Microbes play key roles in maintaining soil ecological functions. Petroleum hydrocarbon contamination is expected to affect microbial ecological characteristics and the ecological services they provide. In this study, the multifunctionalities of contaminated and uncontaminated soils in an aged petroleum hydrocarbon-contaminated field and their correlation with soil microbial characteristics were analyzed to explore the effect of petroleum hydrocarbons on soil microbes.Soil physicochemical parameters were determined to calculate soil multifunctionalities. In addition, 16S high-throughput sequencing technology and bioinformation analysis were used to explore microbial characteristics.−1, high contamination) reduced soil multifunctionality, while low concentrations of petroleum hydrocarbons  might increase soil multifunctionality. In addition, light petroleum hydrocarbon contamination increased the richness and evenness of microbial community (p < 0.01), enhanced the microbial interactions and widened the niche breadth of keystone genus, while high petroleum hydrocarbon contamination reduced the richness of the microbial community (p < 0.05), simplified the microbial co-occurrence network, and increased the niche overlap of keystone genus.The results indicated that high concentrations of petroleum hydrocarbons (565–3,613 mg•kgOur study demonstrates that light petroleum hydrocarbon contamination has a certain improvement effect on soil multifunctionalities and microbial characteristics. While high contamination shows an inhibitory effect on soil multifunctionalities and microbial characteristics, which has significance for the protection and management of petroleum hydrocarbon-contaminated soil. These bThe effects of petroleum hydrocarbons on soil microbes are mainly reflected in two aspects. On the one hand, petroleum hydrocarbons can change the environment which soil microbes live. Petroleum hydrocarbons will increase the soil organic carbon content and C/N ratio . In addiOn the other hand, petroleum hydrocarbons can affect the metabolic activities of soil microbes and reshape the structure and function of the microbial community . PetroleAll of these effects will change the composition, diversity and structure of soil microbes and then affect their ecological functions. However, the responses of resource consumption, growth, reproduction and interactions of different species to environmental changes differ greatly . When stAmong soil microbes, bacteria are the most important for petroleum hydrocarbons degradation and has been extensively studied by a large number of literatures . TherefoAt each site, we measured a range of soil physical, chemical and microbial properties and soil functional parameters. We also quantified the composition and diversity of soil bacteria by using 16S rRNA gene amplicon sequencing. These data were used to construct co-occurrence networks to explore potential biotic interactions and identify microbial keystone taxa. According to further correlation analysis of these parameters, we demonstrated that (1) high petroleum hydrocarbon contamination reduced soil multifunctionality, while light petroleum hydrocarbon contamination increased soil multifunctionality. (2) High petroleum hydrocarbon contamination reduced microbial community diversity, simplifying microbial networks and widening the niche overlap of keystone taxa, while light petroleum hydrocarbon contamination increased microbial community diversity, enhancing microbial networks. (3) The multifunctionality of soils polluted by petroleum hydrocarbon was significant correlation with diversity and co-occurrence of soil microbial community.2.2.1.The study area is located at an abandoned coking plant industrial park  in Beijing, China, which has a long land-use history . AccordiEach soil sample was separated into two parts: one part was packed into a sealed bag for the analysis of soil physicochemical parameters, soil lithology and microbial characteristics, and the other was put in a brown glass bottle for the analysis of petroleum hydrocarbon concentrations. Both the sealed bags and the bottles were labeled according to the sampling site and then transported back to the laboratory in a 4°C storage box. After removing the stones and plant debris, the soil samples in sealed bags were divided into two parts. One part was sieved through a 2 mm screen and stored at 4°C for further physicochemical and lithology analyses, and the other part was stored at −80°C for molecular biological analysis. The soil samples in the brown bottles were stored at 4°C for contamination analysis.2.2.3−), nitrite (NO2−) and ammonium (NH4+), were examined according to previous report . The soil lithology of all samples is listed in In detail, the soil lithology was determined by Laser particle size analyzer , the pH was determined using a pH analyzer with a 1:5 soil/water mixture . The moisture content was determined by oven-drying 2 g fresh soil at 105°C until it reached a constant weight. The TC and TN contents were determined with an elemental analyzer . NHThe details and classified information of petroleum hydrocarbons detected in this study are listed in 2.3.Soil ecological functions include many aspects . Therefore, soil ecological functions should be evaluated according to various soil functional variables rather than a single variable, which is soil multifunctionality. In this study, soil multifunctionality was calculated according to the averaging approach . Soil muwhere 2.4.The E.Z.N.A.® soil DNA Kit  was used to extracted microbial DNA. Extraction procedure strictly followed the kit instructions. After extraction, 1% agarose gel was used to test the quality of DNA extracted. Nextly, region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5’-GGACTACHVGGGTWTCTAAT-3′). The amplification condition was as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and single extension at 72°C for 10 min, and end at 4°C. After amplification, 2% agarose gel was used to test the quality of amplification products. Lastly, the Illumina MiSeq PE300 platform  was used to determinate the sequences of amplification product.The raw sequences were quality filtered by fastphttps://dataview.ncbi.nlm.nih.gov/object/PRJNA890419?reviewer=sbito8l9ubnvsfslt1jistntjbThe raw sequencing data obtained in this research were submitted to the NCBI Sequence Read Archive (SRA) under accession number PRJNA890419, which can be found by the “reviewer” link: 2.5.p value of the microbial community was calculated by Networkx. The co-occurrence network was constructed, analyzed and visualized using Gephi 0.9.3. Network module was divided according to previous literatures , beta diversity  and variance partitioning analysis (VPA) were obtained from the major cloud platform.eratures . The hea2.6.The data were analyzed in Excel 2016 and R 3.6.1. Significant differences of dominant taxa and keystone taxa in highly contaminated, lightly contaminated and uncontaminated soils were obtained by the Kruskal–Wallis rank sum test. Significant differences in the beta diversity of the microbial community in highly contaminated, lightly contaminated and uncontaminated soils were obtained by Adonis. Significant differences in alpha diversity or network topological parameters in highly contaminated, lightly contaminated and uncontaminated soils were obtained by the Wilcoxon rank sum test. The graphics were drawn by the software Origin2020 and R 3.6.1.3.3.1.Soil provides major functions for Earth’s ecosystem . Petrole−1, and the petroleum hydrocarbon concentrations of eight soil samples in the lightly contaminated area were 13–408 mg•kg−1 (The results showed that the petroleum hydrocarbon concentrations of ten highly contaminated soil samples were 565–3613 mg•kg mg•kg−1 . The mul mg•kg−1 .p < 0.05). In lightly contaminated soils, TPH was significantly positively correlated with TN and moisture  according to the petroleum hydrocarbon contamination levels, which explained 89.24 and 51.61% of the total variance at the phylum level and genus level, respectively , Firmicutes (0.95–75.47%), Actinobacteria (0–40.47%), Chloroflexi (0.16–9.59%), Acidobacteria (0–13.51%) and so on . As the Pseudomonas or Tepidiphilus (the sum of the two ranging from 14 to 95.8%) in highly contaminated soils, Paenisporosarcina (ranging from 15.3 to 67.3%) in uncontaminated soils. While the relative abundance of most genus in lightly contaminated soils was uniform (Paenisporosarcina (p < 0.01), norank_f__norank_o__norank_c__MB-A2-108 (p < 0.01) and Paracoccus (p < 0.05) was highest in uncontaminated soils. While the relative abundance of Arthrobacter (p < 0.01), Sphingomonas (p < 0.05) and Pantoea (p < 0.05) was highest in lightly contaminated soils. The relative abundance of Pseudomonas (p < 0.05) and Tepidiphilus (p < 0.01) was highest in highly contaminated soils  and soil lithology (0%) alone . Among v3.3.Soil microbial diversity is crucial to maintaining the multifunctionality of soil ecosystems . The losp < 0.01). Meanwhile, the Chao index of the microbial community in highly contaminated soils was significantly lower than that in the uncontaminated soils  correlated with soil multifunctionality in uncontaminated soil (p < 0.05) with soil multifunctionality in both highly and lightly contaminated soils (p < 0.001) between the soil multifunctionality and the Chao index of the microbial community  and closeness centrality (p < 0.01) of the soil microbial networks (p < 0.05). In uncontaminated soils, there was no correlation between microbial network parameters and soil multifunctionality (p > 0.05) (p < 0.01) with soil multifunctionality  ,D. The dionality ,F, indic3.5.Keystone taxa that have the characteristics of high degree, low betweenness centrality, and high closeness centrality play a critical role in microbial community structure and function and serve as indicators of environmental change . Their nReyranella, Luteimonas, Megasphaera, Enterobacter, Roseburia, Gemmatimonas, Haliangium. The keystone genus in the lightly contaminated soils were Haliangium, Hirschia, Rubrobacter, Rhodoplanes, Lutispora, Sporichthya. The keystone genus in the uncontaminated soils were Sporichthya, Christensenellaceae_R-7_group, Thermoflavimicrobium, Desulfohalotomaculum , which states that when an ecosystem is moderately disturbed, its species diversity will be the highest . This is4.3.Our results showed that long-term high petroleum hydrocarbon contamination led to a decrease in links in microbial positive association co-occurrence networks. While light petroleum hydrocarbon contamination led to an increase in links in microbial positive association co-occurrence networks. This is consistent with previous reports . High peIn addition, the links in co-occurrence networks are related to both interactions among species and environmental filtering . TherefoIt has been reported that the weakening of negative interactions in microbial networks is detrimental to community stability . While t4.4.Luteimonas, Lutispora, Roseburia, Megasphaera, Reyranella and Rhodoplanes can convert refractory petroleum hydrocarbons into simple molecules  Metabolites of keystone taxa provide nutrition for other species. It was reported that olecules . (2) Keymicrobes ; Thermofc enzyme . (3) Keynditions . Enterobbacteria . The potPetroleum hydrocarbon contamination significantly affected the niche breadth and niche overlap of keystone taxa in the community, which is consistent with the report of 5.According to the comparisons of the relationships between the ecological function and microbial characteristics of contaminated soils and uncontaminated soils of aged realistic contaminated fields, we demonstrate that high petroleum hydrocarbon contamination indeed results in a decline in soil ecological function, while light contamination might result in an increase in soil ecological function. 