. The authors also showed that C19orf12 expression increases continuously during the differentiation of healthy pre-adipocytes into fully mature adipocytes [C19orf12 in PLA2G6-deficient flies rescued acyl-chain shortening in phospholipids caused by loss of PLA2G6 [C19orf12 and other NBIA genes are associated with reduced palmitoylation of the transferrin receptor, leading to its impaired recycling and elevated cytosolic iron accumulation in patients’ fibroblasts [So far, the function of C19orf12 is unknown, though the protein has been associated with a potential role in lipid metabolism. In 2011, Hartig et al. showed that roblasts .C19orf12 stimulates lipidation of the autophagy marker LC3-I to its active form LC3-II. The authors also showed that fibroblasts derived from one MPAN patient carrying the homozygous G58S variant were more susceptible to oxidative stress-induced cell death and exhibited increased mitochondrial calcium compared with control cells, suggesting that the mutation impairs mitochondrial function and calcium homeostasis [A role for C19orf12 in autophagy has been proposed by Venco et al. . The stueostasis .C19orf12 have been associated with impaired mitochondrial respiration, reduced mitochondrial length and cellular adenosine 5′-triphosphate (ATP), as well as increased iron, reactive oxygen species (ROS), and lipid peroxidation, conferring MPAN cells an increased susceptibility to ferroptosis, which was rescued upon treatment with the iron chelator DFO [More recently, both recessive and dominant pathogenic variants in ator DFO .Considering the variety of phenotypes associated with C19orf12 deficiency and the need to identify a robust functional target for the assessment of therapeutic options for MPAN patients and provide them with rational treatment, we re-evaluated most of the reported phenotypes in a larger sample cohort.We provide evidence that impaired autophagy is the only significant readout in MPAN fibroblasts and can be used to screen small molecules with potential therapeutic effects in MPAN.C19orf12 can be found in A list of primary skin fibroblasts used in this study and their variants in Commercial cells included NHDF , HEK293FT , HeLa, Hep G2, and SH-SY5Y .2 humidified atmosphere. All cell lines tested negative for mycoplasma contamination using the MycoAlert Mycoplasma detection kit , according to the manufacturer’s instructions.Cells were cultured in high glucose Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , 50 U/mL penicillin/streptomycin , and 200 μM uridine , at 37 °C in a 5% CO6-tag followed by a TEV protease cleavage site [Human C19orf12 long isoform , medium isoform , and short isoform  were amplified by PCR using Pfu polymerase and cloned into pETM-11, an expression vector containing an N-terminal Hisage site . All expE. coli strain Rosetta2 (DE3) and cultured at 20 °C in 2-L flasks containing 500 mL ZYM 5052 auto-induction medium [E. coli strain Rosetta2 (DE3) pLysS and cultured in 2-L flasks containing 500 mL of ZYM 5052 and 100 µg/mL kanamycin, and 33 µg/mL chloramphenicol. Cells were harvested by centrifugation after reaching saturation.The constructs for the long and medium isoforms were transformed into n medium , 100 µg/2, 10 µg/mL DNaseI, 1 mM AEBSF.HCl, 1 mg/mL lysozyme, 0.01% (v/v) 1-thioglycerol, pH 8.0) containing 1% (w/v) DDM, incubated for 10 min on the bench and lysed by sonication. The lysate was clarified by centrifugation  and filtration (0.45 µM). The supernatant was applied to a 3-mL Ni-NTA column , equilibrated in binding buffer  1-thioglycerol, 0.01% (w/v) DDM, pH 8.0). The column was washed 2 times with 25 mL binding buffer. Bound proteins were eluted with elution buffer  DDM, 0.01% (v/v) 1-thioglycerol, pH 8.0). Fractions containing the target proteins were pooled and dialyzed overnight at 4 °C against 1 L SEC buffer  DDM, 0.01% (v/v) 1-thioglycerol, pH 8.0) in the presence of His-tagged TEV protease in a 1:25 molar ratio (TEV:protein). As described above, the cleaved-off proteins were further purified by affinity chromatography, and the flow-through and protein-containing wash fractions were pooled and concentrated to less than 5 mL. This was subsequently subjected to size exclusion chromatography using a HiLoad Superdex 200 16/600 GL column , equilibrated in SEC buffer. The elution fractions containing the C19orf12 protein were collected and concentrated to approx. 