Curculigo orchioides Gaertn (CO) was previously revealed to play a significant role in RA treatment. However, the main active ingredients and molecular mechanisms of CO in regulating RA are still unclear.Rheumatoid arthritis (RA) is an autoimmune disease that can lead to joint destruction and deformity. The active ingredients of CO were obtained from the Traditional Chinese Medicine Systems Pharmacology database and published literature. The targets corresponding to these compounds and the targets linked to RA were collected from public databases. The “ingredient-target” and “protein–protein interaction” networks were constructed to screen the main active ingredients and hub targets of CO in the treatment of RA. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment assays were used to elucidate the potential pharmacological mechanism of CO in RA. Molecular docking was performed to detect the binding between the main active ingredients and hub targets. Collagen-induced arthritis rats were used to validate the hub targets of CO against RA.Network pharmacological topology analysis showed that caffeine, 2,4-dichloro-5-methoxy-3-methylphenol, curculigoside, orcinol glucoside, and orcin were the main active ingredients of CO, and matrix metalloproteinase 9 (MMP9), transcription factor AP-1 (JUN), prostaglandin-endoperoxide synthase 2 (PTGS2), brain-derived neurotrophic factor, and receptor-type tyrosine-protein phosphatase C were the hub targets of CO for RA treatment. Molecular docking revealed that curculigoside and orcinol glucoside had effective binding potential with MMP9, JUN, and PTGS2, respectively. In vivo experiments demonstrated that CO alleviated RA symptoms and inhibited the expression of MMP9, JUN, and PTGS2 proteins.Our study demonstrates the main active ingredients and potential targets of CO against RA, laying an experimental foundation for the development and application of CO as an anti-RA drug. Rheumatoid arthritis (RA) is a chronic autoimmune disease featured by the inflammation of tendon, finally triggering cartilage destruction, bone erosion, and even joint damage, deformity, and disability . The gloCurculigo orchioides Gaertn (CO) is a perennial herb traditionally used to nourish the kidney, strengthen muscles and bones, and dispel cold and dampness [Traditional Chinese medicine (TCM) has gained rising attention in RA treatment due to its excellent effectiveness and low toxicity , 7. Accodampness . CO and dampness , anti-oxdampness , anti-didampness , and antdampness . Our predampness . It is lNetwork pharmacology is a system-level approach integrating pharmacology, network biology, and bioinformatics to elucidate the links among drugs, targets, and diseases . Given thttps://www.tcmspw.com/). We applied Lipinski’s rule of five as the standard to screen the active ingredients of CO [The chemical compounds of CO were acquired from the TCMSP database (ts of CO . Compounhttps://pubchem.ncbi.nlm.nih.gov/) to query the canonical SMILES string, and active ingredients without a canonical SMILES string were deleted. SwissTargetPrediction (http://new.swisstargetprediction.ch) and Traditional Chinese Medicine Systems Pharmacology (TCMSP) databases were utilized to retrieve the potential targets of active ingredients. After removing repeating targets from the two databases, we obtained the potential targets of CO.The obtained active ingredients were imported into the PubChem database (https://www.ncbi.nlm.nih.gov/geo/) with the keyword “rheumatoid arthritis” and species “Homo sapiens.” This dataset contains samples from 7 healthy controls and 16 RA patients. The limma package of R was applied to screen DEGs between RA patients and healthy controls. Adjusted P < 0.05 and |log2Fold Change (FC)|> 1 were set as the screening thresholds. The DEGs were presented as the volcano plot, and the heat map of the expression data was generated.GSE77298 gene expression profiles were downloaded from the GEO database (https://string-db.org/) to build a protein–protein interaction (PPI) network with species restricted to “Homo sapiens” and a confidence score ≥ 0.4. Finally, the PPI network was visualized and analyzed by Cytoscape 3.7.1 software (https://www.cytoscape.org/).The harvested DEGs and targets of active ingredients were overlapped to generate targets related to both RA and CO. Then, the overlapping targets were submitted into the STRING database  with the species set as “Homo sapiens.” The enrichment items with P < 0.05 were regarded as statistically significant.The functions and pathways of potential targets were systematically investigated by Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using the Metascape database (http://www.rcsb.org/) were imported to the PyMOL software to delete the water molecules and separate the ligand. AutoDock Tools 1.5.6 was utilized to convert the small molecules and target proteins to pdbqt format. Autodock Vina software was used for molecular docking [Molecular docking was performed to assess the interaction between the top five active ingredients and the hub targets. Briefly, the 3D molecule structures of ingredients were acquired from the PubChem database. The protein crystal structures of hub targets collected from the RCSB PDB database  and authenticated by Prof. Ling Chen . Bovine type II collagen, complete Freund’s adjuvant (CFA), and incomplete Freund’s adjuvant (IFA) were acquired from Chondex Inc. .The precisely weighed dry CO powder was added to the conical flask, followed by heat reflux extraction with 70% ethanol for 2 h . The extMale Wistar rats (weighing 160–180 g) were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. . The Ethics Committee of Chongqing Traditional Chinese Medicine Hospital approved the experimental protocols (Permit NO. 2020KY-LX). The rats were bred in an air-conditioned room at a constant temperature (23 ± 1 °C) and humidity (60 ± 5%) and were given free access to food and water.n = 8): normal group (NG), model group (MG), tripterygium glycosides , low-dose CO group , and high-dose CO group . Rats in the MG, TGT, LCO, and HCO groups were immunized subcutaneously at the base of the tail with 0.2 mL CII-CFA emulsifier. Seven days later, a booster immune was administered near the primary injection site with 0.1 mL CII-IFA emulsion. The NG rats were injected with an equal volume of normal saline. Drug intervention was performed subcutaneously from day 14 until day 42. The 0.5% sodium carboxymethylcellulose (CMC-Na) solution was taken as a vehicle for administration. Rats in the NG and MG groups were subcutaneously administered with the same volume of saline.Bovine type II collagen was dissolved in an equal amount of CFA or IFA in an ice bath. Rats were randomly assigned into five groups  = viscera weight (mg)/body weight (g).The ankle joints were fixed with 4% paraformaldehyde solution, decalcified with 10% EDTA, embedded in paraffin, and cut into 5-μm-thick slices. Sections were stained with hematoxylin and eosin (H&E) and toluidine blue (TB). A light microscope was used to investigate the inflammatory cell infiltration of synovium, pannus formation, and progressive destruction of articular cartilage.Sections were immersed in 3% hydrogen peroxidase to block endogenous peroxidase, and then incubated overnight at 4 °C with primary antibodies against MMP9 , JUN , and PTGS2 . Specimens were incubated with corresponding secondary antibodies, stained with 3,3-diaminobenzidine (DAB) substrate, and further counterstained with hematoxylin.P < 0.05 denoted a statistically significant difference.GraphPad Prism 8.0 software was applied to analyze all data. The results were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for group comparison and A total of 35 active ingredients conforming to Lipinski's rule were collected through the TCMSP database, and seven active ingredients without canonical SMILES strings were deleted. Curculigoside is reported to have therapeutic effects against RA –23, so aGSE77298 was downloaded to analyze the DEGs between RA patient samples and control samples. A total of 2691 DEGs were identified in RA patients, of which 985 were upregulated and 1706 were downregulated , JUN (5aep), PTGS2 (5f19), BDNF (1b8m) and PTPRC (5fmv) were obtained from the PDB database. The lower the binding energy, the stronger the binding activity. A binding energy < −7.0 kcal/mol represents a good binding affinity . CurculiP < 0.01). Compared with the model group, rats in the HCO and TGT groups showed markedly decreased spleen index (P < 0.01) and thymus index (P < 0.05).Spleen and thymus indices of rats were examined to evaluate the effects of CO on the immune organs Fig. a, b. ComAs displayed by H&E staining, there was no pathological finding of arthritis for the rats in the normal group. In contrast, obvious inflammatory cell infiltration and pannus formation were found in CIA rats. However, the degree of inflammatory cell infiltration and pannus formation were significantly reduced in CIA rats treated with HCO or TGT Fig. a. Semi-qTB staining was performed to analyze the chondral injury of ankle joint. As shown in Fig. P < 0.01) .Immunohistochemical analysis displayed that the expression of MMP9, JUN, and PTGS2 in the ankle joint tissues from the model group was drastically higher than that from the normal group (01) Fig. . HoweverIn this study, network pharmacology combined with molecular docking and experimental validation was employed to investigate the active ingredients of CO and its possible pharmacological mechanisms against RA. Given the results of network analysis, 29 active ingredients and 5 hub targets participated in the anti-RA effect of CO.The 29 active ingredients were mainly phenols and phenolic glycosides, triterpenes and triterpenoid glycosides, and alkaloids. Except caffeine, the other top four active ingredients, including curculigoside, orcinol glucoside, orcin, and 2,4-dichloro-5-methoxy-3-methylphenol are all phenolic and phenolic glycosides, which is consistent with the reported study that CO alcohol extracts are rich in phenols and phenolic glycosides . PreviouIn our present study, molecular docking results showed that curculigoside and orcinol glucoside have a strong binding ability to the hub targets  in the form of hydrogen bonds. PPI network analysis results indicated that MMP9, JUN, PTGS2, BDNF, and PTPRC were the major differential proteins. GO and KEGG analysis demonstrated that the potential targets of CO against RA were mainly related to inflammation responses , 29. TheMatrix metalloproteinases (MMPs), a type of metalloproteinase, play an important role in the degradation of extracellular matrix related to tissue damage in RA . An enhaJUN, a member of activator protein 1 (AP-1), participates in the regulation of inflammatory process in RA by synergistic interaction with NF-κB to activate the pro-inflammatory cytokines , 37. In PTGS2, also known as COX-2, is a key enzyme in prostaglandin biosynthesis and plays a crucial role in inflammatory responses . PTGS2 iIn brief, our study indicated that CO alcohol extracts alleviated the infiltration of inflammatory cells and the formation of pannus in CIA rats. The action mechanism of CO against RA might be related to the downregulation of MMP9, JUN, and PTGS2 expression. Curculigoside and orcinol glucoside are the main bioactive compounds, which have a strong binding ability to hub targets  in the form of hydrogen bonds.In this work, active ingredient screening, potential target prediction and pathway analysis were combined to explore the potential active ingredients and targets of CO against RA. Curculigoside and orcinol glucoside were identified as the main bioactive compounds, and MMP9, JUN, and PTGS2 were confirmed as the potential targets for CO to treat RA. Interestingly, the content of curculigoside and orcinol glucoside in pCO is higher than that of CO according to our previous research, respectively. Further studies are warranted to investigate the effects of curculigoside and orcinol glucoside on RA.