16S high-throughput sequencing technology combined with biological information analysis of microbes in soils with different contamination levels reveals that petroleum hydrocarbon contamination does not always reduce the relative abundance of microbes and the microbial diversity. Light petroleum hydrocarbon contamination promoted microbial diversity and may enhance microbial interactions. When the concentration of petroleum hydrocarbons increased, the co-occurrence network among microbial communities was simplified, and interspecific cooperation may be strengthened, which meant that the community stability decreased. However, the niche overlap of keystone species increased in highly contaminated soils. In general, we cautiously infer that maybe there is a threshold effect of petroleum hydrocarbons on soil microbes, which can be an important reference for the refined management of petroleum hydrocarbon-contaminated sites. However, due to the high heterogeneity of soil structure and the limitation of co-occurrence network, the results of microbial community interactions obtained from our analysis may be inaccurate, which we plan to further explore in the next step.https://dataview.ncbi.nlm.nih.gov/object/PRJNA890419?reviewer=sbito8l9ubnvsfslt1jistntjb.The data presented in the study are deposited in the NCBI repository, accession number is PRJNA890419, which can be found at this link: WJ: investigation, formal analysis, visualization, and writing – original draft. LC: methodology and formal analysis. QT: resources and investigation. YL: investigation and data curation. JD and KY: methodology. QY: validation. SW, JL, and GN: provide sampling site and assist in designing sampling plan. LZ: supervision, methodology, and writing – review and editing. AD: conceptualization, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This research was supported by Beijing Municipal Natural Science Foundation (8232037), National Key R&D Program of China (No. 2018YFC1800905), Key Science and Technology Projects of Inner Mongolia autonomous region (2019ZD001), and China Postdoctoral Science Foundation (2021M690428).SW, JL, and GN are employed by Beijing No.4 Municipal Construction Engineer Co., Ltd.The remaining 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.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1188229/full#supplementary-materialThe Supplementary material for this article can be found online at:


The limonene formulation exhibited antifungal activity against F. graminearum with the EC50 at 1.40 μl/ml, electron microscopy and staining analysis showed limonene formulation could significantly decrease the quantity, length and septa of conidia, caused hyphal break and shrink, damaged the structures of cell membrane, cell wall, vacuoles and organelles in the hypha. Further study revealed the antifungal and antitoxic mechanism of limonene formulation against F. graminearum, limonene formulation significantly inhibited the toxisome and DON formation, was associated with the down-regulation of trichothecenes biosynthesis genes expression and many energy metabolism pathways as well as the inhibition of lipid droplets, the disturbed energy homeostasis and intracellular structures might ultimately inhibit fungal growth and DON production. In addition, limonene formulation enhanced the antifungal activity of triazole fungicides tebuconazole and mefentrifluconazole against F. graminearum, indicated limonene formulation has valuable potential as a bio-alternative fungicide and eco-friendly compound preparation for the effective management of F. graminearum and DON contamination in agriculture.Preventing grain from fungi and subsequent mycotoxins contamination has attracted notable attention. Present study demonstrated the limonene-formulated product Wetcit Preventing food products from fungal infection and mycotoxin contamination have aroused broad public concerns as food security is of particular importance to human and animal health globally . The triFusarium spp., the greatest economic losses are associated with DON and its derivatives  that has become one of the most significant diseases in cereal crops , which is recommended for use as insecticides, fungicides and herbicides , the PH-1 strain was cultured on potato dextrose agar  containing different concentrations of limonene formulation at 25°C for 2 days, then the colony diameter of each plate was measured. The values of effective concentrations that result in 50 and 90% mycelial growth inhibition (EC50 and EC90), were calculated by the Data Processing System (DPS) program .The parental strain used in this study is wild-type strain PH-1 (NRRL 31084) of F. graminearum conidiation, mycelial plugs of wild-type strain PH-1 were inoculated into a flask containing 30 ml carboxymethylcellulose (CMC) broth (50 (1.40 μl/ml) and EC90 (14.0 μl/ml). After incubation for another 3 days, the conidia of each treatment were firstly filtered and then determined by hemocytometer. The conidial length was measured with SPOT software, septa were stained with Calcofluor White Stain, and the conidial morphology was examined with a fluorescence microscope. Each experiment was repeated twice, and each concentration included three replicates.In order to study the impact of limonene formulation on C) broth , then inv/v)] overnight, and then post-fixed with osmium tetroxide [1% (w/v)] for 2 h at room temperature. Subsequently, samples were well dehydrated with gradients of ethanol (30–100%). Then the extracellular and intracellular changes were observed by SEM and TEM analysis, respectively.The alterations of fungal morphological caused by limonene formulation, were observed with scanning electron microscope  and transmission electronic microscopy . Samples treated with or without limonene formulation (1.40 and 14.0 μl/ml) for 2 h were prepared following the steps. Firstly, the mycelia of each treatment were immersed in glutaraldehyde [2.5%  was used to evaluate the influence of limonene formulation on vacuoles. The mycelia of each treatment were stained with 100 μM CMAC solution at room temperature for 30 min, and washed by sterile water for three times. Finally, the stained mycelia were subjected to microscopic analysis using Zeiss LSM780 confocal microscope.To investigate the effect of limonene formulation on the formation of lipid droplets, the hyphae of the treatment were stained with Nile Red, which is the most commonly used fluorescent dye to quantify neutral lipid content . Each saFgTRI1-GFP fusion cassettes were introduced into ΔFgTri1 mutant to generate ΔFgTri1::FgTri1-GFP strain, which was produced in our previous study , FgTRI1, FgTRI4, FgTRI5, FgTRI6, FgTRI12 and FgTRI101, were determined using HiScript II Q RT SuperMix  by qRT-PCR, and FgACTIN gene was used as the endogenous control. The experiments were performed with independent biological triplicates. The RNA extraction and relevant primers to perform qRT-PCR assay were listed in SI and TRI genes were calculated by 2−ΔΔCt formula . Then qRT-PCR assays were conducted to evaluate the impact of limonene formulation on the transcription of DON biosynthesis genes. The mycelia of PH-1 were firstly incubated in TBI media at 28°C for 48 h. Then limonene formulation was supplemented to generate final concentrations of 0.200 μl/ml for another 24 h. The treatments without limonene were used as control. The expression levels of six key trichothecenes biosynthesis genes  analysis was conducted. PH-1 strain was firstly cultured in yeast extract peptone dextrose (YEPD) medium at 25°C, 180 rpm for 36 h, and then supplemented with 0.20 μl/ml limonene formulation for another 2 h. The limonene formulation untreated mycelia were served as control. Samples were quickly collected and froze in liquid nitrogen immediately, then stored at −80°C for further RNA-Seq. The RNA library construction was conducted according the manufacturer’s procedures, and the details were listed in the SI.To further investigate the antifungal mechanism of limonene formulation against F. graminearum reference transcripts using Hisat2 (version 2.1.0), at most two mismatches were allowed during the process. These genes were subjected to alignment against public protein databases; Pfam (Pfam Protein families), Uniprot (Swiss-Prot). It comprised RSEM (v1.2.6) for transcript abundance estimation and normalization of expression values as FPKM (Fragments per kilobase of transcript per million fragments mapped). Differentially expressed genes (DEGs) were identified with DESeq2 with a filter threshold of p-value <0.05 and|log2FoldChange| > 1. ClusterProfilerhttp://www.genome.jp/kegg/) enrichment analysis. The GO and KEGG enrichment analysis were calculated using hypergeometric distribution with a Q value cutoff of 0.05. Q values obtained by Fisher’s exact test were adjusted with FDR for multiple comparisons.After construct RNA libraries, adapters and low-quality reads were filtered by using the cutadapt (version 1.11). When we mapped the clean reads to the F. graminearum and F. fujikuroi, were determined using the mycelial growth rate method. The EC50 of each fungicide was showed in 50 (EC50th) and the interaction level  pathways were up-regulated by 0.20 μl/ml limonene formulation  can be found at: YJ and XC were involved in methodology, experiments, draft and critical revision of article, and final approval. HM was involved in conceptualization and resources. CZ and YL were involved in data acquisition and data consulting. YY conceived and designed the manuscript. JJ was involved in funding acquisition and writing—review and editing. All authors contributed to the article and approved the submitted version.The present study was supported by grants from the National Natural Science Foundation of China (32272577), the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (2021DG700024-KF202203), Fundamental Research Funds for the Central Universities (2021FZZX001-31), Precision training of Zhejiang Academy of Agricultural Sciences. National Key Research and Development Program of China (2022YFD1700500)” here, right before “Fundamental Research Funds for the Central Universities.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.