10 mg/mL and stored at −80 °C.For the purification of long and medium isoforms, cells from 1 L of culture were resuspended in 60 mL lysis buffer  NP-40, 1 mg/mL lysozyme, 0.01% (v/v) 1-thioglycerol, pH 8.0), and lysed by sonication. The lysate was clarified by centrifugation  and filtration (0.2 µM). The purification was performed as described above using buffers without DDM.For the purification of the short isoform, cells from 1 L of the culture were resuspended in 60 mL lysis buffer  and 8696.07 (short isoform) MCells were seeded in black 96-well plates with black bottoms at a density of 20,000 cells/well and left to adhere overnight. The following day, intracellular calcium was measured using the Cal-520 AM kit . Cells were washed with HHBS  and incubated with 10 µM Cal-520 in HHBS supplemented with 10% FBS for 90 min at 37 °C, followed by 30 min at room temperature. Afterward, half of the wells were treated for 3 min with 100 µM ATP  in HHBS, while the other half were incubated with HHBS only. Fluorescence was measured using the microplate reader with the following parameters: excitation/emission = 490/525 nm.g for 5 min, and the supernatant was transferred to a clean plate for protein quantification.Afterward, cells were washed with PBS  and mechanically lysed using 1× mammalian lysis buffer  freshly supplemented with protease inhibitors  by pipetting up and down and scraping the bottom of the wells with the pipette tips. After 15 min of incubation, cells were centrifuged at 435× Cells were seeded in black 96-well plates with black bottoms at 20,000 cells/well density and left to adhere overnight. The following day, ROS were measured using the DCFDA/H2DCFDA—Cellular ROS Assay Kit . Cells were washed once with 1× buffer, then incubated with 25 µM DCFDA in 1× buffer for 45 min at 37 °C. Afterward, cells were washed once with 1x buffer, and half of the wells were treated with 50 µM tBHP in 1× buffer supplemented with 10% FBS. Next, 1× buffer + 10% FBS was added to the other half of the wells. After 3 h of incubation at 37 °C, cells were washed with PBS, and fluorescence was measured in the microplate reader at excitation/emission = 485/535 nm. Cells were lysed for protein quantification.Cells were seeded in transparent 96-well plates at a density of 20,000 cells/well and left to adhere overnight. The following day, cells were lysed, and protein concentration was measured with Bradford. Reduced glutathione in cell lysates was measured using GSH/GSSG Ratio Detection Assay Kit . A GSH standard curve was prepared according to the manufacturer’s instructions in a black 96-well plate, to which the cell lysates were added. The GSH Assay Mixture was added to each GSH standard and cell lysate sample. After 10 min of incubation, fluorescence was measured with the microplate reader at excitation/emission = 490/520 nm.g for 1 min, and the supernatant was transferred to black 96-well plates. According to the manufacturer’s instructions, total ATP was quantified using ATP Bioluminescence Assay Kit CLS II . Luminescence was measured using the microplate reader at 10 s integration time.Cells were seeded in transparent 96-well plates at a density of 20,000 cells/well and left to adhere overnight. The following day, cells were lysed using boiling 100 mM Tris, 4 mM EDTA pH 7.5. After 2 min of incubation, cells were centrifuged at 1000× 2. Cells were washed with medium  and incubated with the same solution for 30 min in a 37 °C incubator without CO2. Oxygen consumption rate (OCR) was measured during 3 cycles of 2 min mixing, 2 min waiting, and 3 min measuring at the basal state and in the presence of 1 µM oligomycin, 0.4 µM FCCP, and 2 µM/2.5 µM of rotenone/antimycin A. At the end of the measurements, cells were washed with PBS and the plate was stored at −80 °C. The following day, the number of cells was counted using the CyQUANT™ Cell Proliferation Assay according to the manufacturer’s instructions.Cells were seeded in Seahorse XF96 Cell