Accumulative evidence shows that an organoid is a more practical and reliable tool in cancer biology research. This study aimed to identify and validate crucial genes involved in non-small cell lung cancer carcinogenesis and development using the transcriptomic analysis of tumor tissues and organoids.Gene set enrichment analysis (GSEA) of tumor tissues, tumor organoids, and normal tissues was performed to reveal the similar and different mechanisms involved in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) carcinogenesis and progression. Differentially expressed gene analysis, prognostic analysis, and gene co-expression network analysis were further used to identify hub genes involved in LUAD and LUSC carcinogenesis and development. Finally, LUAD cell lines and organoids were used to validate these findings.GSEA analysis was performed to reveal the similar mechanisms involved in LUAD and LUSC carcinogenesis and development, such as P53 signaling pathway, base mismatch repair, DNA replication, cAMP signaling pathway and PPAR pathway. However, comparing with LUSC organoids, LUAD organoids showed downregulation of immune-related pathways, inflammation-related pathways, MAPK signaling pathways, and Rap1 signaling pathways, although these pathways were downregulated in LUAD and LUSC tissues by comparing with normal lung tissues. Further gene co-expression network analysis and prognostic analysis indicated CDK1, CCNB2, and CDC25A as the hub tumor-promoting genes in LUAD but not in LUSC, which were further validated in other datasets. Using LUAD cell lines and organoid models, CDK1 and CCNB2 knockdown were found to suppress LUAD proliferation. However, CDC25A knockdown did not inhibit LUAD cell line proliferation but could effectively suppress LUAD organoid growth, indicating that an organoid could be used as an effective tool to study cancer biology in LUAD.The results revealed CDK1, CCNB2, and CDC25A as the hub genes involved in LUAD carcinogenesis and development, which could be used as the potential biomarkers and targets for LUAD.The online version contains supplementary material available at 10.1186/s12967-021-03043-6. Lung cancer is the leading cause of cancer-related death worldwide, with an estimated 1.8 million deaths each year . NSCLC cOrganoids are self-organizing 3D structures grown from stem cells, which can recapitulate the essential aspects of organ structure and function . OrganoiPure lung cancer cell clusters not containing stromal components were obtained by culturing organoids. Therefore, the mechanisms involved in carcinogenesis and tumor progression of lung cancer could be well studied using lung cancer organoid models . Previouhttps://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO) database . The TCGA database contained the data of 525 LUADs with 58 paired normal tissues and 501 LUSCs with 49 paired normal tissues. The GEO project GSE119004 contained the transcriptomic data of two paired LUSC tissues and organoids and six paired LUAD tissues and organoids.The transcriptomic data used in this study was assessed and downloaded from The Cancer Genome Atlas (TCGA) database (https://www.gsea-msigdb.org/gsea/index.jsp). GSEA analysis was performed following the instructions, and the results were displayed by plotting a bubble chart using the “ggplot2” package (Version 3.2.1) in R software (Version 3.5.0).GSEA software and MSigDB gene sets were downloaded from the GSEA database  according to the following criteria: (1) cancer: lung cancer; (2) gene: RAD51, CDK1, CCNB2, CDC25A, and GTSE1; and (3) survival: overall survival (OS).Survival analysis data of patients with lung cancer were searched in the Kaplan–Meier Plotter database  in R software (Version 3.5.0) was used to calculate the Spearman correlation coefficient between two genes. Then, the gene pairs with coefficient > 0.3 and Harvested cultured organoids and cells were lysed in lysis buffer  containing 1% PMSF. The concentrations of proteins of lysates were quantified using a BCA protein assay kit , and these proteins were loaded in each lane. The protein extracts were separated by 10% SDS-PAGE (30 μg/lane) and transferred to a polyvinylidene fluoride membrane. Rabbit polyclonal CDK1 , CCNB2 , and CDC25A  were used at a dilution of 1:1000, and the HRP-conjugated GAPDH monoclonal antibody  was used at 1:5000 dilution. Finally, the protein expression was detected using an electrochemiluminescence (ECL) Western blot detection system.2.The LUAD cell lines A549, PC9, and H2030 were purchased from ATCC . These LUAD cell lines were maintained in the RPMI 1640 medium supplemented with 10% fetal bovine serum  at 37 °C in a humid incubator with 5% CO3 LUAD tissues were separated within 1 h of removal from the patients enrolled in Department of Thoracic Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, and placed in cold Hank’s balanced salt solution with antibiotics . The written informed consents were signed by the three patients. A paired lung normal  and LUAD tissue (LUAD-1), another LUAD tissue (LUAD-2) and a malignant pleural effusion sample of LUAD patient (LUAD-3) were