Deoxyguanosine kinase (DGUOK) deficiency is one of the genetic causes of mitochondrial DNA depletion syndrome (MDDS) in humans, leading to the hepatocerebral or the isolated hepatic form of MDDS. Mouse models are helpful tools for the improvement of understanding of the pathophysiology of diseases and offer the opportunity to examine new therapeutic options.DguokF180S/F180S mutation derived from an N-ethyl-N-nitrosourea-mutagenesis screen. Energy expenditure (EE), oxygen consumption (VO2) and carbon dioxide production (VCO2) were assessed in metabolic cages. LC-MS/MS was used to quantify plasma adrenal steroids. Plasma insulin and leptin levels were quantified with commercially available assay kits.Herein, we describe the generation and metabolic characterization of a mouse line carrying a homozygous Ucp1 was increased. Mutant mice also presented differing mitochondrial DNA content in various tissues and altered metabolic activity in mitochondria, but no further phenotypical or behavioral abnormalities. Preliminary data imply normal survival of DguokF180S/F180S mutant animals.Mutant animals displayed significantly lower body weights and reduced inguinal fat pad mass, in comparison to unaffected littermates. Biochemically, they were characterized by significantly lower blood glucose levels, accompanied by significantly lower insulin, total cholesterol, high density lipoprotein and triglyceride levels. They also displayed an almost 2-fold increase in transaminases. Moreover, absolute EE was comparable in mutant and control mice, but EE in mutants was uncoupled from their body weights. Histological examination of inguinal white adipose tissue (WAT) revealed adipocytes with multilocular fat droplets reminiscent of WAT browning. In addition, mRNA and protein expression of Taken together, DGUOK mutation F180S leads to a lean phenotype, with lower glucose, insulin, and lipid levels rendering this mouse model not only useful for the study of MDDS forms but also for deciphering mechanisms resulting in a lean phenotype. Mitochondrial DNA encodes a restricted number of genes including subunits of the triphosphate adenosine (ATP) synthase, cytochrome oxidase and NADH dehydrogenase, all involved in the oxidative phosphorylation and ATP production . MitochoDGUOK mutations can present in two different forms: one characterized by neonatal-onset hepatopathy and encephalopathy and one by isolated liver disease. In the early-onset form, symptoms appear within the first weeks of life with lactic acidosis and hypoglycemia and progressive liver failure with hepatomegaly, elevated transaminases, cholestasis, and jaundice along with hypotonia, nystagmus and psychomotor retardation [DGUOK mutations have been identified in patients with hepatocerebral or isolated hepatic form of the disease. No clear genotype-phenotype correlation could be established; however, missense mutations seem to result into a later onset of symptoms and slower disease progression, in comparison to frameshift or non-sense mutations [Along the same line, ardation . The proardation . Patientardation , 7. Remiardation , 9. Currardation , 10. Sevutations . Furtherutations . Severalutations , 13–16, DguokF180S/F180S), leading to a mild form of MDDS.Murine models serve as a helpful tool to understand pathophysiology of human disease, allow detailed phenotyping, and can provide the basis for the development of new therapeutic strategies. So far, two Dguok animal models have been described: a complete Dguok knock-out (KO) rat model which did not display a pathological phenotype  and a coN-ethyl-N-nitrosourea (ENU)-mutagenesis screen. This alkylating agent induces point mutations in the spermatogonial stem cells of treated mice. Following this procedure, among others, we established a mouse line of familial hyperaldosteronism, carrying four different point mutations all located on chromosome 6 [DguokF180S/F180S (c.539 A > G) mouse line described herein, LC-MS/MS was used to quantify plasma adrenal steroids and, thereby, no significantly elevated aldosterone values could be documented in mutant animals , designed to be specific for either the wild-type (WT) or the mutant sequence  on a 12 h light/dark photoperiod, in the Laboratory Animal Service Center of the University of Zurich. All animal experiments were approved by the Zurich cantonal authorities (License number 090/2019). Animals were fed normal chow diet  Brain, liver, and bilateral inguinal fat pads, subscapular brown adipose tissue (BAT) and adrenal glands were prepared and resected. Adrenal glands were cleaned from adjacent fat tissue. Livers and right sided bilateral organs were stored in paraformaldehyde solution (PFA) for further histological analyses. Brains, livers and left sided bilateral organs were snap frozen in liquid nitrogen and stored at −80 °C for further uses.Blood glucose levels were determined using the Accu-Chek Aviva glucose meter . Serum electrolytes . Total cholesterol, high density lipoprotein, triglycerides were measured with the AU480 Clinical Chemistry System . Plasma insulin and leptin levels were quantified with following commercially available assay kits: Mouse insulin ELISA Kit , Mouse leptin ELISA kit , according to the respective manufacturer’s instructions.Liquid Chromatography/Mass Spectrometry (LC/MS) for adrenal steroids was performed in EDTA plasma samples in collaboration with the University Hospital Carl Gustav Carus at TU Dresden, Institute of Clinical Chemistry and Laboratory Medicine, Experimental Mass Spectrometry and Trace Elements Lab (Dr. Mirko Peitzsch). The method for analysis of the plasma steroid panel, including validation and assay performance characteristics has been described in detail elsewhere .2 consumption, and CO2 production were recorded over both 24 h periods. Locomotor activity was measured using a 2-dimensional infrared light-beam. Energy expenditure (EE), oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated using the manufacturer’s software and values were additionally corrected for lean body mass (LBM). LBM was calculated according to manufacturer’s software as body weight raised to the power of 0.75. Body weight was daily updated in the software ensuring that EE data normalized to LBM were always taking into account the current body weight of the mice.15-week-old male mice were placed individually in air-tight cages designed for metabolic phenotyping in an open-circuit indirect calorimetric system  for four days as previously described . The aveTwo animals of the same genotype were housed together in a clean IVC cage. After 24 h, 1 g of feces were sampled and frozen at −20 °C until analysis. Samples were then dried under a ventilated hood overnight and placed in the decomposition vessel. The decomposition vessel was then placed in the calorimeter bomb and the samples were processed in adiabatic mode. This analysis was performed by the center of Phenogenomics of the École Polytechnique Fédérale in Lausanne.g the clean supernatant was used for RNA extraction. For adipose tissue, the upper phase containing fat was discarded and not used. Upon extraction, the concentration of RNA was measured with NanoDrop One UV Spectrophotometer . 2 µg of RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor . The cDNA was finally diluted to a concentration of 5 ng/μL. The SsoFast EVAGreen Supermix Mastermix was used to quantify the investigated genes. For the gene expression analysis, 10 ng of cDNA was pipetted to the Mastermix. The Cycle program in the Quant 5 was 5’ @ 95 °C then 40 cycles with following conditions: 15”@95 °C and 30”@60 °C. To ensure the absence of unspecific product, a melting curve analysis between 55 °C and 95 °C was also performed at the end. Quantification of gene expression was adjusted using the Tbp mouse gene expression for the investigated adipose tissues and using Gapdh as housekeeping gene for all other tissues.Whole organs were homogenized in the RNA lysis buffer  using a shaking homogenizer with ceramic beads . Upon 5 min centrifugation at 10.000 Acrylamide gels were cast one day before running the gel or at the same day using standard procedures. Whole organs were homogenized using a shaking homogenizer with 4 mm ceramic beads in RIPA Buffer with protease inhibitor  and then centrifuged. The protein concentration was measured with the Pierce BCA Protein Assay kit  and the absorption measured at 565 nm. Protein was then dissolved to the desired concentration using RIPA buffer with Proteinase inhibitor and 2x loading buffer with DTT . For the inguinal fat pad 30 μg of protein were loaded. For the other organs, 7.5 μg of protein were loaded. Proteins were transferred to a Nitrocellulose membrane  with a Blot transfer machine . The membrane was then blocked with a blocking buffer containing 5% dry milk. Next, the membrane was incubated overnight at 4 °C with the primary antibody (dilution 1:1000). The membrane was washed 3 times and then incubated 1 h at RT with the secondary antibody (dilution 1:10000). Finally, after further washing (3x), the membrane was incubated with the detection reagent  and measured with the Western Blot Imaging System . Primary antibodies , GAPDH - D16H11 Rabbit mAb ) were diluted at 1:1000, while secondary antibodies were used at 1:10000 concentration .DguokF180S/F180S mouse and an unaffected littermate was performed in collaboration with the Center for Microscopy and Image Analysis of the University of Zurich (https://www.zmb.uzh.ch/en.html).Tissues remained in 4% paraformaldehyde overnight and then were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin following standard protocols. Hematoxylin and eosin (H/E)-stained adrenal sections were examined with a light microscope using magnifications of ×40 and ×400. For immunohistochemical staining for Ki-67 paraffin-embedded sections were rehydrated, heated in EDTA 1 mM, pH 9.0, SDS 0.05% in the microwave for antigen retrieval, blocked with 3% H2O2 in methanol for 10 min, and incubated with blocking buffer for 15 min. Ki-67 was immunolocalized overnight at 4 °C by means of a rabbit monoclonal antibody  in a dilution of 1:300 in blocking buffer. After rinsing for 15 min in PBS, SignalStain® Boost IHC Detection Reagent  and Sigma Fast DAB  were used for visualization. Transmission electron microscopy of BAT of a n-number of samples included in all experiments/results presented herein was at least five per genotype. Animals/samples were included in the analysis depending on their availability after breeding. No randomization was applied. The investigators were not blinded to the group allocation during the experiments. Statistical analysis was carried out with the Prism 3.02 (GraphPad Software). Statistical significance was determined using the unpaired t-test for normally distributed parameters and Mann–Whitney test for non-normally distributed parameters. To investigate body mass-dependence of EE, a regression-based analysis-of-covariance (ANCOVA) was performed as previously described [*P < 0.05, **P < 0.01 and ***P < 0.001.Sample size was calculated based on the body weight difference observed between WT and mutant animals of the same sex in a previous pilot experiment  resulting in an n-number of 5 animals per genotype. The escribed , 24. StaF180S/F180S line was examined for its adrenal steroid