culture microplates  at 20,000 cells/well density and left to adhere overnight. On the same day, Seahorse XF96 Sensor Cartridges  were hydrated by adding sterile water to each well of the utility plate. The following day, water was removed, and Seahorse XF Calibrant solution  was added to the Sensor Cartridge and incubated for 45–60 min in a 37 °C incubator without COFluorescence values  and luminescence values  were normalized to proteins with a Bradford assay  using a plate reader . Oxygen consumption rates (OCR) were normalized to cell numbers with CyQUANT™ Cell Proliferation Assay .To control for inter-plate variability, control NHDF cells were seeded in each plate of each independent experiment. Within each plate, fluorescence, luminescence, and OCR values of control and MPAN samples were normalized to proteins or cell number normalized NHDF average values .p values were calculated with an independent sample t-test. All p values were two-sided with a significance level of 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001.In each assay, fold change values are shown in box plots. Within each box, horizontal black lines denote median values. Boxes extend from the 25th to the 75th percentile of each group’s distribution and the whiskers denote adjacent values. Dots outside of the whiskers denote outliers. Cells were seeded in 6-well plates at a density of 90,000 cells/well and left to adhere overnight. The following day, cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4  for 1 h at room temperature, washed three times with 0.2 M sodium cacodylate buffer , and fixed in 1% osmium tetroxide, 1.5% potassium ferrocyanide in 0.1 M cacodylate for 1 h on ice. After several washes in distilled water, samples were “en bloc” stained with 0.5% uranyl acetate in water overnight at 4 °C. Finally, samples were dehydrated in a graded ethanol series . Samples were then infiltrated in a 1:1 ethanol/ Epon 812 solution for 2 h, in 100% Epon twice for 1 h each; then the monolayer cells were covered with a layer of Epon and polymerized in an oven at 60 °C for 48 h. At the end of the polymerization, the plastic was separated from the Epon block by mechanically breaking the plastic, leaving the cell monolayer facing up on the resin block. A portion of the specimen was glued on top of an Epon block and mounted on a Leica Ultracut UCT ultramicrotome. Ultrathin (70–90 nm) sections were then collected on copper grids and stained with uranyl acetate and lead citrate. Grids were examined with a transmission electron microscope  at 120 kV. Mitochondria were segmented manually from the EM images. For each mitochondrion mask of every image, size and shape properties were measured with the eccentricity and maximum Feret diameter functions on Matlab.1  for 3 h at 37 °C, 100 nM Torin and 100 nM Bafilomycin for 3 h at 37 °C, 20 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP)  for 24 h at 37 °C, and 20 µM CCCP and 100 nM Bafilomycin for 24 and 3 h, respectively, at 37 °C. Before each treatment fibroblasts were washed with PBS.Confluent fibroblasts were treated in the following conditions: 100 nM Torin  for 3 h at 37 °C, 100 nM Bafilomycin Ag for 3 min and 30 s. Cell pellets were resuspended in PBS and centrifuged at 1000× g for 5 min at 4 °C. Washed pellets were lysed in RIPA buffer  NP-40, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, pH 8.0) with freshly added protease inhibitors , and incubated for 1 h at 4 °C while constantly spinning. Cell lysates were centrifuged at 15,000× g for 15 min at 4 °C, and supernatants were transferred to clean tubes. Protein concentration was determined with a Bradford assay.Afterward, fibroblasts were washed with PBS, detached using trypsin , and centrifuged at 500× g at 4 °C for 10 min, and then the protein concentration was determined by Bio-Rad protein assay dye reagent .Autoptic or bioptic frozen human tissues were homogenized in 15 volumes of RIPA buffer  in the presence of complete protease inhibitors  using a Dounce homogenizer. Homogenates were incubated on ice for 30 min, cleared by centrifugation at 10,000× in-house) and transferred on a PVDF membrane . After blotting, membranes were washed 3 