collected from three LUAD patients. These tissues were cut after washing using PBS with antibiotics . Then, these tissues were transferred to the EP tubes, and 1 mL of DMEM with collagenase IV (1.5 mg/mL) and hyaluronidase (20 μg/mL) was added. The tissues were incubated in a water bath at 37 °C, and the EP tubes were vibrated using a vortex. The suspensions were passed through 100-μm cell strainers . The strained cells were centrifuged at 1000 rpm for 5 min and resuspended in 80 μL of DMEM. Further, 80 µL of matrix Matrigel was added and mixed well. The mixture was seeded in a 24-well plate (60–80 µL /well). After incubation in the incubator at 37 °C for 20 min, the LUAD organoid culture medium purchased from OmaStem (OmaStem® Lung Cancer Medium (Human), OM14, Guangzhou, China) was added (800–1000 µL /well). The medium was changed every 5 days. For passage, the medium was removed, 1 mL of TrypLE Express Enzyme  was added to digest LUAD organoids, and the cells were resuspended for organoid passages.Three or four pieces of 1-mm2O2 (3%). After blocking nonspecific binding by incubating in 5% bovine serum albumin for 30 min, the sections were incubated overnight at 4℃ with rabbit anti-CK5/CK6 , anti-CK7 , and anti-TTF1 , followed by incubation with a biotinylated secondary antibody (diluted at 1:500) at 37 °C for 60 min. The slides were stained with diaminobenzidine and counterstained with hematoxylin. Finally, all slides were dehydrated by incubating with alcohol and further immersed in xylene for 5 min. All images were taken using an inverted fluorescence microscope .The LUAD tissues and harvested LUAD organoids were fixed with 10% formalin and embedded in paraffin. Four-micrometer-thick sections were made for hematoxylin–eosin (H&E) staining and immunohistochemistry (IHC). For IHC, the sections were first deparaffinized and hydrated. The slides were then immersed in boiling 10 mmol/L sodium citrate buffer (pH 6.0) and microwaved for antigen removal for 10 min. Endogenous peroxidase activity was eliminated after immersing the slides in H5 cells/well. Twenty-four hours after cell seeding, the cells were transfected with siRNAs using Lipofectamine 2000 reagent  following the manufacturer’s protocols. The sequences of siRNAs targeting CDK1, CCNB2, and CDC25A are listed in Additional file The LUAD cells (A549 and PC9) and LUAD organoids were seeded on six-well plates at a density of 3 × 103 cells/well). The cell proliferation ability was examined using a cell counting kit 8 (CCK-8) assay  after 1–7 days following the manufacturer’s protocol. Finally, the absorbance was measured at 450 nm, and every experiment was performed in triplicate.After transfection for 48 h, the cancer cells were plated in 96-well plates  in R. Forest plots also used to display the results. Using GraphPad Prism software , the results from different experiments were compared using the Student e < 0.05 .n = 58), and paired LUAD organoid and LUAD tissues (n = 6). After the intersection, 18 pathways enriched in LUAD tissues compared with normal tissues were obtained and simultaneously enriched in LUAD organoids compared with LUAD tissues , or between all 501 LUSCs and 49 normal tissues (b) in TCGA database.Additional file 3: Figure S2. H&E staining of LUAD organoid (LUAD-3) and normal organoid .Additional file 4: Figure S3. Effects of CDK1, CCNB2, and CDC25A expression on the growth of LUAD-2 organoids. a CDK1, CCNB2, and CDC25A were knocked down in LUAD-2 organoids by siRNA transfection. The effects of CDK1, CCNB2, and CDC25A knockdown on the growth of LUAD-2 organoids were assessed under a microscope  and using CCK-8 assays (c).


Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection  are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine  production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides  were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.Toxoplasmosis, caused by  Toxoplasma gondii, affects one-third of the global population. This infection is most often not characterized as a lethal disease in humans, but it can be fatal in immunocompromised individuals, such as HIV/AIDS, transplanted, or cancer patients, and pregnant women (fetus). Importantly, there is increasing concern about this infection in immunocompetent individuals owing to the neurotropic nature of this parasite, associated development of mental and behavioral disorders, ocular toxoplasmosis causing focal necrotizing retinochoroiditis, and potential myocarditis or polymyositis development , and lymphocytes. These cells produce inflammatory mediators, such as interleukin (IL)-12, tumor necrosis factor (TNF), and interferon-gamma (IFN-γ), triggering the production of microbicidal/microbiostatic molecules  and indoleamine 2,3-dioxygenase enzyme activity that limits parasite replication, highlighting the central role of MOs in the control of parasite growth and dissemination  venom (TsV), is composed of a mixture rich in proteins, peptides, and minor components, such as salts, hormones, amino acids, and sugars   and at Conselho Nacional de Desenvolvimento Científico e Tecnológico/Conselho de