phenotype by LC-MS/MS. Thereby, no significant differences could be observed in the aldosterone levels of this mouse line  and aspartate aminotransferase (AST) levels of ols Fig. , howeverice Fig. . H/E staice Fig. . The Ki-ice Fig. , possiblP < 0.05, Fig. DguokF180S/F180S mutant mice did not differ from controls in terms of absolute EE during both the light and dark phase   Fig. . When anBM) Fig. . FinallyIncreased EE may suggest activation of BAT or browning of WAT in mutant mice. We therefore performed histological analysis of inguinal WAT. As depicted in Fig. DguokF180S/F180S mutant mice  leading to an aberrant metabolic phenotype, characterized by reduced body weight and subcutaneous fat pads, while no other phenotypic abnormalities, such as hepatic or neurological involvement are present that typically characterize MDDS. The mild hepatic impairment documented biochemically and histologically did not seem to phenotypically affect the mice. Thereby, DguokF180S/F180S mice extend the spectrum of MDDS with the possibility to specifically characterize metabolic consequences of the disease.In summary, the mouse model presented herein carries a homozygous DGUOK animal models, the DguokF180S/F180S mutant mouse line provides a metabolic phenotype. Specifically, mutant animals had significantly lower body and inguinal fat pad weights, but no other gross phenotypical abnormalities. Furthermore, DguokF180S/F180S mice presented lower blood glucose values, which is a typical initial sign of MDDS due to Dguok deficiency in humans. However, Pronicka et al. describe an islet cell hyperplasia and a hyperinsulinemia in two patients with Dguok deficiency [DguokF180S/F180S animals presented with significantly lower insulin levels than their unaffected littermates, suggestive of a negative feedback mechanism to compensate for the lower glucose levels. In parallel, these mutant mice displayed low corticosterone values, originating from the adrenal glands, that also presented a very low mitochondrial DNA content. As adrenal steroidogenesis is partly dependent on mitochondrial function, Dguok deficiency could contribute to lack of adrenal counter-regulation thereby accentuating the reduction in blood glucose levels. The DguokF180S/F180S mouse line also displayed significantly lower cholesterol, HDL and triglyceride levels. The lipid profile of affected individuals with MDDS due to DGUOK deficiency has not been described in detail so far, but the Dguok KO mouse line, previously described, presents an opposed biochemical profile, with significantly increased cholesterol levels in the KO animals [DguokF180S/F180S mice displayed up to two-fold increased transaminase levels (ALT and AST), suggestive of a mild hepatic impairment, whereas the Dguok KO mouse model, previously described, presents with a 4- to 5-fold increase in transaminases. Histological signs of hepatic damage are present at different extent in both mouse models [In variance to currently described ficiency , and, sificiency . Unlike DguokF180S/F180S mutant animals had normal or even increased intestinal absorption, excluding a malabsorption of nutrients as causative for their body weight phenotype. Additionally, they did not differ in their absolute EE from control animals. However, in DguokF180S/F180S animals, EE appeared uncoupled from their body mass. The latter may be due to increased browning of white adipose tissue in DguokF180S/F180S animals, as reflected by increased UCP1 protein and mRNA levels in inguinal WAT as well as the appearance of multilocular fat cells on histological examination. These mice did not display any neurological abnormalities, possibly due to the lower but maintained at >50% mitochondrial DNA content in the brain. The fact that they presented a mild hepatic pathology, despite the almost nonexistent mitochondrial DNA in their liver can potentially be explained by the increased hepatic expression of deoxycytidine kinase, the key enzyme for the rescue pathway for dNTP synthesis. Another compensatory mechanism is suggested since we observed an unaffected mRNA expression of mitochondrial genes such as Nd1 in DguokF180S/F180S animals, despite the low mitochondrial DNA content in this tissue. Surprisingly, these animals present increased mitochondrial DNA in the BAT, with an apparently intact mRNA expression of mitochondrial genes, such as Nd1 in this tissue. It has been previously acknowledged, that mitochondria from different cell types are functionally unique, depending on their respective nuclear background, to address the needs of different cells and BAT is recognized as a tissue with high mitochondrial concentration [Dguok KO model, that is characterized by low body weight and decreased subcutaneous fat layer [ntration , 27. Wheat layer .DguokF180S/F180S mice have not yet been systematically observed for the assessment of their life span. According to our preliminary observation, these animals survived up to 30 weeks without further apparent phenotypical or behavioral abnormalities, in line with the preliminary survival estimates of the murine Dguok KO mouse line [DguokF180S/F180S mouse line also possesses sufficient compensatory pathways ensuring sufficient mitochondrial DNA levels, that do not further influence their phenotypical appearance and survival.use line . The difDGUOK mutations, the phenotypical abnormalities, the time of onset and the course of the disease present with a large variety among genetically affected patients. Both human and mouse Dguok genes contain 277 amino acids and present homology with 75% identities and 85% positive residues [In MDDS due to residues . The posresidues . In contDguokF180S/F180S mutant mouse line, that displays a lean phenotype, with reduced subcutaneous fat pads, characteristics of WAT browning, and increased EE. Furthermore, mutant animals are characterized by lower blood glucose, insulin, and lipid levels. This mouse line presents differential mitochondrial DNA quantities in various tissues and altered metabolic function in the mitochondria, but no further phenotypical abnormalities observed in MDDS forms. These data are suggestive of the presence of compensatory mechanisms in the context of this specific mutation, ensuring sufficient mitochondrial DNA levels that do not further influence phenotype and survival. This mouse model could serve in the future not only in the study of MDDS forms but also in the understanding of mechanisms resulting in a lean phenotype.Taken together, we herein describe the generation and metabolic characterization of a Supplemental Material


Ibutilide has already been used for cardioversion of persistent atrial fibrillation (PsAF) after radiofrequency catheter ablation (RFCA). The purpose of this study was to determine the effect of Ibutilide-guided cardioversion on clinical outcomes after individualized ablation of PsAF.From October 2020 to September 2021, consecutive patients with PsAF accepted for RFCA were prospectively enrolled. After individualized ablation including pulmonary vein isolation plus left atrial roof line ablation and personalized linear ablation based on left atrial low-voltage zones, patients were divided into the spontaneous conversion (SCV) group, direct current synchronized cardioversion (DCC) group and Ibutilide group according to different cardioversion types during ablation. The rates of freedom from atrial tachyarrhythmia (ATT) among the three groups were evaluated after follow-up.P = 0.042} and smaller left atrial diameter (LAD) . A 12-month freedom from ATT rate was 83.3% in SCV group, 69.4% in DCC group, and 79.2% in Ibutilide group, respectively . During the follow-up [17 months (IQR: 15–19)], the rate of freedom from ATT of SCV group (83.3%), and Ibutilide group (72.9%) were both higher than that of DCC group . Moreover, Kaplan–Meier analysis showed a significantly higher sinus rhythm (SR) maintenance in Ibutilide group than in DCC group . After adjusting for risk factors of AF recurrence, the hazard ratio for AF recurrence of the DCC group with reference to the Ibutilide group was 4.10 . Furthermore, subgroup analysis showed that freedom from ATT rate in effective Ibutilide subgroup was significantly higher than noneffective Ibutilide subgroup .In this study, 110 patients were enrolled, including 12 patients with SCV, 50 patients receiving DCC and 48 patients receiving Ibutilide cardioversion after individualized ablation. Among the three groups, the SCV group had shorter AF duration {12 months [interquartile range (IQR) 12–16], For the treatment of the patients with PsAF, Ibutilide-guided cardioversion after individualized RFCA may be benefit for maintenance of SR compared to conventional DCC, especially for the patients who are effective for administration of Ibutilide. Atrial fibrillation (AF) is the most frequently occurring cardiac arrhythmia and its presence increases the risk of embolic stroke, heart failure, cognitive impairment and mortality . DespiteMore durable lesions are produced by ablation guided by ablation index (AI) that combines contact force, power, and time . Meanwhihttp://www.chictr.org.cn, No.ChiCTR2200066444, Retrospectively registered).Two-center, prospective observational study was conducted at the First Affiliated Hospital of Guangxi Medical University and Xiangtan Central Hospital, and consecutively enrolled the patients with PsAF who underwent their first RFCA according to the expert consensus on catheter ablation for AF  between The study design is shown in After single transseptal puncture, heparin (100 IU/kg) was administered intravenously with the activated clotting time maintained for 300–350 s. A 20-pole multielectrode catheter  and an open-irrigated ThermoCool SmartTouch Surround Flow (STSF) catheter  were used for LA voltage mapping and ablation. LA voltage mapping was performed under AF rhythm. Considering temporal electrogram amplitude variability during AF, each point was manually selected. During LA mapping, the multielectrode catheter was stably attached to the tissue in the same site for at least 5–8 s to assess consistency of intracardiac electrocardiogram patterns to avoid or minimize errors. To ensure highest accuracy of intracardiac electrocardiogram criteria, endocardial voltage maps had a minimum target of 1,500 endocardial points and a maximum point density of 1 mm. LVZs was determined by the presence of ≥3 adjacent points exhibiting voltage <0.5 mV during AF –21. The All antiarrhythmic drugs (AADs) were stopped in patients for at least 5 half-lives, and amiodarone was discontinued for 1 month at least prior to ablation, unless the arrhythmia was uncontrolled according to physician preference. The patients could receive uninterrupted warfarin therapy with INR 2.0–3.0, or the patients who previously treated with novel oral anticoagulants (NOACs) stopped 24 h before the ablation procedure and restarted 6 h afterward. The preoperative electrolyte results of all patients indicated normal blood potassium and magnesium. In the Ibutilide group, Ibutilide 1 mg diluted in 20 ml of 0.9% sodium chloride was administered via a slow intravenous injection for over 10 min. If AF rhythm was still persisted 10 min after the first Ibutilide administration, second dosage was given. Once AF converted to SR, AT, occurrence of ventricular tachycardia, or any