times with TBS-T  and blocked for 5 min using EveryBlot buffer . Membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies or primary antibodies against housekeeping genes for 1 h at room temperature. Membranes were washed three times with TBS-T between each incubation and before detection. Each antibody was diluted in EveryBlot buffer at the following dilutions: anti-LC3 , anti-p62 , anti-C19orf12 , anti-SDHA , anti-actin , anti-tubulin , anti-rabbit , anti-mouse , and anti-guinea pig . Immunological detection was performed using an ECL solution  and images acquired at Fusion FX (Vilber) instrument. Quantification was performed using Fiji software v. 2.3.0/1.53f [Protein samples were reduced using 4× Laemmli buffer  and heated at 50 °C for 10 min. Separation of proteins occurred via SDS-PAGE. Samples were loaded on polyacrylamide gels  at 7000 cells/well density and left to adhere. Subsequently, cells were treated with 20 μM CCCP for 3, 6, or 24 h and fixed with 4% PFA. Cells were permeabilized using a 0.1% NP-40 solution in PBS, and primary and secondary antibodies were diluted in a 2% BSA 0.1% NP-40 solution in PBS at a 1:200 and 1:500 dilution, respectively. LC3 was immuno-stained with the corresponding antibody  and detected with a fluorescent-labeled anti-rabbit secondary antibody . Nuclei were stained with DAPI , and images were acquired by confocal microscopy (Leica TCS SP5). The quantification of LC3 puncta total fluorescence was performed using the Fiji software v. 2.3.0/1.53f using the formula integrated density/area of cell × average mean of background fluorescence to calculate the corrected total cell fluorescence (CTCF).Fibroblasts were seeded as described in the section above. Cells were simultaneously treated for 24 h with 20 µM CCCP and an autophagy-modulating compound . Data were aggregated into the categories of patients and controls. No significant alterations emerged in the patient group under basal conditions, while a significant difference was detected after treating cells with ATP A,B. Howe finding . This wa finding , we searG58S/G58S and C19orf12p.99fs*102 fibroblasts [ATP is a critical metabolite and provides energy in various cellular processes. Cells produce ATP through oxidative phosphorylation in mitochondria and glycolysis in the cytosol. Based on a recent report indicating reduced ATP in C19orf12 knock-out (KO) M17 cells in addition to C19orf12roblasts , we evalMitochondrial contribution to respiration and cellular ATP production was assayed with the Seahorse assay in a subset of MPAN fibroblasts. We evaluated oxygen consumption rate (OCR) at the basal and the maximal respiration state, i.e., after stimulation with carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and the ATP-linked respiration. No significant alterations were detected in the patient group E–G or inT11M/G69Rfs*10, C19orf12D18G/L132Q, and C19orf12 G69Rfs*10/G69Rfs*10 fibroblasts for investigation  using Thiol Green as a proxy of the endogenous oxidative stress defense. No significant decrease of GSH was identified in patient cells , support1, an autophagy inhibitor, and the combination of both , we depolarized mitochondria of C19orf12G69Rfs*10/G69Rfs*10 fibroblasts and tracked LC3 puncta fluorescence by confocal microscopy at different time points  were not consistently impaired in all patients. We did not explore iron and ferritin levels, however, the absence of increased ROS, and normal GSH levels, spoke against eliciting ferroptosis in MPAN fibroblasts.The absence of a consistent mitochondrial impairment in MPAN fibroblasts in our study is probably due to the fact that we used cells from multiple individuals, thus increasing interindividual variability and producing data with larger variation, when compared with studies that used repeated measures in smaller data sets, as in . While oG58S/G58S mutations after ATP stimulation. A similar result was reported in a previous study [G58S/G58S fibroblasts in the current and two previous studies [A significant increase in intracellular calcium was identified only in the cell line with C19orf12us study  using fius study ). We exc studies ,14 were In the current study, we also point out