The silver nanoparticles were loaded onto the mat to enhance their photocatalytic activity. These mats were characterized using different techniques, including Fourier transform infrared (FTIR), scanning electron microscope (SEM), and transmission electron microscope (TEM). The water contact angles were used to study their hydrophilic and hydrophobic behavior. The Langmuir isotherm model and adsorption kinetics were studied to evaluate their adsorption capabilities against methylene blue (MB). Sample 2 followed the Langmuir isotherm model (R2 = 0.9939). Adsorption kinetics exhibited pseudo-second order behavior (R2 = 0.9978) due to their maximum correlation coefficient values. MB has excellent adsorption at room temperature and the formation of the monolayer at the surface of the adsorption mat. An enhanced PO43− and MB adsorption was observed, providing recyclability up to 4–5 times. Hence, the fabricated nanofibrous mat would be a potential candidate for more effective wastewater treatment applications.The effluents of industrial wastewater contain several toxic organic and inorganic pollutants that may contaminate clean and freshwater sources if untreated or poorly treated. These toxic pollutants include colors; hazardous compounds; surfactants; cosmetics; agrochemicals; pharmaceutical by-products; and agricultural, pharmaceutical, and medical contaminants. Treating wastewater has become a global problem. Many projects have been started in the last two decades to treat wastewater, resultant water pollution, and associated waste management problems. Adsorbants based on graphene oxide (GO) are viable wastewater treatment materials due to their adaptability, photocatalytic action, and capacity for self-assembly. Here, we report the fabrication of nanofibrous mats from polycaprolactone (PCL), carboxymethyl arabinoxylan (CMARX), and carboxyl- Modern industrialization has developed all the necessary products to facilitate the increasing population. The effluent from the wastewater often remains untreated or poorly treated, causing increased water pollution and contaminating freshwater resources. The rapid expansion of the mining, paper, and battery industries, as well as agriculture, medical, and food industries, have contributed to water pollution due to their inorganic and organic contaminants. Recent industrial growth has resulted in heavy metals, hazardous substances, colors, and pharmaceutical waste accumulating in water bodies ,2. The w2) nanoparticles were incorporated into nanofibers to produce reactive oxygen species (ROS) that will aid in degrading dyes and other toxins, removal activities have improved [Natural polymers have drawn increasing attention because of their distinctive qualities, which include ease of chemical functionalization, stability in both heat and cold, and fouling resistance. Thus, various contaminants with biosafety were studied for water contaminant removal using natural polymers . Naturalimproved .Graphene oxide (GO) is known due to its excellent physicochemical characteristics. It is a 2D material and can easily functionalize polymeric nanofibrous in small quantities . The perf-COOH (Sample 1/PCG) with standard operating protocols and parameters. The PLA/CMARX/GO-f-COOH was thermally processed, and AgNO3 was used to anchor over the nanofibrous mat as PCL/CMARX/GO-f-COOH@Ag (Sample 2/PCG@Ag). The recently developed composite nanofibrous mats have desirable physicochemical and mechanical stability. Treating wastewater by removing toxic and unwanted materials is cost-effective, and newly developed nanofibrous materials have never been reported. The manufactured nanofibrous mats are cost-effective, environmentally friendly, and may be used to treat wastewater.In this study, we have developed a composite nanofibrous mat; the nanofibrous mat was fabricated by electrospinning technique using PCL/CMARX/GO-Plantago ovata seed husks were bought from the local market of Johor Bahru, Malaysia, to extract arabinoxylan (ARX), graphene oxide (GO), sodium mono-chloroacetate (ClCH2COONa), sodium hydroxide (NaOH), polycaprolactone (PCL), and silver nitrate (AgNO3). Chloroacetic acid (CH3COOCl), Rhodamine B (Rh-B), and methylene blue (MB) were supplied by Sigma-Aldrich, Selangor Malaysia. The hydrogen peroxide (H2O2) and hydrochloric acid (HCl) were purchased from Aladdin Reagent, Shanghai, China.Plantago ovata seed husk was soaked overnight in 2.5 L deionized water. Then, it was blended with NaOH solution (2.5%) for 5 min, and vacuum filtration separated the insoluble husk particles from gelation. The gelling part was treated with acetic acid to coagulate. The obtained gel was washed with plenty of deionized water to neutralize, and the obtained gel was freeze-dried to obtain dried ARX powder. Then, 2 g ARX was placed into ethanol for 1 h with continuous stirring at room temperature by adding ClCH2COONa and NaOH (20%) solution. The reaction mixture was allowed to be stirred at 55 °C for 5 h to synthesize the CMARX. After 5 h, the reaction media was washed with an 80% water/methanol mixture (v/v) to obtain CMARX. The synthesized CMARX was treated with acetic acid to neutralize and was washed with ethanol. It was dried in a vacuum oven to obtain a fine powder of CMARX.Arabinoxylan is a natural polymer, and it is extracted from the seed husk of psyllium using a reported procedure with little modification . A totalf-COOH) and freeze-dried at −50 °C [The graphene oxide was functionalized with carboxyl (GO-t −50 °C . The solThe prepared solutions were filled in the syringe and fixed with a stain-free steel needle (diameter of 0.6 mm). The constant flow rate of solution from the syringe was adjusted to 0.25 mL/h and operated under a strong voltage difference of 20 kV. The needle and collector were separated by 25 cm, and the collector was covered with aluminum foil to collect the resultant nanofibers. After collection, the nanofibers were gathered on aluminum foil and dried in the oven. These nanofibers were oven-dried at 150 °C for 2 h for heat-induced crosslinking between −OH and −COOH through esterification. Therefore, PCL ensured the structural integrity, CMARX provided desirable gelation, and GO enhanced the functional behavior via π–π stacking. Thanks to the carboxylic functional groups, the silver nanoparticles were in a very bad environment. The combined synergistic catalytic effect of these individual behaviors has produced wastewater treatment benefits for dye degradation.The nanofibrous mats were dipped into ascorbic (1 mL) acid-added silver nitrate solution (30 mg/mL), and the reduced silver nanoparticles initiated their anchoring onto the nanofibrous mat. The dipping of nanofibrous mats was repeated after different intervals  to anchor maximum silver nanoparticles. These silver nanoparticles were removed from the nanofibers by washing them in deionized water after 3 h. These nanofibrous mats were dried at 85 °C to prepare a dried nanofibrous mat for wastewater treatment.−1 wavenumber. The surface morphology was analyzed using scanning electron microscopy (JEOL-JSM 5410 LV) with an accelerated voltage of 10 kV. The nanofibrous mats were dried well and gold-sputtered before analysis. The aggregates of silver nanoparticles were observed through the transmission electron microscope . A UV–Vis spectrophotometer  was used to observe dye adsorption.The functional groups and structural analysis of nanofibrous mats were conducted using Fourier transform infrared spectroscopy . The FTIR analysis was conducted from a 4000 to 400 cm−, SO42−, HCO3−, and PO43−) was used to study their impact on the effectiveness of MB removal. Composite nanofibrous mats were tested for their ability to be recycled using a diluted HCL solution (2 mM) at an ambient temperature for 5 h. The nanofiber mat’s surface charge then inverts from negative to positive, allowing MB to be removed from the adsorbent surface via electrostatic repulsion. Five cycles of regeneration were tested using the same methodology. The composite nanofibrous mats were washed twice with water for the subsequent adsorption cycle. The concentration of MB was once more measured to determine the adsorption capacity of composite nanofibrous mats.The various concentrations of methylene blue (MB), a reference dye used to study wastewater treatment, were examined using a UV–Vis spectrophotometer at 664 nm. A known concentration of a standard solution was used to calibrate the UV–Vis spectrophotometer. The recently developed composite nanofiber mat was present during the batch-mode adsorption studies of MB. Similar research has been carried out on the effects of MB concentrations (200–1000 mg/L) on adsorption efficiency. Under optimal conditions, a constant concentration of (1 mM) of inorganic anions (Cl−1 are due to C=O–C (stretching), C–O–C (asymmetric stretching), C–O, C–C (stretching), –CH2 (symmetric and asymmetric) vibrations, respectively [−1 is due to carboxyl –C=O (stretching). The vibration peak at 1651 cm−1 is due to C=C stretching vibration. The graphene oxide characteristics peaks of oxygen-based functional groups at 1552, 1414, and 1374 cm−1 are attributed to functional groups C=O (stretching), C–O (stretching), and C=O (stretching) vibrations, respectively [−1 and 919 cm−1 are due to saccharine structure and pyranose ring, as these confirm the presence of CMARX [−1 is characteristic of O–H stretching absorption and describes intramolecular and intermolecular hydrogen bonding. The FTIR analysis of the PLA has been provided in the The functional groups and structural behavior of fibrous mats were presented using FTIR spectral profile, as shown in ectively . The vibectively ,37. The of CMARX . The broThe wetting behavior is essential for interacting with water and determining hydrophilicity and hydrophobicity in order to understand the composite nanofibrous mats. In general, a material’s hydrophilic nature is represented by a lower contact angle, while a larger contact angle represents its hydrophobic nature. The various oxygen-based functional groups in the composite nanofibrous mats provide them with hydrophilic properties through hydrogen bonding . The redIt has been found that the electrospun nano-fibrous mats’ mechanical properties are crucial for treating wastewater. 2 = 0.9861) is greater than that of the pseudo-second order (R2 = 0.9793) for Sample 1. The correlation coefficient of the pseudo-second order (R2 = 0.9861) is higher than that of the pseudo-first order (R2 = 0.9793) for Sample 2. However, the correlation coefficient of the pseudo-first order (R2 = 0.9917) was lower for Sample 2 and higher for the pseudo-second order (R2 = 0.9978) for Sample 1, and these values are summarized in The rate constants for the entire adsorption process were calculated by studying the kinetics of dye adsorption. As shown in 2) demonstrate that these isotherms of adsorption followed the Langmuir isotherm model, which confirms that the monolayer adsorption is linked to the heterogeneous surface of adsorbents. Compared to Sample 1, which had a lower adsorption capacity (53.0472 mg/g), Sample 2 exhibits an increased adsorption capacity (1129.5809 mg/g). This shows that Sample 2 enhanced the adsorption ability of MB, which is integrated with the value of KL, as the adsorbent’s higher KL determines excellent adsorption efficiency at low concentrations. According to The adsorption isotherms contain crucial information about the sorption process. The highest adsorption capacity was determined by this study’s correlations between adsorbent and adsorbate concentrations. Through the use of the well-known non-linear isotherm models, i.e., Langmuir and Freundlich, this study evaluated the performance of adsorption. According to the Langmuir fitted model, the adsorbent’s uniform solid surface was coated with a monolayer as the adsorption mechanism. There is no evidence for the interaction of MB with nearby binding sites. The multi-layer adsorption mechanism on the adsorbent’s heterogeneous solid surface was seen using the Freundlich model, as shown in −, SO42−, HCO3−, and PO43−) to achieve this. We investigated their influence on MB adsorption capacity by the mat, as shown in − ions had a detrimental impact on the mat’s MB adsorption behavior, which could be due to their competition with foreign anions. The chloride anions are more reactive to be adsorbed than the positive dye. However, anions have a lower binding constant to MB, and SO42− and HCO3− had little impact on the mat’s ability to adsorb MB. It is interesting to note that the availability of hydrogen phosphate ions increased