adverse reaction, Ibutilide was immediately stopped. Adverse reactions were observed in all patients within 6 h after Ibutilide administration. After the procedure, all patients received AADs (amiodarone or propafenone) for up to 3 months, anticoagulation therapy (warfarin or NOACs) for at least 3 months, and proton pump inhibitors for 6 weeks.P-wave dispersion (Pd) were recorded after ablation. Pd was defined as the difference between the maximum and minimum P-wave duration recorded from the 12 electrocardiogram (ECG) leads in SR after the operation. AF recurrence after ablation was defined as occurrence of atrial tachyarrhythmia  lasting >30 s after the initial 3-month blanking period as per current guidelines , transoesophageal echocardiography (TEE), and enhanced cardiac computed tomography were routinely collected at enrollment. Left atrial appendage ostium diameter (LAAOD) that was the maximum opening distance, and left atrial appendage depth (LAAD) that was the longest depth of left atrial appendage were measured at different angles under TEE, respectively. Ablation characteristics such as ablation parameters, fluoroscopy dose, procedure time (defined as the time from sterilization to the end of the procedure), and idelines . All theThe primary endpoint of the study was freedom from recurrence of any ATT (>30 s), after a blanking period of 3 months, at 1 year and at the end of follow-up after the initial ablation procedure. The secondary endpoints were to investigate the independent risk factors related to AF recurrence after RFCA and the assessment of complications of RFCA among the groups at the end of follow-up.P-value <0.05 was considered statistically significant. C statistic and bootstrapping for model validation were performed using “survival package” and “boot package” in R software (https://cran.r-project.org/), respectively. All other analyses were performed using IBM SPSS version 26.0 (IBM Corp.) and GraphPad Prism 8 .Continuous variables were tested for normality using the Kolmogorov–Smirnov test. Continuous variables in each group were expressed as mean ± standard deviation or as median with interquartile range (IQR) according to the distribution type of the dada, and compared using one-way analysis of variance (ANOVA) or Kruskal–Wallis test, respectively. Categorical data were expressed as frequencies (percentages) and compared with the chi-squared test or Fisher's exact test, as deemed appropriate. The risk factors of ATT recurrence were analyzed by univariate analysis and multivariate Cox regression analysis. The independent predictors of AF recurrence after catheter ablation were determined by multivariate Cox proportional hazard regression. C statistic was performed to assess the discrimination of multivariate Cox model . Becausen = 3], roof dependent [n = 1], mitral isthmus AFL [n = 2], and focal AT from LA posterior wall [n = 1], were eliminated by targeted ablation. There were 50 patients in DCC group, and 48 patients in Ibutilide group, respectively. In the Ibutilide group, 27 patients (56.3%) converted to SR (Ibutilide conversion subgroup), 13 patients (27.1%) converted to AT/AFL (AT/AFL subgroup), and the other 8 patients (16.6%) underwent successful DCC (DCC subgroup). The 13 AT/AFL including typical AFL [n = 9], mitral isthmus AFL [n = 3], focal AT from LA anterior wall [n = 1], were successfully terminated by activation mapping and ablation in AT/AFL subgroup. One patient in DCC group died of traffic accident and was lost to follow-up, and 109 patients with PsAF completed follow-up at the end of the study. The median of follow-up duration was 16 months (IQR: 13–18) in SCV group, 17 months (IQR: 15–19) in DCC group and 17 months (IQR: 15–20) in Ibutilide group, and there was no significant difference among the three groups (P = 0.387). AF duration was different among the three groups (P = 0.042), with the minimum duration in SCV group. The smallest LAD was in SCV group compared with other groups (P = 0.023). The other baseline clinical characteristics did not statistically different among the three groups ; 63.6% males] were consecutively enrolled. A flow chart depicting the classification of the patients is shown in e groups .P = 0.157).The details of ablation procedures and complications are shown in P = 0.027). Fluoroscopy dose (P = 0.028) and procedure time (P = 0.001) were different among the three groups.LA roof line ablation was performed in all the patients, and the bidirectional conduction block rate was 11/12 (91.6%) in SCV group, 47/50 (94%) in DCC group, and 46/48 (95.8%) in Ibutilide group, respectively. LA anterior wall line ablation was underwent in 2 patients (4%) in DCC group and 3 patients (6.3%) in Ibutilide group, and their block rates were 1/2 (50%) and 2/3 (66.7%), respectively. The success rate of LA BOX ablation was 2/3 (66.7%) in SCV group, 10/13 (76.9%) in DCC group, and 11/13 (84.6%) in Ibutilide group. Cavotricuspid isthmus ablation was fulfilled in 6 patients (50%) in SCV group, 10 patients (20%) in DCC group, and 15 patients (31.3%) in Ibutilide group, all of which achieved bidirectional conduction block. The rate of bidirectional conduction block of mitral isthmus line was 1/2 (50%) in SCV group, 3/5 (60%) in DCC group, and 6/8 (75%) in Ibutilide group. Superior vena cava ablation was successfully completed in one patient in SCV group and one patient the DCC group. The shortest Pd was in SCV group [43 ms (IQR: 35–52)] compared with other groups (P = 0.624). No other complications, such us pericardial tamponade, embolic events, atrioesophageal fistula and malignant arrhythmias, happened in the patients enrolled in this study. There were no differences in the patients receiving AADs (amiodarone or propafenone) during the follow-up among the three groups.Two patients in DCC group and 2 patients in Ibutilide groups suffered from a groin hematoma that was resolved with compression. The rate of hematoma among three groups was not different (P < 0.001), Scr (P < 0.001), LAD (P < 0.001), LAAD (P < 0.001), LVA (P < 0.001) and Pd (P < 0.001) were predictors of AF recurrence. Cardioversion type was also included in the multivariate analysis because it may be clinically related to AF recurrence. Ultimately, the multivariate Cox regression, indicated that cardioversion type , LAAD , LVA  and Pd  were independent risk factors for AF recurrence after RFCA. C statistic showed that the model had good discrimination of AF recurrence after RFCA . Then, we performed a series of 1,000 bootstrap samples and revealed that the model discrimination was similar . These results demonstrate that the ability of discrimination of the model is reliable. For further analysis of the relationship between the cardioversion types and AF recurrence, multivariate-adjusted Cox models were constructed. The Ibutilide group was defined as the reference. In the crude model that was not adjusted for risk factors of AF recurrence, compared with the Ibutilide group (reference = 1), the DCC group  in SCV group, and 4.10  in DCC group, compared to Ibutilide group (reference), respectively . Simultaneously, Kaplan–Meier analysis also showed a significantly higher SR maintenance in Ibutilide group than that in DCC group , compared to that in SCV group (83.3%) and Ibutilide group (79.2%), but no statistical significance were reached among the three groups. Meanwhile, Kaplan–Meier analysis also showed that there was no markedly difference in the rate of freedom from ATT among the three groups , P = 0.001], and LAAD  than the other two subgroups  and AT/AFL subgroup (92.3%) than that in DCC subgroup  The HPSD-AI guided individualized ablation strategy, including PVI plus LA roof line ablation and personalized linear ablation based on LVZs design for patients with PsAF, are safe and effective. (2) Ibutilide-guided cardioversion is noninferior to SCV but superior to conventional DCC in maintaining SR after index ablation after a median of follow-up 17 months. (3) In patients with PsAF undergoing RFCA, Ibutilide-effective therapy significantly reduced the risk of AF recurrences compared to Ibutilide-noneffective treatment. (4) The cardioversion types including SCV, ibutilide cardioversion or DCC was independently associated with AF recurrence after RFCA.PVI is the cornerstone of AF catheter ablation, particularly for paroxysmal AF . HoweverIn this study, we also implemented the concept of an individualized ablation strategy for patients with PsAF. For example, PVI plus LA roof line ablation was followed by a personalized linear ablation designed based on LVZs. The results showed that LVZs was predominantly identified from LA posterior wall. Therefore, the LA BOX ablation was the primarily additional line ablation in our study. Studies have indicated that the LA posterior wall serves as one of the most key regions in pathophysiology of PsAF and is the most common non-PV site that contains AF re-entry drivers , 26. FurNontransmural tissue injury and discontinuity of ablation path are the pathological basis of AF recurrence after RFCA. The reason why additional ablation strategies adjunctive to PVI failed to significantly improve ablation outcomes in patients with PsAF is partly due to PV reconnection and gap-related ATT caused by extensive ablation , 30. MetInterruption of the maintenance mechanisms for PsAF with atrial arrhythmogenic substrates and triggers is the key to improve the success rate of ablation. However, it is very difficult to find exact mechanisms for maintenance of AF during RFCA. Therefore, the majority of patients with PsAF needs DCC or pharmacological cardioversion, even if PVI and extra ablation are completed. Ibutilide exerts a class III antiarrhythmic effect through blocking potassium (IKr/HERG) channels and activating slow delayed inward sodium current that emerges in early repolarisation, and is commonly used for AF termination . The MAGOur study was different from the previous two studies , 35. We Several limitations should be mentioned. First, this is a non-randomized and observational study with a small sample size, especially in Ibutilide subgroup, which may engender the potential to bias to our results. Therefore, our findings may not be generalized to all the patients with PsAF. Second, the rate of AF recurrence might have been underestimated because some asymptomatic patients did not see a doctor in time to complete an ECG or Holter. Third, the homogeneity of different surgeons could not be guaranteed, which may cause a potential influence on the effect of ablation. Fourth, although there was no difference in the median follow-up time among the three different cardioversion groups, the undemanding follow-up protocol after 12 months may lead to a bias of the results. Fifth, there is no consistent standards for constructing LA voltage mapping under SR or AF –21, 23, In the treatment of PsAF, Ibutilide-guided cardioversion after individualized RFCA is safe, and provides a benefit for maintaining of SR compared to conventional DCC, especially in the patients administrated with ibulide-effective therapy. This may be because Ibutilide may identify the PsAF patients with less advanced structural and electrical remodeling, and help to uncover the obscured critical sites maintaining AF followed by targeted ablation to terminate AF.