that the effects of AD MPAN mutations on mitochondrial cellular phenotypes are indistinguishable from those of AR mutations, providing evidence that the overlap of clinical and pathology aspects in AD and AR MPAN patients  is also By investigating autophagy in naïve MPAN fibroblasts, we corroborated the finding that C19orf12 intervenes in the initiation of the autophagy process by supporting the conversion of LC3-I to LC3-II without binding directly to LC3  and, posAs the conversion of LC3-I to LC3-II relies on the conjugation of LC3-I to a phosphatidylethanolamine (PE) moiety, defective lipidation of LC3 in MPAN may point to a primary role for C19orf12 in the conjugation of PE to LC3-I, or in the synthesis of PE between ER and mitochondria. Considering C19orf12 localization at the mitochondria, ER, and ER-mitochondria contact sites, C19orf12 could tether mitochondria and ER to allow efficient transfer of the PE precursor, phosphatidylserine, from ER to mitochondria, its conversion to PE in mitochondria and transfer back to the ER for phosphatidylcholine production. By integrating studies in cellular models with dynamic lipid metabolism and structural biology, we hope to unambiguously clarify the function of C19orf12 soon.C19orf12 result in impaired activation of LC3. Therefore, any pharmacological treatment restoring LC3 activity could be used as a therapeutic compound for MPAN. To this end, we screened 14 autophagy-modulating compounds in MPAN fibroblasts and identified five that were able to increase the LC3 levels in patient fibroblasts: carbamazepine, LY294002, ABT-737, oridonin, and paroxetine. Of note, LY294002 and paroxetine may also have an inhibitory effect on autophagy [Despite the fact that the primary function of C19orf12 is still to be defined, mutations in utophagy ,21, suggutophagy ,23. Stilutophagy , and theutophagy ,25.Although the five hits take part in various biological processes , based on a literature search, they have a shared effect in stimulating the initial steps of the autophagy process. Oridonin and LY294002 inhibit the mechanistic target of rapamycin complex 1 (mTORC1) by inhibiting the PI3K/Akt pathway or activating p53, respectively ,26. CarbAlthough fibroblasts cannot recapitulate tissue-specific metabolic aspects, conserved processes such as autophagy can be reliably investigated due to the conservation of the autophagy machinery and the prominent expression of the 141 aa isoform in fibroblasts, as well as in all other investigated cell types and tissues. In conclusion, while further research is needed to identify the primary function of C19orf12, our study moves one step closer to understanding the consequences of C19orf12 dysfunction at the cellular level and to establishing a rational therapy for MPAN.


Funneliformis mosseae (FM) and Claroideoglomus etunicatum (CE) were used as experimental AMF. In a pot experiment, plants “N1” and “Xu28” were inoculated with FM or CE, and applied with or without K fertilizer to uncover the effects of K application and AMF inoculation on the root morphology and nutrient absorption of sweet potato during their growing period. Results demonstrated that AMF inoculation-improved root morphology of sweet potato highly relied on K application. With K application, AMF inoculation significantly increased root tip number of “N1” in the swelling stage and optimized multiple root morphological indexes  of “Xu28” and CE had the best optimization effect on the root morphology of “Xu28”. In addition, CE inoculation significantly promoted root dry matter accumulation of “Xu28” in the swelling and harvesting stages, coordinated aerial part and root growth of “Xu28”, reduced the dry matter to leaf and petiole, and was beneficial to dry matter allocation to the root under conditions of K supply. Another promising finding was that CE inoculation could limit K allocation to the aboveground and promote root K accumulation of “Xu28” under the condition with K application. The above results lead to the conclusion that K and CE displayed a synergistic effect on root development and K acquisition of high-KIUE “Xu28”. This study could provide a theoretical basis for more scientific