the pH of the solution, which in turn enhanced the adsorption capability after the response of PO43−. As a result, there may be an acceleration of the electrodynamic activity between the mat and the MB adsorption, which would improve adsorption efficiency.Several inorganic salts have been used to process and manufacture dyes, and several toxic inorganic anions have been found in industrial wastewater ,46. InduCompared to other nanofibrous mats (Sample 1), the composite nanofibrous mat (Sample 2) has shown greater adsorption and reusability. The results of the newly developed composite nanofibrous mat revealed it to be an efficient absorbent to treat wastewater . The com2 = 0.9978) than the pseudo-first order model. However, the adsorption equilibrium was reached after 12 h for Sample 2 and 18 h for Sample 1, respectively. Moreover, the adsorption isotherm confirms that Sample 2 has an exceptional ability to remove MB (1129.5809 mg/g) under normal conditions in comparison to Sample 1 (48 mg/g), and it also follows the Langmuir model. However, the inorganic anion PO43− has more enhanced adsorption than other anions . Sample 2 has exhibited exceptional recyclability during up to 5 cycles with improved adsorption capacity. Hence, all the findings support the confirmation that Sample 2 is the best sample and has the ability to treat wastewater, including organic and inorganic pollutants. As a result, Sample 2 is a promising, environmentally friendly, and long-lasting nanofibrous adsorbent for wastewater treatment.The composite nanofibrous mat is fabricated from PCL, GO, and silver nanoparticles to remove the dyes to treat wastewater treatment with excellent properties. Sample 2 has an exceptionally smaller diameter than the PCL nanofiber mat, according to the analysis of the SEM morphology. Additionally, increasing the mechanical stability and hydrophilicity of Sample 2 and silver nanoparticles causes them to become more hydrophilic (with a contact angle of 56.30° to 32.40°), effectively increasing MB adsorption. Adsorption kinetics confirmed that the pseudo-second order model has a maximum value to linear correlation coefficient (R


However, electrical and mechanical function are often studied separately—despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function.The Langendorff-perfused Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol.At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the “pulse deficit” phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion.Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts. With thIsolated mouse heart models have been widely used to test the effect of gene mutations on cardiac physiology due to the fast and easy manipulation of the mouse genome. However, in most of the isolated mouse heart models used to investigate IR described in scientific literature, contractile and electrical cardiac function are studied separately. Yet, there is a complex relationship between electrical and mechanical function in cardiac physiology. Electrical excitation triggers the contractile activity of the heart (through excitation-contraction coupling), but mechanical activity can also affect the electrical activity  –12. IndeTherefore, we developed an isolated mouse heart perfusion system to simultaneously assess cardiac electrical and mechanical function. We showed that our model can recapitulate pathophysiological electro-mechanical interactions, and we tested this approach in a specific example of a global ischemia-reperfusion protocol. We finally suggest that this approach might be useful to better understand the complex mutual influence between electrical and mechanical cardiac function.2.Equipment and reagents used in this study are listed in 3.3.1.All performed experiments were in accordance with EU legislation (directive 2010/63/EU) and the Swiss animal welfare law and have been approved by the Swiss animal welfare authorities .−/−) and transgenic K18-hACE2 mice were purchased from the Jackson laboratory and cross bred to obtain K18-hACE2-Ldlr-/-mice. Eight-week-old female and male animals were housed in groups at 22°C with a 12 h day/night cycle, food/water supply ad libitum and 12 weeks of Western-type diet (WD) to induce a cardiovascular disease phenotype. One week before the end of the WD feeding period, mice were also treated with 50 µg CpG oligodeoxynucleotides + 100 µg Polyinosinic:polycytidylic acid [Poly(I:C)] antigens every second day and three times in total to induce an inflammatory setting. The animals were euthanized three days after the last injection. These mice represent the control group of larger set of animals investigated during a Covid-19 research project, which will be published separately. However, to establish and validate the method of simultaneous mechanical and electrical function determination in Langendorff-perfused mouse hearts, as well as to allow proper understanding and replication of our experimental set up, we aimed to generate a separate paper to concentrate on technical aspects of the experimental system.Low-density lipoprotein receptor knock-out mice  perfusion, followed by 25’ normothermic, global ischemia, and 60’ aerobic reperfusion .3.3.1.P wave and QRS complex of the extracardiac electrocardiogram. Representative traces are shown in Mice were administered 100 µg/g bodyweight ketamine  and 8 µg/g bodyweight xylazine  by intraperitoneal injection. The pedal pain withdrawal reflex was used to ensure sufficient depth of anesthesia. Mice were brought into a supine position and the thoracic cavity was accessed via midline skin incision and bilateral thoracotomy. The heart was then carefully explanted and transferred to a petri dish containing 50 ml ice-cold 0.9% NaCl and 0.5 ml of heparin . While fully submerged in the solution, the aorta was cannulated with a blunt 22 G needle using a stereomicroscope . The cannulated heart was then quickly connected to the perfusion system. The 1.1 F intracardiac octapolar catheter was gently inserted into the right atrium through a small incision and was then advanced to the right ventricle with an angle following the axis of the heart. Then, two silver wires were put in place at the root of the aorta and base of the heart. Proper positioning of the electrodes was visualized by an overlay of the intracardiac electrocardiogram signals with the 3.3.2.2PO4 1.2, CaCl2 1.5, MgSO4 1.2, (D+) Glucose 11.1, NaHCO3 25, Pyruvate 2) using the non-recirculating Langendorff circuit at a constant pressure of 60 mmHg. The system was then switched to the recirculating Langendorff circuit for all aerobic periods and perfused with the same modified Krebs-Henseleit buffer supplemented with 1.2 mM palmitate and 3% albumin at a constant pressure of 60 mmHg. To make this buffer the palmitate was initially dissolved in 40% ethanol containing 0.5–0.6 g Na2CO3 per g of palmitate. After evaporating the ethanol by boiling the mixture, it was quickly added to a 3% albumin Krebs-Henseleit solution to bind the palmitate to the albumin with continuous stirring. This solution was then filtered with a sterile filter system. During ischemia, hearts were immersed in a warm (37°C) ischemic bath with energy-substrate-free modified Krebs-Henseleit buffer   was used to monitor and record temperature, electrical signals, left ventricular pressure, and flow data throughout the perfusion protocol. Two-minute time windows were then analyzed after 20 min of baseline and 10, 20, 30, 40, and 60 min of reperfusion using LabChart Pro v8  as shown in 3.3.3.Left ventricular systolic pressure (LVSP) as well as left ventricular diastolic pressure (LVDP) were measured  to measure heart rate, PR and QT intervals. Intra-cardiac EP signals and intra-atrial and intra-ventricular conduction times were determined using the 1.1 F octapolar intracardiac catheter as shown in the representative traces in 3.4.P < 0.05 was considered statistically significant and tests used in each experiment are specified in the figure legends.Statistical analyses were performed using Prism 9.5.1 (GraphPad Software). Data are expressed as mean ± the standard error of the mean (SEM) unless stated otherwise [4.4.1.ex-vivo perfused mouse hearts. From the octapolar catheter inserted in the right atrium and ventricle  and ventricle (channels 1–3). Finally, the ventricular effective refractory period (VERP) can be measured by pacing the ventricles with a S1–S2 stimulation protocol   . At basefemales) . On averart rate . As expe4.2.ex-vivo perfused hearts to see how the mechanical function reacts to a change in the electrical activity.The advantage of simultaneous assessment of electrical and mechanical function is that it allows a better understanding of the electro-mechanical interaction, i.e., how electrical activity impacts on contraction, and vice versa. We, therefore, looked at spontaneous arrhythmia occurrence in The combined use of the intracardiac octapolar catheter with the ECG allowed us to better define arrhythmia, such as extrasystole, and whether the ectopic signal originates from the atria  or the ventricles  . At base−1), and a significant decrease in LV work  and dP/dt min   . InteresmHG.s−1) .4.3.ex-vivo Langendorff perfused hearts can be combined with any experimental conditions. Here we decided to perform global ischemia for 25 min followed by 1 h of reperfusion. After ischemia, the mechanical function dropped drastically with a significant decrease in DP , LV work , dP/dt max  and dP/dt min  at 10 min reperfusion  and decreases in LV work  and dP/dt min   . In addimHg.s−1) . TherefomHg.s−1) .These results suggest that the electro-mechanical interaction is preserved after global ischemia-reperfusion and that the decrease in mechanical function  after is5.ex-vivo perfused mouse hearts. We also demonstrate that this approach can be combined with varied experimental conditions such as global ischemia-reperfusion. In addition, although this Langendorff setup was designed for mouse hearts, this should be readily transposable to larger mammal hearts such as rabbits with some adjustments for size.This manuscript presents an approach to simultaneously assess electrical and mechanical function in 5.1.ex-vivo perfused hearts are investigated separately  can affect the mechanical function. Inversely, one can see how changes in the mechanical function  can influence electrical properties.Here we investigated the effect of brief episodes of bigeminy on the cardiac mechanical function. Comparison between episodes of bigeminy and episodes of sinus rhythm within the same hearts showed a significant improvement in DP and maximal velocity of contraction and relaxation in the subsequent beats following the extrasystole . In cont5.3.ex-vivo Langendorff-perfused hearts. Here we showed an example of global ischemia-reperfusion with continuous monitoring of both electrical and mechanical function. Interestingly, except for the increased VERP, global ischemia-reperfusion did not affect electrical parameters, even at an early stage of reperfusion (10 min) (2+ overload due to increased Na+/H+ exchange and subsequent reverse mode of the Na+/Ca2+ exchanger (2+ responsiveness where Ca2+ dynamics (Ca2+ transient generation) is normal, but the contractile response is reduced  . Diastol(10 min) , 40. On (10 min) . This de(10 min) . It is m(10 min) –44. Uponxchanger , 45, 46, reduced , 47, 48.Interestingly, during reperfusion, we still observed the same contractile response during bigeminy as what was previously seen at baseline. Indeed, the extrasystole-triggered contractions were only 50% as strong as in sinus rhythm, while the subsequent contraction had an increased DP and maximal velocity of contraction and relaxation . Therefo6.Our approach was designed for heart perfusion with the Langendorff method, a technique for which the perfusion is forced into the coronaries. Hence, the left ventricle is not exposed to blood/perfusate-induced mechanical strains. Therefore, it will be interesting to try adapting our approach to a perfusion system of working hearts to recreate a more physiological electro-mechanical interaction.7.Our study presents an approach for simultaneously assessing electrical and mechanical function in Langendorff-perfused mouse hearts on a beat-to-beat scale. We show that this approach can be combined with any experimental conditions in Langendorff-perfused heart with an example of global ischemia-reperfusion. With this approach, we hope to better understand the complex mutual influence of cardiac electrical and mechanical function under physiological and pathophysiological conditions.