Soft tissue sarcomas (STS) represent less than 1% of all tumors and are thought to be derived from mesenchymal progenitor cells. This heterogeneous class of tumors include over 100 histological subtypes with varying clinical presentation and genetics. Their heterogeneity reflects the ability of mesenchymal precursors to develop into an array of tissue types including muscle, bone, cartilage, and fat.There are no routine standard screening tests for sarcoma, but once a tumor is detected the prognosis is based on the disease stage, histopathology, size, and genetics. Standard of care includes surgery, radiation and chemotherapy. A percentage of sarcomas have actionable driver mutations, but most do not. STS are typically “cold”, with a low tumor burden and are not likely to respond to immunotherapy. Despite improvements in therapy, the 5-year survival rate remains low, reported as 58%  due in part to higher frequencies of advanced metastatic disease.This Research Topic includes nine multidisciplinary manuscripts. One overall theme is that targeted therapies can be very effective for specific STS with specific actionable mutations, but only a limited percentage of sarcoma patients have these mutations. Even patients that respond well to targeted therapy often relapse, and then require subsequent non-targeted therapy. Therefore, additional therapies that are both for targeted sarcomas and for sarcomas lacking obvious actionable genetics are required that can prevent relapse/prolong PFS.Fuchs et al., reviewed FDA approved drugs for STS. Pazopanib is FDA approved for non-adipocyte STS based on clinical prolongation of the PFS from 1.6 months to 4.6 months. The OS increase did not reach statistical significance (10.7 vs 12.5 months). Other drugs reviewed were approved for more targeted populations and have longer response rates. (1) Pexidartinib  for tenosynovial giant-cell tumors , 39% overall response rate (2) imatinib for dermatofibrosarcoma protuberans , 5% CR, 55% PR, (3) crizotinib for ALK positive inflammatory myofibroblastic tumors , (4) tazemetostat (EZH2 inhibitor) for epithelioid sarcoma , (5) nab-sirolimus for perivascular epithelioid cell tumor , (6) tropomyosin receptor kinase inhibitors for TRK fusion positive cancers. Also reviewed were targeted kinase inhibitors that do not have FDA approval for STS but are in clinical testing.Maleddu et al. reviewed only locally aggressive mesenchymal tumors, particularly Desmoid fibromatosis, giant cell tumor of bone, and tenosynovial giant cell tumor. These tumors have limited ability to metastasize and therefore carry a better prognosis than metastatic tumors. As noted by Fuchs et al. as well, treatment for these includes targeted chemotherapy approaches that can be effective. New therapies and paradigms have focused on utilizing less toxic regimens and identifying patients that do not require more toxic regimens.Fuchs et al. reviewed new therapeutics for synovial sarcoma (SYN). SYN is defined by the translocation of t(X:18)  forming an oncogenic fusion protein, with about 1000 cases per year in the US . First-line therapies are anthracycline- or gemcitabine-based regimens, resulting in a median PFS time of about 5 months and overall survival time between 14-16 months. LMS is not typically associated with specific mutations, but with complex karyotypes. Highlighted in the review are new therapies based upon: (1) DNA repair deficiencies because LMS is often associated with defects in DNA-repair. This includes a Ph2/3 trial assessing temozolomide and Olaparib in uterine LMS. (2) Kinase inhibitors, including cabozantinib and anlotinib. (3) Metabolic vulnerabilities due to the frequent loss of argininosuccinate synthase 1. (4) New chemotherapeutic combinations including a Phase 3 trial of unesbulin with dazacarbazine. Unesbulin is an orally bioavailable inhibitor of tubulin polymerization. Possibly, the combination of unesbulin (causes G2M arrest) and dazacarbazine  causes replicative stress, particularly in tumors such as LMS that are deficient in DNA-repair.Bernardo et al. demonstrates that both photon and proton irradiation are equally effective in a sarcoma mouse model. In clinical trials, first line therapy is the combination of radiotherapy with doxorubicin, although the rates of recurrence remain high. Clinical trials are not conducted to optimize radiation/chemotherapy regimens or to compare types of radiation, increasing their value in preclinical studies. Second, Marritt et al. present preclinical data demonstrating efficacy of STING agonists in models of undifferentiated polymorphic sarcoma UPS. Third, Li et al. describe a case report of a favorable response of an ALK-fusion positive protein to the ALK TKI ensartinib. Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is often associated with ALK fusion proteins.Three papers discuss new therapies tested in preclinical studies or in a case report. First, Ganguly et al.). Tumor size, baseline metastases and SAP were prognostic factors to predict survival, but social factors were not. Similar parameters are likely to be relevant to STS tumors.Finally, because osteosarcomas show genetic similarities to soft tissue sarcomas, we have included a paper describing a prognostic score predicting responsiveness to high dose methotrexate (In summary, new therapies are being identified and developed for sarcomas. Some of these are targeted therapies leveraging the gains made in personalized medicine. Others are focused on novel chemotherapeutic combinations leveraging the complex karyotype of sarcoma on the background of DNA repair deficiencies. Exciting new immuno-oncology approaches are in preclinical and clinical stages. Some of these approaches may provide new therapies to overcome resistance and provide more possible treatments to mix and match in varying sequence so as to provide measurable benefit to STS patients.MW: Conceptualization, Writing – original draft, Writing – review & editing. MR: Writing – original draft, Writing – review & editing. MI: Conceptualization, Writing – review & editing. BT: Conceptualization, Writing – review & editing.


Men are disproportionately often perpetrators of physical domestic violence (DV). Gender role constructs, such as traditional masculinity ideologies (TMI), are broadly accepted as an explanation for this effect. Emotional competence further constitutes an important role in TMI and the prevention of DV. However, the interactions between these constructs remains unclear.The present study aims to investigate associations of TMI with aggression, DV perpetration, and emotional competence, while also examining emotional competence as a potential moderator.Mage = 43.9 ± 15.3) from German-speaking countries in Europe completed an anonymous online survey that assessed TMI, aggression, and DV perpetration as well as alexithymia, emotion regulation, and self-compassion as indicators of emotional competence.A sample of 428 cisgender men (Strong TMI were associated with high levels of aggression and overall reduced emotional competence, as reflected by high levels of alexithymia, frequent use of expressive suppression, and low levels of self-compassion. Strong conformity to TMI was associated with a higher likelihood for DV perpetration when considering relevant sociodemographic covariates. Moderation analyses revealed that expressive suppression buffered the association between TMI and DV perpetration.Men with strong TMI report high levels of aggression and impaired emotional competence. While strong conformity to TMI was associated with more frequent perpetration of DV, higher expressive suppression seems to buffer the association between TMI and DV perpetration. The present study highlights the importance of addressing gender ideologies when working on aggression, DV perpetration and emotional competence in men. A largeTo date, the greatest risk factor to engage in injurious and severe DV is being male, as the vast majority of violent offenders and severe DV perpetrators are men . While bInterpersonal processes of social reinforcement and observational learning are thought to promote conformity to gender-aligned behavior. Thus, TMI inform boys and men about socially reinforced and gender-aligned ways to experience and express emotions . The priHowever, DV may not only be associated with TMI, but also closely associated with emotional competence. While TMI is related to difficulties in processing and expressing emotions , and emotionally dysregulated behavior , emotionThus, it is highly interesting that low emotional competence skills – a crucial protective factor against the general use of violence – shows a strong negative association with the endorsement of TMI . For exaFurther evidence of the association between TMI and impaired emotional competence can be found in the Normative Male Alexithymia Hypothesis . ThereinEmotion regulation serves to successfully adapt to one’s environment by modifying emotional responding . ConsideAnother functional form of emotional competence, which has become increasingly important in resilience and well-being research, is self-compassion . Self-coTaken together, processes of social learning encourage gender-specific responses to different emotions . Through(1) Strong TMI are associated with higher levels of aggression and more frequent perpetration of DV.(2) Strong TMI are associated with impaired emotional competence .(3) High emotional competence  has a buffering effect on the association between TMI and DV perpetration.2.2.1.Andromind Self-Test (AST), accessible via the website andromind.ch, which informs men about gender role norms and men-specific mental health issues. Participation was based on self-selection as the subjects were recruited through advertisement on social media. Only participants providing informed consent and accepting the data privacy agreement could proceed to the survey. Subsequent inclusion criteria consisted of age (being 18 years or older), sufficient German skills to complete the online questionnaire, and being male while also identifying with one’s birth-assigned gender. Participants who did not provide complete data for the examined constructs were excluded from the analysis. A detailed overview of the exclusion process is presented in Open Science Framework (OSF) and approved by the University of Zurich Ethics Commission.The present pre-registered cross-sectional study used data from an anonymous online survey called 2.2.2.2.1.Participants’ sociodemographic information was assessed, of which a detailed overview can be found in the Supplementary . Partici2.2.2.Restrictive Emotionality, Self-Reliance Trough Mechanical Skills, Negativity Toward Sexual Minorities, Avoidance of Femininity, Importance of Sex, Dominance, and Toughness. Participants are asked their agreement with different statements about TMI  using a 7-point Likert scale (1 = strongly disagree to 7 = strongly agree). For the English version, Cronbach’s α ranges from 0.79 to 0.90 with α = 0.92 for the total score. The German version of the MRNI-SF has adequate validity and reliability . The CMNI-30 comprises 10 underlying dimensions of conformity to TMI, namely Emotional Control, Winning, Playboy, Violence, Heterosexual Self-Presentation, Pursuit of Status, Primacy of Work, Power over Women, Self-Reliance, and Risk-Taking. For the original English version internal consistencies ranging from α = 0.72 to α = 0.94 have been reported . Thus, higher scores indicate higher levels of alexithymia. The current study used a slightly shorter German-language version consisting of only 18-items , which showed the highest reliability and validity in a representative German population sample . Higher scores mean more frequent use of cognitive reappraisal and expressive suppression, respectively. For the German translation, α = 0.74 (suppression) and α = 0.76 . In the present sample, the reappraisal subscale possessed a reliability of α = 0.73 and ω = 0.83, and the suppression subscale showed a reliability of α = 0.63 to ω = 0.70.The Emotion Regulation Questionnaire ERQ;  measures2.2.6.elf-Kindness, Self-Judgement, Common Humanity, Isolation, Mindfulness, and Over-Identification. Participants are asked how often they behave in a certain manner  with possible responses on a 5-point Likert scale . Adequate internal consistency has been reported for both the English version with α ≥ 0.86  using a 5-point Likert scale (0 = extremely uncharacteristic of me to 4 = extremely characteristic of me). A psychometric evaluation of the German translation confirms support for validity and reliability with α ranging from 0.75 to 0.88 . Thus, higher scores indicate greater social desirability in participant’s response style. The short form of the English version possessed a composite reliability of ρx = 0.83  and within lifetime . Furthermore, to include as a control variable in the analyses participants were asked about their experience of physical DV, again for the past-month  and lifetime . For all questions, physical violence was specified as “including behaviors such as hitting someone with a fist or flat hand, kicking someone with the feet, strong painful holding/grabbing/squeezing of someone, choking someone by the neck, pulling someone’s hair, or hitting someone with objects such as a belt.”Physical DV was assessed in five questions described in the following, using a dichotomous Physical Aggression scale of the BPAQ .The self-constructed item assessing one of the main outcome variables of the present study, namely lifetime perpetration of physical DV showed an agreement rate of 84.2% over a four- to eight-week period among 57 participants who completed the survey twice. Convergent validity was further determined by a positive point-biserial correlation between lifetime perpetration of DV and the 2.3.First, descriptive statistics were calculated, and psychometric properties of the study questionnaires were assessed by their internal consistencies  and distribution of their raw scores  and binomial logistic (MLE) regression analyses were conducted with TMI  as individual