application of AMF in sweet potato cultivation and will help further clarify the outcomes of plant-K–AMF interactions.Sweet potato is a typical “potassium (K)-favoring” food crop and strongly dependent on arbuscular mycorrhizal fungi (AMF). Recent studies show the importance of K and AMF to morphology optimization and nutrient uptake regulation of sweet potato; meanwhile, the interaction exists between K and K use efficiency (KIUE) in sweet potato. To date, only a few studies have shown that AMF can improve plant K nutrition, and whether the benefits conferred by AMF on plant are related to K remains unclear. In this study, low-KIUE genotype “N1” and high-KIUE genotype “Xu28” were used as experimental sweet potato;  Ipomoea batatas (L.) Lam.], belongs to the Convolvulaceae family, is widely grown in tropical and subtropical climates worldwide  could live in a mutualistic symbiosis with plant roots of the majority (> 80%) of terrestrial plant species . Sweet pFunneliformis mosseae (FM) and Claroideoglomus etunicatum (CE) were used as the experimental AMF mainly considering the existing studies ; and 15.1 mg kg–1 organic matter. The pH of the soil was 7.13 in a 1:2.5 (w/v) soil–water suspension ratio.The soil was sandy loam fluvo-aqic (Eutyic Cambisol) collected from Jiangyan City, Jiangsu Province . Before The two sweet potato genotypes “N1” and “Xu28” were selected based on K uptake and K-use efficiency in the previous studies , 2018, wTrifolium repens as host plant in sterilized fine sand and vermiculite culture in a greenhouse of the Jiangsu Academy of Agricultural Sciences  over 3 months (–1), external hyphae, infected root in addition to fine sand and vermiculite.The original AMF inoculum FM (BGC XJ02) and CE (BGC XJ03C) were purchased from the Bank of Glomales in China (BGC), Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences . AMF spores were germinated with 3 months . The AMF2SO4 per kg air-dried soil) were applied in the presence or absence of AMF (FM or CE) for two sweet potato genotypes (“N1” or “Xu28”). Each plastic pot  was filled with 7 kg air-dried soil; the soil contained 0.82 g urea, 1.55 g superphosphate, and either without K2SO4 (K0) or with 1.12 g K2SO4 (K1). In total, 16 (cultivar × K treatment × AMF inoculation) treatments include (1) N1/−K/−FM, (2) N1/−K/+FM, (3) N1/−K/−CE, (4) N1/−K/+CE, (5) N1/+K/−FM, (6) N1/+K/+FM, (7) N1/+K/−CE, (8) N1/+K/+CE, (9) Xu28/−K/−FM, (10) Xu28/−K/+FM, (11) Xu28/−K/−CE, (12) Xu28/−K/+CE, (13) Xu28/+K/−FM, (14) Xu28/+K/+FM, (15) Xu28/+K/−CE, and (16) Xu28/+K/+CE. For +FM or +CE, the AMF inoculum (70 g) of FM or CE was inoculated into each pot at the depth of 10 cm, respectively. For −FM or −CE, the same amount of AMF inoculum (70 g) of FM or CE was sterilized three times  and filtered through a 25-μm membrane, respectively. The obtained microbial filtrate (50 ml) of FM or CE inoculum was inoculated into the pot of −FM or −CE, respectively. Each pot contained one plant, and there were 12 pots of each of the 16 (genotype × K treatment × AMF inoculation) combinations.A greenhouse experiment was conducted in the greenhouse of the Jiangsu Academy of Agricultural Sciences  over 4 months from August to November 2021. Two different K treatment ], swelling stage , maturing stage , and harvesting stage . Three sweet potato plants from three individual pots were destructively sampled and regarded as three independent biological replicates.Plant samples were separated into leaves, stems, stalks, and roots for fresh matter (FM) measurement, and fresh root samples were used for the evaluation of root morphological characteristics and the analysis of mycorrhizal colonization. Additionally, the root parts were divided into fibrous roots and tuberous roots at harvesting stage. Then, leaves, stem, petiole, and root were oven-dried at 105°C for 1 h and then at 75°C to constant weight for DM measurement, then ground and passed through a 0.5-mm sieve for K concentration analysis.Root morphology of sweet potato was characterized using an Epson Perfection V850 Pro scanner  at 300 dpi. Root morphological characteristics, including the root length (RL), total root length (RL), root surface area (RA), average