All patients were classified as having supine hypertension (SH), orthostatic hypertension (OHT), delayed orthostatic hypotension (dOH), or orthostatic hypotension (OH) according to consensus criteria. The patients were assigned to one of three groups: extreme BP dysregulation (BPextreme), mild BP dysregulation (BPmild), and no BP dysregulation (BPnone) according to their orthostatic BP subtypes. The autonomic functions of 237 patients were re-assessed after approximately 3 years. Among initially enrolled subjects, 61.8% of the patients showed orthostatic BP dysregulation: 29.6% in the BPextreme group and 32.2% in the BPmild group. At follow-up, the BPextreme group increased in number, while the BPmild group diminished. Two-thirds of the initial BPextreme patients maintained their initial subtype at follow-up. In comparison, 40.7% of the initial BPmild patients progressed to the BPextreme group, and 32.4% and 14.7% of the initial BPnone group progressed to BPextreme and BPmild groups, respectively. Cardiac denervation was most severe in the BPextreme group, and a linear gradient of impairment was observed across the subtypes. In conclusion, various forms of positional BP dysregulation were observed during the early disease stage. SH and OH increased with disease progression, while OHT and dOH decreased, converting primarily to SH and/or OH.In Parkinson’s disease (PD), cardiovascular dysautonomia accumulates with disease progression, but studies are lacking on the natural history behind each subtype except orthostatic hypotension. This study investigated the early natural history of orthostatic blood pressure (BP) subtypes in PD. Two hundred sixty-seven early PD patients were included. Their cardiovascular functions were assessed by head-up tilt-test and  BP dysregulation is a continuum in its respective body position, demarcated by operational criteria. These positional BP fluctuations can take the form of supine hypertension (SH), orthostatic hypertension (OHT), delayed orthostatic hypotension (dOH), or orthostatic hypotension (OH)5.Parkinson’s disease (PD) is a neurocardiologic disorder that includes a spectrum of positional blood pressure (BP) dysregulations ranging from hypertensive to hypotensive states7, and pathobiological inter-relay between dOH and OH has been suggested8. Delayed OH is a mild form of OH5. These findings suggest that the range of positional hemodynamics represents different levels of adrenergic derangement. Also, each orthostatic subtype can influence the clinical outcomes negatively12, highlighting the need to investigate the characteristics of each.The pathophysiology of orthostatic BP dysregulation is multifaceted and involves central and peripheral catecholaminergic deficits6; dOH, SH, and OHT have received relatively little attention until recently5, and their inter-relationships have been ignored. This research aimed to describe the features of orthostatic subtypes in early de novo PD. We focused on the interrelationships of these subtypes through disease progression and on their early changes to minimize confounders of comorbidities and dopaminergic medication use.Many studies have focused on the neurobiology and outcome of OH in PDextreme, and 86 (32.2%) were classified as BPmild at enrollment. Overall, 61.8% of the total population had orthostatic BP dysregulation. The mean age at diagnosis was 66.9 ± 9.1 years, and 128 (47.9%) were female. Disease duration at diagnosis was 13.3 ± 10.8 months, and patients were monitored clinically through an average period of 61.4 ± 21.8 months  did not differ across subgroups, and there were no differences among groups in the types of drugs used to treat PD symptoms at each patient’s endpoint. Antihypertensive classes were not different, with fewer than six participants in each subgroup taking beta-blockers.The clinical and autonomic features of the patients are summarized in Tables vs. follow-up ∆HR/∆SBP3min < 0.5, 95.8% vs. 96.1%).At initial evaluation, 23 PD patients (8.6%) were hypertensive in the supine position; 48 18.0%) patients were OH. Twenty-two (8.2%) were determined to have delayed OH, and 64 (24.0%) had orthostatic hypertension. The two OH groups were not different in age at diagnosis, disease duration, or H/M ratio (Supplementary Table 8.0% patiextreme group had more severe motor impairment than the BPnone group (21.1 ± 12.2 vs. 17.5 ± 7.6). The median H&Y stage was two; this grade was maintained throughout the follow-up. PIGD and AR phenotypes were the dominant forms. The motor phenotype was significantly associated with the initial orthostatic subtype, particularly BPextreme related to PIGD .The initial converted MDS-UPDRS Part II score was 6.6 ± 4.3, and this did not differ among the orthostatic subgroups. This lack of difference remained at follow-up between-group comparisons. The initial motor score, part III, was 18.5 ± 9.1, and its severity was similar between groups. This score increased to 19.5 ± 11.0 at the follow-up exam, and the BPextreme group had the smallest tracer uptake ratio overall and the highest washout rate (WR). Trend analyses of the tracer uptake ratio revealed a gradient of increase or decrease in the groups .The overall H/M uptake ratios for both early and delayed groups were below the pre-defined reference. The BPups Fig. . Early aextreme group  increased, while patients with BPmild group decreased. About one-third of patients in the initial BPextreme group changed to an orthostatic BP dysregulation subtype, while the rest retained their subtype at follow-up , while 42.2% remained free of orthostatic stress . A linear ascending trend of delayed H/M at both initial and follow-up was observed across turnover patterns of BPnone subtype  = 15.9, p < 0.001, Fig. Positional ∆BP types progressed in different distribution patterns after 29.3 ± 9.4 months  showed shorter disease duration before diagnosis. There were no associations between motor phenotypes and subtype transformation patterns.Initial BPon Table . No discextreme (SH and/or OH with any other forms), BPmild (dOH or OHT), and BPnone (PD without orthostatic BP dysregulation). The BPextreme group was initially associated with PIGD but lost its relationship during follow-up. The number of patients in the BPextreme group increased, while that of those with BPmild decreased after 2–3 years. A large proportion of the BPmild group progressed to BPextreme. The transformations of orthostatic subtypes suggested a dynamic classification along its adrenergic failure: BPnone → BPmild → BPextreme.Early PD, the diagnosis of which was re-affirmed at an average follow-up of five years, was investigated for positional adrenergic failure. Cardiovascular dysautonomia was sub-grouped into BP13. That group estimated the point prevalence at the final visit to be 32.8%, but the cumulative incidence of OH was centered at a median of 1.7 years . Therefore, most newly detected OH occurred within 2.5 years and then approached a plateau. This approximates our 32.5% prevalence at an average of 29.3 ± 9.4 months. The age of the BPextreme group was the oldest among the groups. Previous studies reported the OH group to be the oldest; this difference could be explained by the status of OH as the major constituent of the BPextreme group13.In this study, the PD population was in the early stage of disease at enrollment. The patients were at the mid-phase of the disease when their diagnosis was reviewed for the study. The point prevalence of PD with OH (PD + OH) at initial presentation and follow-up was comparable to that in a previous study by Hiorth et al.16. This difference could be attributed to how supine BP was calculated. This study used the average of four separate measurements, which might have resulted in a lower estimate of SH. The study focused on accurately identifying SH, as it can co-occur with neurogenic OH in PD2, making its separation crucial for the study’s orthostatic subtyping. The definitions of OH and OHT were influenced by supine BP, which served as a reference for comparing standing BP. The study aimed to provide a conservative estimation of the prevalence of different subtypes, leading to a lower prevalence of SH. However, the baseline prevalence of OHT in this study was similar to that of other previous work4.The prevalence of SH and SH + OH was lower in the present study than in previous estimatesextreme) represent the extremes of hemodynamic adrenergic failure in its respective supine and upright positions by a potentially shared pathophysiology7. Delayed OH and OHT (BPmild) previously were conceptualized to be situated between PD without cardiovascular dysautonomia (BPnone) and BPextreme along a spectrum of adrenergic failure8. The characteristics of delayed OH and OHT also did not differ in the present study; thus, they were grouped to represent BPmild. The H/M uptake ratios and washout rates reflected postganglionic cardiac sympathetic denervation and its progressing catecholaminergic malfunction19. Trend analyses revealed increasing uptake ratios and decreasing washout rates, implying a gradient of adrenergic failure across the groups. Higher H/M uptake ratios and lower washout rates correlated with more preserved sympathetic integrity. These gradients persisted during follow-up. The initial establishment and maintenance of the adrenergic gradient during disease progression support the argument of BPmild as an intermediary phase, indicating possible temporal evolution among the subtypes (BPnone→ BPmild→ BPextreme).PD Patients with SH and/or OH  was correlated with the most impaired sympathetic tone. In contrast, the BPnone→BPnone (the most preserved form) demonstrated the least impairment. This association between the severity of sympathetic tone and the grade of orthostatic turnovers could provide another explanation of the underlining pathophysiologic nature of BP dysregulations (BPnone→ BPmild→ BPextreme).The BPnone increased significantly across the initial subtypes. This implies that increasing remnant sympathetic function, as indicated by increased linear H/M uptake ratio, played a role in such transformation. Within the context of PD as a neurocardiologic disease, we interpreted regression to BPnone to be a result of different resiliencies of extant sympathetic functions among subtypes, rather than actual resolution6. Some of the transformations into normal BP regulation might result from the hypotensive effect of L-DOPA, particularly in the SH + OHT group (BPextreme) which needs further clarification with a larger population in future research.The proportion of patients resolving to BPextreme, showed the largest overlap with the PIGD or AR motor phenotype. Motor impairment was greater in this extreme group than in the others at follow-up. We presumed that this was not a coincidence since BPextreme and non-tremor-dominant PD correlated with poor outcomes21, and BPextreme was suggested to demonstrate diffuse pathology22. Future longitudinal studies are warranted to assess the association between motor prognosis and orthostatic subtype.The non-tremor-dominant type was the most prevalent motor phenotype in this cohort despite the absence of a significant difference. Both PIGD and AR types increased during follow-up, while others decreased. The most impaired subtype, BPThis study has some strengths. First, we