predictor variables and emotional competencies , aggression (ABPQ), and lifetime perpetration of physical DV as individual outcome variables. Each predictor – outcome combination was analyzed with three sequential regression models, including the following steps: (1) no covariates, (2) including participant’s age, education, sexual orientation, relationship status, and social desirability (MC-SDS) as covariates, and (3) including participants’ lifetime experience of physical DV as an additional covariate.In a second part, a potential moderating effect of emotional competencies on the association between TMI, aggression, and perpetration of physical DV was examined by introducing an interaction term  into the respective model, irrespective of whether significant main effects were present. As in the first part, each predictor – outcome combination was analyzed with three sequential regression models, (1) no covariates, (2) including sociodemographic covariates, and (3) including lifetime experienced DV.R software environment , conformity to TMI 1.8 , alexithymia 2.5 , cognitive reappraisal 4.2 , expressive suppression 3.9 , self-compassion 3.0 , aggression 1.4 , and social desirability 0.4 . Participant-level mean scores of each questionnaire are reported in 3.3.As presented in 3.4.As presented in Binomial logistic regression analyses with lifetime perpetration of DV as an outcome variable  revealedAdditional linear regression analyses examining the association between TMI and emotional competencies, controlled for covariates and multiple testing, revealed that strong endorsement of TMI was linked to frequent expressive suppression  and redu3.5.n = 221) self-compassion showed a weaker association between endorsement of TMI and aggression  as compared to men with low  self-compassion . However, this effect did not persist after controlling for covariates and was not present for conformity to TMI as a moderator.In order to examine a potential moderating effect of emotional competencies on the association between TMI and aggression as well as TMI and perpetration of physical DV, separate interaction terms  were added to the respective regression models, again controlled for sociodemographic covariates, and corrected for multiple testing. A moderating effect of self-compassion on the association between endorsement of TMI and aggression was found , where mn = 221) of alexithymia the association between conformity to TMI and perpetration of physical DV  was weaker as compared to men with low  alexithymia . The moderating effect did not remain significant after correcting for multiple testing and was not found for endorsement of TMI as a moderator.Alexithymia moderated the association between conformity to TMI and perpetration of physical DV , indicatn = 216) expressive suppression showed a significantly weaker association between endorsement of TMI and the likelihood of having ever used physical DV  as compared to men with low  expressive suppression . Similarly, men with high expressive suppression also showed a significantly weaker association between conformity to TMI and the likelihood of having ever used physical DV  as compared to men with low expressive suppression . However, the moderating effects did not withstand a correction for multiple testing. Additional sensitivity analyses for these moderation analyses that include heterosexual-identified men only , cognitive reappraisal , and expressive suppression . Also, no moderation effect was found for the association between TMI and perpetration of physical DV in regard to cognitive reappraisal  and self-compassion .No moderation effect was found for the association between TMI and aggression in regard to alexithymia . AW and LE are supported by a grant from the Swiss National Science Foundation (Grant PZPGP1_201757 awarded to AW). Open Access costs financed by University Library Zurich.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P < 0.05). However, LA strain was similar among 45 male and 35 female strength athletes (P > 0.05), while male athletes presented with a higher LA volume when compared to female controls (P < 0.05). ROC curve analysis showed that LA contraction longitudinal strain (LASct) was the best predictor in evaluating athletes' LA function. Athletes' heart rate and left ventricular mass index were significantly correlated with 4D LA function parameters.4D LAQ can be used for early detection of the changes in LA function in young strength athletes. There was no significant difference in LA strain between male and female athletes. The LASct was the most effective index for evaluating athletes' LA function.Athletes might suffer from potentially fatal heart disease, which has always been a concern in cardiovascular medicine. The changes in left atrial (LA) size and function are related to the occurrence of arrhythmia. In the present study, four-dimensional automatic quantitation (4D LAQ) was used to explore the changes in LA function of young strength athletes. Eighty professional young strength athletes and sixty healthy young adults matched in age were selected for the study. The LA volumes and strains were automatically analyzed by 4D LAQ. The receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of strain in athletes' LA function. Pearson correlation analysis was performed to assess the potential association between conventional echocardiographic indexes and 4D parameters related to athletes' LA function. Compared to the control group, LA longitudinal and circumferential strain in the athlete group decreased, while LA volume increased ( High-intensity exercise training can lead to changes in hemodynamics and heart structure, which is known as “athlete's heart” . With inA total of 80 professional strength athletes participating in wrestling, boxing, judo, and weightlifting were randomly selected from the Henan heavy competitive sports management center. Of these, 45 were male and 35 were female . Inclusion criteria included the following: strength athletes with training experience of ≥ 5 years, those training for ≥ 30 h per week, and athletes who have never stopped training. A total of 60 young adults  who were declared to be healthy after a physical examination, those matching in age, and athletes with similar body mass indexes (BMIs) were randomly selected as the control group. The exclusion criteria in both groups included cardiovascular disease, such as history of atrial arrhythmia, hypertrophic cardiomyopathy, and severe valvular disease, and poor image quality for myocardial speckle tracing analysis. The study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University. All subjects signed the informed consent form.Transthoracic echocardiographic examinations were performed at rest in the left lateral decubitus position with a high-quality echocardiograph  equipped with a 3.5-MHz M5S 2D and 1.5–4.0-MHz 4-V transducers. Heart rate (HR) was recorded continuously via a three-lead electrocardiogram.Dynamic two-dimensional grayscale images were acquired in the apical four-chamber, apical two-chamber, and LV long-axis views. Images were imported into the EchoPAC software  and analyzed off-line. Left atrial diameter (LAD) measurements were made in the parasternal long-axis view (2D-Mode) at end systole. LVEF was calculated by the Teichholtz method. Pulsed-wave Doppler echocardiography was used to measure early (E) and late (A) blood flow velocities through the mitral valve. The peak early diastolic velocity (e’) was measured on the septal corner of mitral annulus using tissue Doppler. The E/A and E/e’ were calculated.The 4D probe was used to collect the apical four-chamber full-volume dynamic images, where the volume frame rate was adjusted to be > 40% of the subjects' heart rate, and the subjects were asked to hold their breath to obtain the dynamic images. After the images were imported into the workstation, the 4D Auto LAQ option was selected and the software automatically identified and wrapped the LA intima, which could be adjusted manually to reduce the error. The 4D LA images were then obtained by selecting “Result”. The following LA parameters were analyzed by the software: maximum atrial volume (LAVmax), minimal atrial volume (LAVmin), volume before atrial contraction (LAVpre-a), left atrial ejection fraction (LAEF), left atrial reservoir longitudinal strain (LASr), conduit longitudinal strain (LAScd), contraction longitudinal strain (LASct), reservoir circumferential strain (LASr-c), conduit circumferential strain (LAScd-c), and contraction circumferential strain (LASct-c). Left atrial active ejection fraction (LAaEF) and left atrial passive ejection fraction (LApEF) were calculated using the following respective formulas: LAaEF = (LAVpre-LAVmin)/LAVpre × 100% and LApEF = (LAVmax-LAVpre)/LAVmax × 100%. The 4D LAQ technique for the analysis of the 4D LA parameters in the study subjects is shown in Fig. All ultrasound images were acquired by an experienced sonographer (a chief physician). All data were analysed by two sonographers (one attending physician and one current master's student), and the average values were taken for three consecutive measurements.The analyses were performed using the statistical software, SPSS21.0 . Normality for the continuous variables was determined using the Kolmogorov–Smirnov test. Normally distributed continuous data with equal variance were compared between two groups using the independent samples t-test. Otherwise, the corrected t-test was used. The Mann–Whitney U test was used to compare the non-normally distributed variables in the two groups. Pearson correlation analysis was performed to assess the potential association between conventional echocardiographic indexes and 4D LA parameters. In addition, the receiver operating characteristic (ROC) curve was used to identify the optimal diagnostic indices for LA function changes. The absolute values for LAScd and LASct were used for Pearson correlation and ROC analyses.P < 0.05). The LV ejection fraction (LVEF), E/A, and E/e' did not differ between the two groups (P > 0.05). Compared to the female athlete group, BSA, BMI, SBPand LAD in the male athlete group were significantly higher and E/e' was lower (P < 0.05). There were no significant differences in age, HR, DBP and LVEF (P > 0.05).A total of 45 male and 35 female athletes, and 60 control subjects  were enrolled in the present study. The demographic data and traditional echocardiography results for all participants are presented in Table P < 0.05). Although LApEF in the athlete group was lower than that in the control group, the difference was not statistically significant (P > 0.05). Compared to the female athlete group, LAVmax, LAVmin, LAVpre-a, and LAEV were higher in the male athlete group (P < 0.05). There were no statistical differences in LAVI, LAEF, LAaEF, LApEF, and LA strain values between the male and female athlete groups (P > 0.05). The LAD showed a significant inverse relationship with LA longitudinal strain and LAEF and positively correlated with LAVI. However, HR was positively correlated with LA longitudinal strain and LAEF and negatively correlated with LAVI. All differences were significant (P < 0.05) (Table The comparisons of LA volumes and strains are shown in Table 5) Table . The are5) Table .Table 24The intraobserver intraclass correlation coefficients (ICCs) were 0.962, 0.985, 0.958, 0.881, 0.974, and 0.985, while the interobserver ICCs were 0.922, 0.930, 0.921, 0.872, 0.937, and 0.977 for LASr, LAScd, LASct, LASr-c, LAScd-c, and LASct-c, respectively, indicating good intra- and inter-observer agreement in the LA strain measurements.It has been widely accepted that regular exercise has significant benefits, such as enhanced physical performance and metabolic homeostasis . HoweverThe condition known as “athlete’s heart” has different effects depending on the type of exercise. Endurance “athlete’s heart” mostly manifests as eccentric hypertrophy, while strength “athlete’s heart” is mainly characterized by concentric hypertrophy . Heart rThe first application of 4D LAQ to determine the LA characteristics in strength athletes and to perform early analysis of LA strain in male and female athletes represents the present study’s main strengths and innovation. The 2D-STE technique has been used to investigate the LA properties of athletes in most studies. However, it has a strong space and time dependence and easily causes speckle loss of tracking, and the analysis process is complex . The 4D In the present study, the athlete group showed a lower LA strain and larger LAV, which helps to explain the weakening in LA myocardial deformation due to excessive atrial volume load after training. It was also found that the LAV in male athletes was greater than that in female athletes. This discrepancy is probably a result of the androgen effect on cardiac protein synthesis . HoweverSeveral limitations in the context of this research should be discussed. First, athletes with a family history of heart disease may not be able to provide all relevant information due to the lack of knowledge. Second, further studies with a larger sample size are needed in the future, as the present study analyzed only a small number of power athletes. Third, the reference values of 4D strain parameters in athletes obtained in the present study may differ from the actual strain reference values, and it is necessary to conduct further national multi-center studies to establish this reference range.The present study found that long-term high-intensity training can lead to LA remodeling in athletes, and 4D LAQ technology can quantitatively evaluate atrial myocardial function and detect these changes early. Since athletes can present with LAE prior to structural changes in the LV, further remodeling of the heart can be prevented from occurring early by analyzing changes in LA morphology and function. Gender had an impact on LAV rather than LA strain. LASct had the highest efficacy in evaluating LA function in athletes. LAD had a significant negative correlation with LA longitudinal strain and LAEF.