root diameter (RD), root volume (RV), root tip number (RT), root branch number (RB), and root cross number (RC), were estimated using WinRHIZO Pro 2017a Root Analysis System  .Mycorrhizal root colonization intensity was assessed. The fresh fine roots (0.5 g) were cleared with 10% KOH for 60 min at 90°C, acidified in 2% HCl for 30 min, and then stained with ink  and the vinegar method at 90°C for 15 min. Then, about 90 root segments were randomly selected from each sample and mounted on the microscope slides. Mycorrhizal colonization of root was determined using a microscope at 40× magnification  .2SO4 and H2O2, then the K concentration was determined by flame photometry (Dried plant samples passed through 0.5-mm sieve (0.5 g) were digested with a mixture of Hotometry . The plaP value is >0.05, the data have the characteristic of normal distribution. If the absolute value of kurtosis is <10 and the absolute value of skewness is <3, the data can be accepted as normal distribution basically. The normal distribution histogram and regression analysis was observed intuitively to check whether this set of data roughly conforms to the normal distribution. For the homogeneity test of variance, when the P value is >0.05, the data conforms to homogeneity of variance. The statistical evaluation between two treatments was compared using the independent-samples t-test, and asterisks denote significant differences between two treatments . The statistical evaluation of more than two treatments was compared using one-way ANOVA, followed by Tukey’s multiple-comparison test (P < 0.05), and different lowercase letters denote significant differences. Two-way ANOVA was used to test the statistical significance of K application (K), FM inoculation (FM), CE inoculation (CE), K application × FM inoculation interaction (K × FM), and K application × CE inoculation interaction (K × CE) using the general linear model (GLM). Three-way ANOVA was used to test the statistical significance of Genotype (G), K application (K), AMF inoculation (AMF), Genotype × K application interaction (G × K), Genotype × AMF inoculation interaction (G × AMF), K application × AMF inoculation interaction (K × AMF), and Genotype × K application × AMF inoculation interaction (G × K × AMF) using the GLM. Pearson’s correlation relationship was analyzed using Origin 2021 and displayed as heatmap format.Means and standard errors (SE) were calculated using SPSS Statistics 20.0 software  . Values Funneliformis mosseae and Claroideoglomus etunicatum could establish symbiotic relationship with sweet potato for both “N1” and “Xu28”  for plant root parameters and nutrient uptake  in relation to the cultivars of Ficus carica, suggesting diverse root strategies for exploiting the soil resources  varied in their effects on plant biomass and nutrient acquisition of different host plant (Genetic and environmental factors collectively determine plant growth and yield . Similart uptake . The intt uptake . On the t uptake . As showesources . In thisesources , indicatst plant . In thisst plant , CE rathst plant , indicatConsistent with the optimized root morphology of “Xu28” , CE inocThis study showed that K application differentially regulated the root architecture of low-KIUE genotype “N1” and high-KIUE genotype “Xu28”, FM and CE inoculation were more inclined to optimize root morphology of “N1” and “Xu28”, respectively. AMF inoculation-improved root morphology of sweet potato highly relied on K application. Particularly, K application and CE inoculation displayed a synergistic effect on root development and K acquisition of high-KIUE genotype “Xu28”. This study provides a theoretical basis for more scientific application of AMF in sweet potato cultivation and helps to further clarify plant–nutrient–AMF interactions.The original contributions presented in this study are included in this article/JY and JW: conceptualization. JY, LW, CX, HZ, and CS: methodology. JY, KS, and XZ: investigation. JY: writing—original draft preparation. JY, YZ, and JW: writing—review and editing and funding acquisition. GZ, YZ, and JW: supervision. All authors contributed to the article and approved the submitted version.