attempted to reduce selection bias by including PD patients who were re-assessed after at least two years and clinically followed for an average of five years. Another strength was the distinction of the OHT group, allowing for a hypertensive span in both supine and upright positions. The inclusion of an OHT group is rare in descriptions of cardiovascular dysautonomia in patients with PD, and the present study provides a baseline for future studies. In addition, various labile BP subgroups were studied. Finally, this study demonstrated the change in each subtype from baseline, allowing observation of the dynamic changes of BP dysregulations.early clinical course of the total follow-up (approximately the first one-third of the disease duration) was investigated. This retrospectively confirmed prospective nature of the study design prevented the authors from estimating the transition to atypical PD, such as multiple system atrophy, because they were not considered during clinical retrospection. The experimental design represents one major bias, but in our belief, it is also a strength because no single biomarker could replace the clinical follow-ups of neurologists in confirming PD. Although more extended periods of follow-ups are necessary, the average total follow-up duration allowed the neurologists to assert their re-affirmation of PD. Future prospective research, inclusive of all parkinsonism, is warranted to assess the development of multiple system atrophy.However, this study also had several limitations. One limitation is that the follow-up head-up tilt test period was relatively short. One purpose of this study was to investigate whether transformation between orthostatic subtypes could indicate the severity of neurobiology, avoiding the influence of confounders such as the administration of dopaminergic medications. Had the study design included results of patients with advanced PD, the changes of subtypes could have been significantly affected by the effects of medication. However, prolonged follow-up, including those in the advanced stage would be informative since there lacks of prospective investigation of the prevalence of diverse orthostatic subtypes that reflect clinical reality. The exclusion criteria also could have biased the results as it might prohibit the enrollment of PD patients who indeed had orthostatic adrenergic failure, regardless of confounders. In a study looking into the natural course of a disease, the excluded patients could provide precious information that mirrors the clinical circumstances; thus, offering generalizability in its interpretation. Another limitation is that clinical severity was not related to laboratory parameters. The study would be more complete if the longitudinal clinical symptoms, other than motor phenotypes, were associated with longitudinal changes. Future study encompassing both clinometric and laboratory findings is needed. Lastly, we did not include any suspected cases with clinical manifestations of atypical PD in the study. We investigated only PD patients whose diagnosis was assured by the two neurologists. Their diagnoses were confirmed by reviewing the patients’ clinical course after an average follow-up period of 61.4 ± 21.8 months. This signified that only clinically confirmed PD patients with much confidence were enrolled initially, and their In conclusion, various forms of positional BP dysregulation existed in the early PD patients in this study. Although the exact pathobiology of each type and their interrelationships are not fully understood, OHT and delayed OH might be a preliminary phase before progressing to SH and OH. Orthostatic subtypes might represent the severity of adrenergic dysfunction in early PD regardless of dopaminergic medication use.This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital. All subjects provided written informed consent to participate. The research was conducted in accordance with relevant guidelines and regulations.23, and its diagnosis was supported by positron emission tomography studies using 18F-N-(3-fluoropropyl)-2beta-carbon ethoxy-3beta-(4-iodophenyl) nortropane24. Patients showed decreased dopamine transporter uptake in the striatum, primarily in the posterior putamen. Patients were monitored every 2–6 months for at least 24 months and a maximum of 10 years  current intake of medications known to influence autonomic functions, such as alpha-blockers or tricyclic antidepressants.Baseline characteristics of age; sex; body mass index; disease duration at diagnosis; follow-up duration; history of hypertension, diabetes mellitus, or dyslipidemia; and smoking status were investigated at the initial assessment. Patients with any of the following were excluded: (1) any symptoms or signs of atypical and/or secondary parkinsonism, (2) family history of PD among first-degree relatives, (3) history of diabetic neuropathy, (4) history of symptomatic stroke that could affect general cognition and performance, (5) history of heart failure that required cardinal symptoms and signs of exercise intolerance  and fluid retention (such as edema)n = 237), 175 were assessed using the UPDRS; the remainder were evaluated using the MDS-UPDRS. The Hoehn and Yahr (H&Y) stage was scored at every assessment. The subtotal scores of parts II and III of the UPDRS were scaled into the MDS-UPDRS for comparisons26.The 267 patients were initially evaluated using the Unified Parkinson’s Disease Rating Scale (UPDRS), and ten were also assessed using the Movement Disorder Society (MDS)-UPDRS. At the follow-up examinations .Two motor phenotyping methods were applied to stratify the PD patients. These methods were tremor dominant (TD´) versus postural instability/gait difficulty (PIGD) or tremor dominant (TD˝) versus akinetic-rigid (AR) typeThe average examination interval was 29.5 ± 9.7 months . After 20 min of supine rest, head-up tilt testing (20 min at 60°) was performed using a Manumed Special Tilt1-section tilt table . Supine BP and heart rate were recorded every 5 min before tilting to 60°, and the same measurements were performed at 0, 3, 5, 10, 15, and 20 min during head-up tilt, and as deemed necessary to ensure subject safety.30.After excluding the first supine BP (at 0 min), average supine systolic/diastolic BPs and heart rates  were calculated from the measurements at 5, 10, 15, and 20 min. Supine hypertension (SH) was diagnosed if the average supine systolic and/or diastolic BP (SBP/DBP) was ≥ 140/90 mmHgmin) at three or five minutes in the tilted position was selected to identify orthostatic hypotension (OH). The orthostatic BP decreases in systole (ΔSBP) and diastole (ΔDBP) were calculated from average supine BPs. When patients were supine-hypertensive, ΔSBP and/or ΔDBP ≥ 30/15 mmHg within five minutes was used to diagnose OH; otherwise, ΔSBP and/or ΔDBP ≥ 20/10 mmHg were used31. Patients were classified as delayed OH (dOH) when they met the above criteria after 10 min.The lowest SBP/DBP (BPmax) among the tilted measures at 3, 5, 10, and 15 min (average supine BP minus highest orthostatic BPmax). Patients with PD with SH were described as having orthostatic hypertension (OHT) if ΔSBPmax and/or ΔDBPmax was ≤−20/10 mmHg. Patients with PD without SH were categorized as OHT when orthostatic BPmax was ≥140/90 mmHg or ΔBPmax was ≤−20/10 mmHg32. Positive ΔSBPmax and/or ΔDBPmax signified a decrease in upright ΔBP, and negative values indicated an increase in orthostatic ΔBP.The orthostatic ΔBP was re-calculated from the highest BP  were classified as BP3min)34. The measurement was only applied to those with PD with OH as the original description.Orthostatic HR at three minutes was selected to estimate the change in HR (ΔHR) from average supine HR, and its values were utilized to calculate ΔHR/ΔSBP at three minutes , and data were collected at 30 minutes (early) and 120 min (delayed) after injection of 111 MBq of 123I-MIBG. A static image was obtained with a 128 × 128 matrix. Regions of interest (ROIs) were drawn manually around the heart and mediastinum. Tracer uptake was measured within each ROI to calculate the heart-to-mediastinum (H/M) ratio for early and delayed time points. The washout rate (WR) was calculated as [(early H/M ratio – late H/M ratio)/early H/M ratio] × 100. Reference values for normal H/M ratio were 1.70 for early and 1.78 for delayed ratios36.Patients were evaluated initially at the time of diagnosis, and the changes of tracer uptake were re-assessed after 30.0 ± 9.6 months  for Mac, a graphical user interface for R. Analysis of variance (ANOVA) or Kruskal-Wallis test was performed for continuous or ordinal variables when appropriate. Categorical variables were examined by Fisher’s exact test. Cochran-Armitage test and Jonckheere-Terpstra test were executed in R . Multiple comparisons were adjusted as appropriate. Statistical significance was defined as a two-tailed p-value < 0.05.Statistical analyses were conducted with jamovi software (version 2.3.18; retrieved from Further information on research design is available in the Supplemental informationReporting Summary


Fmr1 and Shank3 mutants) forms of ASD at late infancy and early adulthood. Using receptor autoradiography for ionotropic excitatory (AMPA and NMDA) and inhibitory (GABAA) receptors, we mapped the receptor binding densities in brain regions known to be associated with ASD such as prefrontal cortex, dorsal and ventral striatum, dorsal hippocampus, and cerebellum. The individual mouse lines investigated show specific alterations in excitatory ionotropic receptor density, which might be accounted as specific hallmark of each individual line. Across all the models investigated, we found an increased binding density to GABAA receptors at adulthood in the dorsal hippocampus. Interestingly, reduction in the GABAA receptor binding density was observed in the cerebellum. Altogether, our findings suggest that E/I disbalance individually affects several brain regions in ASD mouse models and that alterations in GABAergic transmission might be accounted as unifying factor.Autism spectrum disorder (ASD) comprises a wide range of neurodevelopment conditions primarily characterized by impaired social interaction and repetitive behavior, accompanied by a variable degree of neuropsychiatric characteristics. Synaptic dysfunction is undertaken as one of the key underlying mechanisms in understanding the pathophysiology of ASD. The excitatory/inhibitory (E/I) hypothesis is one of the most widely held theories for its pathogenesis. Shifts in E/I balance have been proven in several ASD models. In this study, we investigated three mouse lines recapitulating both idiopathic (the BTBR strain) and genetic ( Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition involving altered social communication and presence of repetitive behaviour. Several other co-occurring conditions such as attention deficit hyperactivity disorder, depression, sleep disorder, epilepsy, anxiety, and intellectual disability are often associated with ASD . The preFmr1 knockout (KO) mice are the best characterized model to study fragile X syndrome. This model also shows promising behavioural and physiological features to be used as a validated model for ASD  mice are an inbred strain showing face validity for idiopathic ASD . Fragile for