The results show that the PLCL/BG2/PDA scaffold possessed a good compression modulus and brilliant hydrophilicity. The proliferation, adhesion and osteogenesis of hBMSCs were improved in the PDA coating groups, which exhibited the best performance. The results of the SD rat cranium defect model indicate that PLCL/BG2/PDA obviously promoted osteointegration, which was further confirmed through immunohistochemical staining. Therefore, PDA decoration and the sustained release of bioactive ions  from BG in the 3D-printed PLCL/BG2/PDA scaffold could improve surface bioactivity and promote better osteogenesis and angiogenesis, which may provide a valuable basis for customized implants in extensive bone defect repair applications.Large size bone defects affect human health and remain a worldwide health problem that needs to be solved immediately. 3D printing technology has attracted substantial attention for preparing penetrable multifunctional scaffolds to promote bone reconditioning and regeneration. Inspired by the spongy structure of natural bone, novel porous degradable scaffolds have been printed using polymerization of lactide and caprolactone (PLCL) and bioactive glass 45S5 (BG), and polydopamine (PDA) was used to decorate the PLCL/BG scaffolds. The physicochemical properties of the PLCL/BG and PLCL/BG/PDA scaffolds were measured, and their osteogenic and angiogenic effects were characterized through a series of experiments both   Reconstruction of critical size bone defects resulting from trauma, accidents and bone necrosis has historically been a great challenge for patients and surgeons worldwide . Althougin vitro , 13. Thein vitro .4O10 scaffolds can increase osteogenic activity [Bone tissue engineering scaffolds with controllable degradation rates, tunable biological performances and adjustable mechanical strength can be prepared by hydrogel composites, bioceramic powders, polymer melts and polymer/bioceramic composites , 18, 19.activity , 29. Stuactivity , 31.o-phenol hydroxyl groups, are present on the PDA surface. PDA can be closely bound with nearly all types of external components to enhance the stability of composites, which is attributed to its unique chemical structures [Polymerization of lactide and caprolactone (PLCL) can result in a copolymer prepared from lactide and caprolactone monomers. The degradation rate of PLCL could be controlled by the monomer proportion to reduce the acid degradation products. Because of its marvelous biodegradability and biocompatibility, it has been studied for cell adhesion and tissue defect repair . Becauseructures . Recent ructures , 38, 39.in vitro and in vivo. The results demonstrate that the PLCL scaffolds with bioactive glass and PDA could greatly facilitate bone regeneration.To sum up, there were various organic–inorganic hybrid scaffolds for different tissue regeneration have been developed by many research groups , 34, 35.PLCL was purchased from Daigang Biomaterials . The ratio of the copolymer composition (LA:CL) was 80:20 (mol:mol) with a Mw of 215.56 kDa. Implant grade 45S5 (BG) was purchased from Dongguan Hannuo Biotechnology Co., Ltd. Other agents were all of analytical grade.Before 3D printing, we first prepared PLCL/BG composites. Briefly, PLCL and BG with varying weight ratios  were prepared. PLCL was dissolved in DCM at room temperature, and then the BG particles were added to the PLCL solution under ultrasonic dispersion (10% w/v). Finally, PLCL/BG composites were obtained with absolute ethanol precipitation and dried in a vacuum oven at 40°C for 48 h. Subsequently, the composite scaffold model was designed by using CAD/CAM software and exported in STL format. A bioplotter printer was used to prepare the desired scaffolds. A metal nozzle (22G) was employed. The target scaffolds were named PLCL, PLCL/BG2, PLCL/BG5 and PLCL/BG8 according to the BG content in the composite scaffold.To prepare the PDA-coated PLCL scaffold and PLCL/BG composite scaffolds, an immersion procedure was used . DopaminThe chemical structure of the PLCL composites was tested by FTIR technology (Tensor ll). After uniform sputtering with a gold layer on the scaffold, SEM  with an acceleration voltage of 3 kV was used to observe the morphologies of the porous composite scaffolds. An electromechanical universal testing machine  was applied to investigate the compressive strength of various porous structures. The porosity of the porous scaffolds was calculated according to reference . TGA . Mesenchymal stem cell medium with 10% fetal bovine serum and a 1% penicillin mixture was used to culture the cells. Every 2–3 days, the culture medium was changed. hBMSCs were grown until they reached 80–90% confluence. Trypsin–EDTA was used to digest the cells in the tissue flask bottles, which were then placed in another Petri dish. Cells from Passages 2 to 5 were used for further experimentation. All the cells were placed in a constant temperature incubator with 5% CO2 at 37°C for subculture.hBMSCs were purchased from Oricell5 cells/well. Cytotoxicity was assessed using the CCK-8 assay kit and a multifunction microplate reader  at an absorbance of 450 nm.Before the cell seeding procedure, scaffolds with a diameter of 8 mm and height of 2 mm were sterilized with ethylene oxide gas and then placed in a 48-well cell culture plate. For cell toxicity assessment, hBMCSs were cultured on 3D-printed porous scaffolds and PDA treatment scaffolds for 1, 3 and 7 days. The cell density of seeding was 2.5 × 10The hBMSCs and scaffolds were cultured for 3, 7 and 14 days in stem cell culture medium. At different culture time points, the scaffolds were removed and washed three times with sterile PBS. A 2.5% glutaraldehyde solution was used to fix the scaffolds in cacodylate buffer for 1 h, and the scaffolds were dehydrated with graded ethanol for 15 min each. Finally, the scaffolds were dried, and the interaction between the cells and scaffolds was observed by a SU8010 scanning electron microscope .Cell viability and proliferation with the porous scaffolds after coculture for 1, 3, 7 and 14 days were evaluated by a live/dead assay. At different time points, the scaffolds were removed, washed with sterile PBS, and stained with ethidium homodimer-1  and Calcein AM (0.5 µl/ml) at 37°C for 15 min. Then, the scaffolds were cleaned with sterile PBS two times and tested by a laser scanning confocal microscope .hBMCSs were cultured with porous scaffolds as mentioned above. Various scaffolds were cocultured with cells for 7, 14 and 21 days. Lysate was used to lyse the cells at different time points. The DNA amount and alkaline phosphatase (ALP) activity were measured using a PicoGreen assay and ALP activity kit (Beyotime Biotechnology), respectively. The mineralized nodules in each scaffold were measured by an alizarin red (AR) staining kit .® 480 II). The primers of the osteogenic-related genes are listed in The osteogenic differentiation of hBMSCs was further studied. The expression of osteogenic-related genes, including Runx2, Col-1, OCN, CPN, BMP2 and ALP, was measured by real-time polymerase chain reaction (RT-PCR), and the coculture time of the scaffold and cells was 7, 14 and 21 days, respectively. A 20 µl system was used for the subsequent experiment. The reagent used to isolate the total RNA was purchased from Beyotime. The concentration of the isolated RNA was measured using a spectrophotometer (LightCyclerThe SD rats (≈320–360 g) were provided by Zhejiang Provincial Laboratory Animal Center. According to the Laboratory Animal Care and Use Guidelines, all SD rats were treated ethically. All SD rat surgical processes were carried out according to agreements authorized by the Animal Care and Use Committee of Wenzhou Institute University of Chinese Academy of Science, Zhejiang, China .The scaffolds were sterilized using ethylene oxide gas. The skull defects were created as described in reference . After aMicro-CT analysis  was conducted after the skull tissue was collected after 4, 8 and 12 weeks, and corresponding histomorphometric analyses were conducted. The scanning was processed at a resolution of 18 µm, and 3D Creator software was used to reconstruct tomograms to obtain the images of the skull. Bone-related parameters, such as the trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), bone volume/tissue volume (BV/TV) and bone mineral density (BMD), were measured by CTAn image analysis software according to the micro-CT images obtained above.For further histological analysis, the collected skull tissue at different cycles  was fixed with paraformaldehyde and decalcified with 10% EDTA. Paraffin was applied to embed the bone tissue. The embedded tissue was sectioned at a 5-μm thickness in the sagittal direction by a slicer. Both the control group and experimental group were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) to evaluate new bone regeneration in the defect area. Furthermore, immunohistochemical (IHC) staining of angiogenesis-related CD31 and osteogenesis-related osteocalcin OCN was used to measure angiogenesis and osteogenesis in the skull defects, respectively. IHC was semi-quantified by calculating the fraction of positively stained areas in ROI Image J .n ≥ 3). One-way ANOVA tests via Student’s t tests were conducted, and P < 0.05 was considered a significant difference. .All data from this study are shown as the means with standard deviations  expression analysis were conducted . The hemThe results are consistent with the analysis of H&E and MT staining. CD31 and OCN positive areas and mean optical density of the PDA groups were significantly increased compared with 4 and 8 weeks . The PLCin vitro and promote new angiogenesis formation in vivo. The 3D porous composite structure fully adopted the functional properties of PLCL, bioceramics and PDA. Natural bone tightly integrated with the implant, and new bone ingrowth toward the center of the scaffold accelerated the fast and lasting osseointegration of the PLCL/BG/PDA porous structure. In general, post-treatment of PLCL/BG2 composite scaffolds with PDA modification offers better possibilities for the evolution of bioactivity connected customized porous scaffolds and various growth factor/organic/inorganic composite implants to achieve bone defect repair in the future.In summary, we successfully constructed a 3D-printed PLCL/BG scaffold with PDA decoration to achieve valid bone repair and regeneration in a rat cranium defect model. The released bioactive ions  from BG could accelerate the osteogenic proliferation and differentiation of hBMSCs rbad062_Supplementary_DataClick here for additional data file.


Pisum sativum L.) and lentil (Lens culinaris L.) are economically important and widely grown. However, in recent years, root rot, caused by a variety of fungal and oomycete pathogens, including Aphanomyces euteiches, has become a limiting factor on yield. In this study, we examined the impacts of nitrogen (N) fertilization and a commercial arbuscular mycorrhizal fungal (AMF) inoculant on pea and lentil plant health and agronomic production at three locations in Saskatchewan: Swift Current, Indian Head and Melfort. The AMF inoculation had no impact on root rot severity, and therefore is not considered a reliable method to manage root rot in pea and lentil. In contrast, N fertilization led to reductions in root rot in Swift Current, but not the other two sites. However, N fertilization did reduce nodulation. When both pea and lentil are considered, the abundance of A. euteiches in soil increased from pre-seeding to mid-bloom. A negative correlation between soil pH and disease severity was also observed. The high between-site variability highlights the importance of testing root rot mitigation strategies under multiple soil conditions to develop site-specific recommendations. Use of N fertilizer as a root rot management strategy merits further exploration, including investigation into its interactions with other management strategies, soil properties, and costs and benefits.In the Canadian prairies, pulse crops such as field pea ( In the the year . Becausethe year . N fertithe year . Howeverthe year . N playsuteiches . Increasuteiches , the lifuteiches  and the uteiches  can alteThe application of a commercial arbuscular mycorrhizal fungal (AMF) inoculant is another potential management strategy to help decrease root rot in pulse crops. In natural systems, AMF form symbiotic relationships with plants, increasing nutrient uptake, improving plant health and suppressing disease . In agriA. euteiches inoculum levels in soil planted to pea or lentil, 2) pea and lentil root health  and 3) pea and lentil crop yield.The current study explores whether and how N fertilization and an AMF commercial inoculant influence root rot and agronomic production in field-grown pea and lentil crops on the Canadian prairies. We used a combination of disease ratings and qPCR to analyze rhizosphere and root samples from three locations in Saskatchewan. The specific objectives of this study were to determine the effects of N fertilization and an AMF commercial inoculant on 1) 22.1A. euteiches as well as other root rot pathogens, including Fusarium spp. All sites were seeded to field pea (P. sativum) in 2016 and 2017 to encourage inoculum build-up for these pathogens.Field experiments were conducted in 2018 at three locations in Saskatchewan: 1) Agriculture and Agri-Food Canada (AAFC) Research Farm in Melfort (soil type: Orthic Black Chernozem silty clay loam), 2) a commercial field located approximately 15 km south of Swift Current (soil type: Orthic Brown Chernozem with a silt loam), and 3) AAFC Research Farm in Indian Head (soil type: Redo Black Chernozem with a heavy clay). All field sites had high levels, sufficient to cause root rot symptoms, of 2)2], side-banded) and two AMF inoculant treatments . The 12 treatments were replicated four times at each location, for a total of 48 plots per site. All plots were fertilized with phosphorus at 17 kg/ha and received 5.2 kg/ha of Cell-Tech single action granular rhizobial inoculant (100 million (1 x 108) viable cfu/g Rhizobium leguminosarum bv. viciae), applied in-furrow, and the row spacing was 25 cm. The plots were 4 x 8 m in Melfort, 2 x 8 m in Swift Current and 4 x 11 m in Indian Head. Seeding occurred on May 4 (Indian Head), May 16 (Swift Current) and May 23 (Melfort). Pea was seeded at 200 kg/ha and lentil at 67 kg/ha in Melfort and Swift Current, and at 194 and 54 kg/ha, respectively, in Indian Head. In Melfort, in season herbicide application consisted of imazamox (8 g/acre) and bentazon (171.6 g/acre) (June 18) and bentazon (436.8 g/acre) (July 6) applied to pea; 37 g/acre each of imazethapyr and imazamox, 13.4g/acre of tepraloxydim and 0.20 L/acre of the surfactant Merge (June 18) to lentil; and sethoxydim (202.5g/acre) (June 29) on both crops 7.0 g/ac). In Swift Current, glyphosate (270 g/acre) and carfentrazone (7 g/acre) were applied pre-seeding (May 9); imazamox (8.1 g/ac) and quizalofop (19.0 g/ac) were applied for in-crop weed control (June 13) and Diquat (167.9 g/ac) was used for desiccation (August 9). At Indian Head, imazamox and imazethapyr (6.1 g/ac each) with sethoxydim (6750 g/ac) and Merge surfactant (0.5% v/v) were applied for in-crop weed management (June 19).The impacts of N fertilizer, AMF inoculation, and crop [field pea (‘CDC Amarillo’) or lentil (‘CDC Maxim’)] on root rot, nodulation, biomass and yield were examined with a three-factorial experiment using a randomized complete block design. Each block contained three N fertilization rates  and two soil cores from the back right corner of each plot . The four soil cores were homogenized in the field to form one composite sample per plot. A 10 g subsample was immediately removed and flash frozen in liquid N in the field and then stored at -80°C prior to molecular analysis. In the laboratory, the remaining soil was passed through a 2 mm sieve, and a 20 g subsample of the sieved soil was used to determine soil moisture gravimetrically. Another 200 g of sieved soil from each plot was air-dried for chemical analysis. Crop yield of each plot was collected at harvest.Soil samples were collected before seeding of each trial in early to mid-May  and again during the growing season at early flowering . At both sampling times, four soil cores (2.5 cm in diameter and 20 cm deep) were collected from two 1 m sampling locations in each plot. This included collecting two soil cores from the front left corner of each plot , phosphate phosphorus (PO4-P) and potassium were determined using sodium bicarbonate extractions and colorimetric analysis using Technicon Autoanalyzer , 3) A. euteiches and Fusarium root rot (Aphanomyces severity [AS]), and 4) nodulation. The average ratings of the 10 plants from a plot were used for statistical analysis. SSS was rated on a 1-5 scale based on the discoloration and stunting  were dug up, keeping their roots intact, and stored at 4°C for processing. Roots were subsequently washed and individually rated within two days for 1) shoot symptom severity (SSS), 2) stunting . A ratinstunting  and usinstunting . The aboA. euteiches in the soil, DNA was extracted from each sample (0.25 g x 2 per sample) using a DNeasy PowerSoil Kit (Qiagen) and quantified via qPCR as described in detail by In order to quantify the abundance of 2.3-1), and AMF inoculation  on A. euteiches abundance, crop disease symptoms , root symbioses (nodulation and AMF colonization), and agronomic production (crop biomass and grain yield). Crop, N fertilization rate, and AMF inoculation were included as fixed factors, and replicate was included as a random factor in the models. Initial assessment revealed when all sites were analyzed together the data did not meet the assumptions of the linear mixed model. As a result of this and different soil types at these trial locations .To determine whether there were differences in soil properties prior to seeding at the three field trial locations, we used non-parametric tests  due to the data not meeting the assumptions of an analysis of variance (ANOVA). Principal component analysis (PCA) was also used to visualize the differences in composition of the soil properties at the three locations. Linear mixed models were used to test the effects of crop (lentil and pea), N fertilization rate  were significantly different  was consistently higher in pea than lentil at all three sites  at the Swift Current site for both crops relative to 60 kg ha-1, and a strong drop in lentil grain yield with increasing N rates at the Melfort site. These results, along with the lack of N fertilization effect on lentil yield at the Indian Head site are not consistent with previous studies that showed N to increase seed yield of lentil  at all sites. Similarly, f lentil . Gan et A. euteiches infection when pea plants were inoculated with AMF (A. euteiches (Rhizophagus irregularis), which may have limited the effectiveness of this treatment and highlights the need for further research in field-based experiments to better understand the potential of AMF inoculants for controlling root rot pathogens.Inoculation with AMF had no significant impact on shoot or root symptoms in our field study. In contrast, previous studies under controlled, greenhouse conditions have found reduced above and belowground symptoms caused by with AMF . This mauteiches . Additiouteiches . Differeuteiches . One reputeiches . Our fieA. euteiches present in soil pre-seeding were correlated with AS and FS in both pea and lentil . The larger root systems that pea plants tend to have compared to lentil may have contributed to this effect by providing more tissue in which A. euteiches could potentially reproduce. Other edaphic factors also likely played a role in the varying levels of A. euteiches abundance at these locations by influencing the germination and/or further fungal structure formation during the growing season.The levels of elopment . The qPCg season . Growingfe cycle . It was A. euteiches and FS for both crops. This is consistent with Fusarium and Aphanomyces were present. In contrast, a negative relationship was found between these variables for pea at mid-bloom. A possible explanation is that, root rot ratings such as FS may have limitations in determining specific pathogens responsible for the root rot complex , symbiotic N fixation was replaced by direct absorption from the soil. As a result, N had no significant effect on grain yield. This suggests N fertilization remains a viable option for managing root rot despite its reduction in nodulation. N application decreased percent nodulation at Swift Current and Melfort in both pea and lentil  (GX_GR210) | MMN (usda.gov)), equating to $49.17 CAD per hectare to apply the 60 kg/ha rate used in this study, $0.34 CAD/kg for yellow pea ) and the pea yields from Indian Head ($2207 and 2931.7 kg/ha for the 0 and 60 N rates), the 60 N rate would yield $246.40 more for an input cost of $49.17 per hectare. Future research should focus on determining the mechanisms by which the protection against root rot occurs, as the current methods can not confirm the explanation of “woodiness” of roots previously proposed by A. euteiches infection and root rot suppression would also be useful. Variation in our results demonstrate the importance of testing pea and lentil root rot treatments in multiple site-year trials, including different soil types and pre-existing field conditions, for robust conclusions. As shown by this study, both environmental and soil characteristics can affect treatment efficacy substantially.The AMF inoculant used in this study is likely not a reliable method of managing The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LB conceived the idea, and developed and conducted the field trials with WM and GP. LB was responsible for conducting the field sampling and laboratory analyses. MH was responsible for assessing plant disease ratings. LB, MT, AM, and MH conducted the statistical analyses and contributed to writing the manuscript. All authors contributed to editing the manuscript and approved the final version.