ASD . SH3 andwith ASD .Tight balance between excitatory and inhibitory synaptic transmission at neural circuits is crucial for normal brain development and function. Accordingly, shifts in the excitation/inhibition (E/I) balance have been implicated in the development and maintenance of ASD. In recent years, the theory of E/I imbalance in ASD has gained a lot of attention. It has been postulated that the autistic brain may be overactive because of a ‘signaling imbalance’ with too much excitatory signaling or too little inhibition at synaptic or circuit levels. This may also in part provide an explanation to the high propensity of people with ASD to develop seizures or epilepsy . CumulatA  is one of the main inhibitory receptors at synapses. There are in total 19 known subunits, which differentially combine in heteropentamers. Different subunits and their combinations contribute to the regional and functional diversity of the receptor, being most commonly composed of two alpha, two beta‚ and one gamma subunits , whereas reduced GABAA receptor binding density was observed in the cerebellum (Cer) concomitantly. Quantitative evaluation of GABAA, AMPA, and NMDA ionotropic receptor distribution will thus contribute to develop a better understanding towards underpinning the selective role of these receptors in alterations of E/I balance.Ionotropic receptors are ligand-gated ion channels, made up of multiple subunits. GABAsubunits . NMDA ansubunits . In this+ Itpr tfT/J, stock #002282), C57BL6/J (stock #000664), Fmr1 , and Shank3b  mice were purchased from Jackson laboratories and housed in a pathogen-free facility with 12 h light/dark cycle, food, and water available −/yad libitum. Fmr1 (Fmr1 KO) mice were generated by (Shank3b (B6.129-tm2GfngShank3/J) mice were generated by replacing exons 13–16 with a neomycin resistance cassette (−/yFmr1 (Fmr1 KO), and −/−Shank3b (Shank3b KO) were used as test animals. C57BL6/J mice were used as controls for BTBR mice, wildtype littermates for Fmr1 KO and Shank3b KO. Breeding was approved by the local authorities. Since ASD shows a higher prevalence in male individuals, only male mice were used in the experiments  in the coronal plane with a cryostat microtome . Slices containing Cer were cut similarly in the sagittal plane. The following bregma points were chosen for the analysis: 1.93 mm for the PFC, between 1.53 mm and 0.97 mm for DS and VS, −1.55 mm for the DH. Sagittal sections cut 0.72 mm lateral to the midline were considered for the Cer. For the location of the regions of interest, we referred to Paxinos and Franklin .3]-labeled ligands for AMPA, NMDA and GABAA receptors. Slices containing Cer were incubated only with the [H3]-labeled ligands for GABAA receptor. Indeed, as already shown, [3H]MK-801 yields no signal in the Cer at the concentrations used in this study  for 1 min. After washing in running water for 10 min, the slices were put for 10 s in Eosin Y (Thermo Fisher). Then, the slices were dehydrated in increasing ethanol concentrations (96 and 100%) each for 2 min. Finally, the slices were placed for 3 min in xylol, and cover slipped with Cytoseal XYL (Thermo Fisher). Pictures were scanned at 4× magnification with a Leica microscope , digitized and transferred to the MCID program.A receptors was adapted from the protocols described in . AMPA is an agonist of the homonymous receptor, MK-801, also known as dizocilpine, is an uncompetitive antagonist of the NMDA receptor and Muscimol is an agonist of the GABAA receptor. In the first step, the pre-incubation, endogenous ligands were washed off. In the following main incubation, the tritiated ligands were incubated both in the presence of a competitor, in order to determine the unspecific binding, and without it, in order to assess the total binding. Finally, the slices were rinsed. The slices incubated with [3H]AMPA were additionally dried with a warm air stream for 2 s and afterwards with a cold air stream. Slices incubated with [3H]MK-801 and [3H]Muscimol were dried with a cold air stream. A detailed description of the protocols used is reported in The receptor binding density for AMPA, NMDA, and GABAribed in . The tri3H] plastic standards  were exposed together with the tritium-labeled sections to a [3H]-sensitive film  for 12 ([3H]AMPA and [3H]Muscimol) and 15 ([3H]MK-801) weeks. The autoradiograms and the standards were scanned in equal lighting conditions with the digital CoolSNAP camera  and digitized with the MCID image analysis system . The standards were used to calculate the relationship between the gray values of the autoradiograms and the concentration of radioactivity. Total binding was calculated on the autoradiograms on both hemispheres in the regions of interest after tracing their boundary on the hematoxylin–eosin staining  and Microsoft Excel. Normal distribution of the data was assessed through the D’Agostino-Pearson test. Outliers were screened with the Rout test. Student’s multiple ribed in . p < 0.0A receptors in PFC, DH, DS, VS, and Cer. It was obtained by means of quantitative in vitro receptor autoradiography of 4- and 12 week-old BTBR, Fmr1 KO and Shank3b KO mice. Representative HE-stained sections were used to trace the boundaries of the above-mentioned regions of interest, which were then overlapped on the autoradiograms for AMPA, NMDA, and GABAA receptors prior to analysis  and a similar tendency was observed at 12 weeks  of age. However, no significant alterations were observed in PFC  and DH  at both time points (p = 0.025), but neither in VS (p = 0.102) nor in both striatal subregions at adulthood . There was no change in AMPA or NMDA receptor binding in PFC  and DH  at both time points (A receptors was not changed in PFC at both 4 weeks (p = 0.092) and 12 weeks (p = 0.158). In DS and Cer a tendency to reduced receptor binding density was found at 4 weeks  but was not observed at adulthood . GABAA receptor binding density in VS showed no change at both time points . Notably, a strong propensity to increased GABAA receptor binding density was found at 4 weeks (p = 0.069), and it became significantly increased at 12 weeks (p = 0.003) of age in DH (A receptors in DH (increased).In BTBR mice, receptor binding density to AMPA receptors was significantly increased at DS and VS at 4 weeks  but not 4 weeks (p = 0.156). In DH we found the opposite, with a tendency to reduced receptor binding density at 12 (p = 0.059) but not at 4 weeks (p = 0.156). DS and VS showed no significant alterations at either time point  . In DS and VS, age dependent alterations are highlighted, with no change in receptor binding profile at 4 weeks  but reduced receptor binding densities at adulthood . DH showed reduced binding densities to NMDA receptors at both time points   but showed significant reduction at 12 weeks (p = 0.049). Similarly, no change was observed in DH at 4 weeks (p = 0.373), whereas it was strongly increased at adulthood (p = 0.025). Binding profile of DS and VS remained unchanged at both time points . Strikingly, binding density to GABAA receptor was significantly reduced in Cer at both 4 (p = 0.0009) and 12 weeks (p = 0.049)  and GABAA binding in Cer (decreased).= 0.632) ,B. Bindi= 0.058) ,D. Regar= 0.049) ,F. ColleA receptor binding density can be observed both in the BTBR and Fmr1 KO mice. At 4 weeks, it was decreased in Cer (strong tendency in BTBR and significant change in Fmr1 KO mice), whereas it was increased in DH at adulthood. This evidence highlights that the ionotropic neurotransmitter receptors analyzed might be discretely altered in different regions.Interesting similarities regarding GABAShank3b KO mice no change was found for AMPA receptor binding density for all regions analyzed, both at 4  and 12 weeks , respectively  but a significant reduction at 12 weeks . However, no significant alterations were observed at both time points in PFC  and DH  , DS , and VS . Finally, no change was observed at 4 weeks in DH (p = 0.852) and Cer (p = 0.852), whereas at 12 weeks the receptor binding density showed strong tendencies to increase in DH (p = 0.068) and decrease in Cer (p = 0.068), respectively  ,D. Conceectively ,F. As alectively , knockdoA receptor binding density at adulthood in the DH was discovered as point of commonality among all the ASD mouse models analyzed in this study. This also leads to a disbalance in the binding to ionotropic excitatory and inhibitory receptors estimated through the calculation of the corresponding binding density-related E/I ratio (A receptor binding density was reduced in Cer both in Shank3b (strong tendency) and Fmr1 KO mice at adulthood.Remarkably, increased GABA/I ratio . MoreoveA) receptors at the synapse by the means of receptor autoradiography in three different ASD mouse models. For the analysis, we chose two developmental time points corresponding to late infancy (4 weeks) and early adulthood (12 weeks). It has indeed been shown that synaptic proteins go through dynamical regulation during postnatal development  and inhibitory ..A) recepA) and metabotropic receptors, signaling pathways, intrinsic neuronal excitability, homeostatic synaptic plasticity, interneurons, and glial cells   balance theory assumes that several psychiatric diseases, among which ASD, are due to a dysregulation of the excitatory and inhibitory factors existing at cellular, synaptic and circuit level, leading to a detrimental overall circuit activity , 16. Seval cells , 46. Notal cells . Moreove . mR12 weeks . At 10 w and Cer . Analysidulthood . In a pr 8 weeks . Both α2l day 22 . The strreceptor , 79, 80.hibition , 81. In erature) , whereased in DH . Subsequsyndrome , 83.SHANK3 account for up to 0.7% of cases of ASD and a multitude of mouse models have been generated so far  and in-vitro on ASD mouse models, among them Shank3b KO mice (via receptor autoradiography)  . Of reledulthood . Discrepstigated , 3F, 4F KO lines , 4F. Thedulthood .A receptor subunits in parts of the PFC and Cer -positive interneurons. Briefly, they are a class of GABAergic cortical and hippocampal interneurons expressing the calcium-binding protein parvalbumin and fine-tuning the E/I balance in the brain . Reportsin-vitro approaches such as patient-derived induced pluripotent stem cells, organoids and assembloids can further pave the way in both modelling neuropsychiatric conditions and testing potential drugs acting on these membrane receptors.Future studies should be directed at investigating the role of the E/I imbalance  or PV-poThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because organ removal from mice for scientific purpose (the brain in this study) does not require approval by an ethics committee in Germany.SC, LN, PL, CJS, and MJS planned the autoradiographic experiments. SC, LN, and PL conducted the autoradiographic experiments and analyzed the data. SC and LN drafted the manuscript. PL, DK-B, CJS, and MJS critically revised and edited the manuscript. All authors contributed to the article and approved the submitted version.LN was supported by an internal grant of the University Medical Center, Mainz (Stufe I). MJS was supported by the German Research Foundation  and the Werner Reichenberger Foundation. DK-B was supported by the Heisenberg program of the DFG (grant KR 5329/1-1).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.


Obesity has been associated with an adverse prognosis and reduced efficacy of endocrine therapy in patients with hormone receptor-positive (HR+) breast cancer (BC). This study determines the prognostic and predictive effect of body mass index (BMI) on the disease-free survival (DFS) of postmenopausal HR+ BC patients.2), overweight (25.0–29.9 kg/m2), or obese (≥30.0 kg/m2). The primary endpoint was DFS, evaluated from randomization  or 3 years after randomization onwards  using multivariable Cox regression analyses. P-values were 2-sided.Patients were identified from the DATA study (NCT00301457), a randomized controlled trial evaluating the efficacy of 6 vs 3 years of anastrozole after 2 to 3 years of adjuvant tamoxifen in postmenopausal women with HR+ BC. Patients were classified as normal weight (BMI: 18.5–24.9 kg/mP-interaction = .009). The effect of extended anastrozole on aDFS was similar in normal weight , overweight , and obese patients  (P-interaction = .24).This study included 678 normal weight, 712 overweight, and 391 obese patients. After a median follow-up of 13.1 years, overweight and obesity were identified as negative prognostic factors for DFS (hazard ratio (HR) = 1.16; 95% confidence interval (CI) = 0.97 to 1.38 and HR = 1.26; 95% CI = 1.03 to 1.54, respectively). The adverse prognostic effect of BMI was observed in women aged younger than 60 years, but not in women aged 60 years or older (In this study among 1781 HR+ BC patients, overweight and obesity were adverse prognostic factors for DFS. BMI did not impact the efficacy of extended anastrozole. One in five women worldwide are estimated to be obese by 2025 . Obese pIn the general population, however, the association between obesity and all-cause mortality tends to differ between younger and older adults . The majOver the years, the impact of BMI on the efficacy of (extended) endocrine therapy has also been studied in postmenopausal women with HR+ BC. Tamoxifen seems to be equally effective across BMI classes . HoweverThe present post hoc study was performed within the framework of the DATA study, a randomized controlled trial that evaluated the efficacy of 6 versus 3 years of anastrozole after 2 to 3 years of adjuvant tamoxifen in postmenopausal women with HR+ BC ,21. The The DATA study (NCT00301457) was an open-label, phase 3, randomized controlled trial in which postmenopausal women with HR+ BC received either 6 or 3 years of anastrozole  after completing 2 to 3 years of adjuvant treatment with tamoxifen without signs of disease recurrence . From 202) were excluded because of the small number of patients.For the current analysis, all patients with a baseline BMI measurement were selected. Underweight patients . Written informed consent was obtained from all patients.2), overweight (BMI: 25.0-29.9 kg/m2), or obese (BMI: ≥30.0 kg/m2). Follow-up was performed by the treating physician every 6 months during the first 6 years after randomization and yearly thereafter. A mammogram was done once a year. Database lock: March 7, 2022.Height and weight were measured by the treating physician or self-reported by the patient at randomization and were used to calculate BMI. We categorized BMI according to the World Health Organization criteria: normal weight . Secondary endpoints were overall survival (OS), BCSM, and other-cause mortality (OCM). The following events ended a period of DFS: BC recurrence, second primary BC, second primary cancer , or death from any cause. A period of OS ended as a result of death from any cause. All BC-related deaths were included in the analysis of BCSM, whereas all non-BC-related deaths were included in the analysis of OCM.Baseline characteristics were compared by BMI class and assigned treatment. The chi-squared test and the Mann-Whitney U test were used to assess differences in categorical or continuous variables. The presence of a trend was evaluated using the Cochran-Mantel-Haenszel test and the Jonckheere-Terpstra test.The prognostic effect of BMI was evaluated irrespective of assigned treatment from date of randomization onwards. The predictive effect of BMI on the efficacy of 6 versus 3 years of anastrozole was assessed from 3 years after randomization onwards . Patients with a DFS event or patients who were lost to follow-up within 3 years after randomization were excluded from the adapted analyses.DFS and OS were examined using Kaplan-Meier survival analyses and Cox regression analyses, when adjusting for potential confounders. BCSM and OCM were determined with competing risk methodology, using the Fine and Gray model when adjusting for potential confounders. Differences between BMI classes and treatment groups were assessed with the log-rank test and the Gray’s test. In the absence of an event, patients were censored at the last follow-up visit in all analyses. Death not related to BC was considered a competing event in the analysis of BCSM. BC-related death was considered a competing event in the analysis of OCM. Missing data of confounders were imputed.Prognostic analyses were stratified by age (<60 versus ≥60 years). The cutoff point of 60 years, ie, 57 years at BC diagnosis, was chosen to distinguish younger postmenopausal women from older postmenopausal women, as some younger patients were premenopausal at BC diagnosis ,23. The P-values were 2-sided and considered statistically significant at a value of .05 or less.All statistical analyses were performed with SPSS (version 25) and Stata (version 17).Overall, 1781 patients were included in the analysis on the prognostic effect of BMI . Of thesAfter a median follow-up period of 13.1 years (interquartile range [IQR]: 12.5-13.9) beyond randomization, 706 patients had developed a DFS event and 484 patients had died. Details about endpoint events per BMI class are presented in P = .10 and adjusted HR = 1.26; 95% CI = 1.03 to 1.54; P = .03, respectively)  in normal weight, 59.5% (95% CI = 55.7% to 63.1%) in overweight, and 52.4% (95% CI = 47.2% to 57.4%) in obese patients . OverweiP = .10) and 1.16  for overweight and obese patients (The 13-year OS rates were 77.6% (95% CI = 74.1% to 80.6%) in normal weight, 71.5% (95% CI = 68.0% to 74.7%) in overweight, and 67.7% (95% CI = 62.5% to 72.2%) in obese patients . When coP = .15) and 1.36  for overweight and obese patients   and was nearly statistically significant for the association between BMI and OS (P-interaction = .07) (P-interaction = .02), but the association between BMI and BCSM was similar in both age groups (P-interaction = .56).Age showed to be a statistically significant effect modifier of the association between BMI and DFS (n = .07) . SpecifiP = .04). All other baseline characteristics were equally distributed between treatment groups.Overall, 1589 patients were included in the analysis on the predictive effect of BMI: 613 (38.6%) normal weight, 628 (39.5%) overweight, and 348 (21.9%) obese patients . SupplemP = .073) for adapted DFS (aDFS) and a HR of 0.93  for adapted OS (aOS), respectively , 6 versus 3 years of anastrozole resulted in a HR of 0.86 , overweight , and obese patients  (P-interaction = .24)  .P = .06), overweight , and obese patients  (P-interaction = .03)  . These rResults of both adapted BCSM (aBCSM) and adapted OCM (aOCM) were comparable to those of aOS in every BMI class .In this study, we evaluated the prognostic and predictive effect of BMI in 1781 postmenopausal women with nonmetastatic HR+ BC from the phase III DATA study. We confirmed the results from previous studies, showing a negative association between obesity and DFS in the overall study population. Interestingly, however, subgroup analyses demonstrated that the negative association between obesity and DFS was statistically significant in patients aged younger than 60 years, but not in patients aged 60 years or older. The effect modification by age is of potential interest.We observed that obesity was associated with a decrease in DFS and OS in younger postmenopausal HR+ BC patients only. However, while obesity was also associated with an increase in OCM in younger patients only, it seemed to increase the risk of BCSM irrespective of age. These findings indicate that different mechanisms might apply to the association between obesity and OCM and the association between obesity and BCSM. Primarily, the increased risk of OCM in younger obese patients may be the result of developing cardiovascular or metabolic diseases at a younger age . In our In our study, we did not observe a reduced efficacy of extended anastrozole therapy in overweight or obese postmenopausal women with HR+ BC. In the analysis of aOS, however, we did observe a potential difference in treatment effects between BMI classes. In fact, 6 versus 3 years of anastrozole was associated with a non–statistically significantly increased risk of death in normal weight patients , whereas it was associated with a non–statistically significantly decreased risk of death in overweight patients . Obviously, as the number of patients and events per subgroup was low, this difference in treatment effects could simply be a chance finding. Alternatively, one might speculate that normal weight patients receiving extended aromatase inhibition are at an increased risk of developing endocrine resistance, as an increased risk of BCSM was also observed. We did not, however, observe a decrease in the DFS of normal weight patients receiving extended aromatase inhibition, although this might also be the result of a decrease in the incidence of second primary cancers in the extended therapy group. In addition, one might speculate that normal weight patients receiving 6 years of anastrozole experience an increased risk of death due to adverse events, ie, cardiovascular events or bone fractures. In a previous report of the DATA study, it was shown that the incidence of cardiovascular events and bone fractures during the first 6 years after randomization did not differ between patients receiving 6 versus 3 years of anastrozole, but the incidence of osteopenia or osteoporosis was higher in the extended therapy group . ConsideThe major strength of our study is the use of data from patients who participated in a randomized controlled trial, in which endpoints were well defined and consistently measured during follow-up. Another strength of our study is the long-term follow-up period of more than 13 years after randomization. Our study also has some limitations. Patients may experience changes in body weight after BC diagnosis . We obtaIn this study among 1781 postmenopausal women with HR+ BC, we have shown that obese patients experienced an increased risk of BCSM irrespective of age. These findings highlight the need for maintaining a healthy BMI in all patients with HR+ BC. In addition, we did not observe a reduced efficacy of extended anastrozole therapy in overweight and obese patients. Therefore, we conclude that (extended) aromatase inhibitor therapy can also be considered in overweight and obese patients with HR+ BC.pkad092_Supplementary_DataClick here for additional data file.