Gestão do Patrimônio Genético .C57Bl/6 female mice (8–10 weeks old) were obtained from the animal care facilities of Universidade Federal de Minas Gerais (UFMG), Minas Gerais, Brazil. This research was conducted according to the Brazilian Guidelines on Animal Work and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The UFMG Animal Ethics Committee (CEUA) approved all experiments and procedures (Permit Number 449/2015). The research activity was registered at ®, Ribeirão Preto, SP, BR), filtered , and stored at -20°C.TsV was extracted from scorpions at the Ezequiel Dias Foundation (FUNED) serpentarium. For assays, lyophilized TsV was resuspended in sterile water for injection (SAMTECg for 20 min. The supernatant was collected and loaded onto a sephadex G-50 (Superfine) column, pre-equilibrated with the aforementioned buffer. Peaks (F1 – F7 fractions) were eluted at a flow rate of 0.5 mL/min based on protein detection at 280 nm, followed by lyophilization and storage at -20°C until use.To obtain F1, F2, F3, F4, F5, F6, and F7 fractions, TsV was separated by a combination of gel filtration, ion-exchange and reverse phase chromatography. Initially, 250 mg of the sample was solubilized in 2 mL of ammonium bicarbonate buffer (0.5 M/pH 7.4) and centrifuged at 5000 × To obtain the subfractions 6A and 6B, the peak corresponding to fraction F6 was subjected to HiTrap-SP HP cation exchange chromatography with a gradient of 0.01 M sodium acetate buffer  and solution B  at flow rate 1mL/min, resulting in 17 peaks detected at 214 nm. Each fractionation peak was subjected to reverse phase chromatography  in a gradient system containing 0.1% v/v trifluoroacetic acid solution in water (Solution A) and 0.1% v/v trifluoroacetic acid solution in acetonitrile (Solution B) to obtain the homogeneous molecules, followed by MALDI-TOF mass spectrometry analysis. To further characterize the molecules (peptides), their partial sequence was elucidated by Edman degradation using a Shimadzu PPS-21A automated protein sequencer.de novo sequencing method) and the following partial amino acid composition (~47%) was found: (KEGYAMDHEGCCFSCFIGPAGF CDGYCCC…); to identify it, a protein BLAST search was performed using the default parameters against the non-redundant (nr) database (via homology modeling using the software package Modeller 9v16 (3CO) N-terminus and C-terminal carboxamide (NH2) were generally named “Pep1” and “Pep2a”; a third peptide sequence, named “Pep2b”, was designed by removing the N-terminal acetyl group of Pep2a.The Sub6-B molecule was sequenced , filtered, and maintained at -20°C until use.The C-terminal amidated peptides were synthesized on a Rink amide resin using the Fmoc (9-fluorenylmethyloxycarbonyl) strategy as described elsewhere . N-termi® fluid), and after 72 h, MOs were harvested from the peritoneal cavity by washing with sterile and cold 1× PBS. The cells were centrifuged , counted, plated (2 x106 cells/well) onto culture plates , placed or not a round coverslip onto bottom of each of the wells, and maintained in an incubator at 37°C and 5% CO2 in the presence of Roswell Park Memorial Institute 1640 culture medium  supplemented (RPMIc) with 5% v/v fetal bovine serum , 1% v/v L-glutamine (Gibco), and 1% v/v penicillin/streptomycin (Gibco) for 3 h, followed by a wash, addition of RPMIc, and overnight incubation for subsequent infection/stimulation. The tachyzoite forms of T. gondii (RH strain) were cultured in the kidney epithelial cell line LLCMK2 (Macaca mulatta– “Rhesus”) in the presence of RPMIc. The parasites were centrifuged twice , counted, and used to infect the MOs (at a 1:1 parasite/host cell ratio), for 2 h, followed by a wash (RPMIc). The uninfected and infected MOs were stimulated or not with TsV  or TsV fractions 6 and 7  or Sub6-A, Sub6-B (100 and 50 μg/mL) or peptides  for 4, 24 or 48 h. Intracellular tachyzoites counting was determined by light microscopy in macrophages/coverslips fixed and stained with hematoxylin/eosin method , and mounted onto microscope slides (triplicates) using Entellan mounting medium . Total 300 cells were counted for each coverslip discriminating between uninfected and infected cells. Moreover, at 24 and 48 h after stimulation, supernatants were collected for subsequent viability assay and nitric oxide and cytokine quantification. For parasite pre-stimulation experiments, the tachyzoites were incubated in the presence of TsV or F6, both at 100 µg/mL, or RPMIc alone for 1 h, followed by washing with RPMIc, and used for MO infection (as described above).Mice were injected with 2 mL of sterile sodium thioglycolate solution at 3% v/v (DifcoTM6 cells/well into 24-well culture plate), and incubated for 3 h . Then, the cells were washed with RPMIc and incubated overnight. Subsequently, the cells were first washed with RPMIc medium either treated with 2% v/v triton X100  (death cell positive control), TsV (100 µg/mL), F6 (100 µg/mL), or F7 (50 µg/mL). At 48 h after incubation, cell viability was performed using the colorimetric method MTT -2,5-diphenyl-2H-tetrazolium bromide) in adherent cells, as previously described  were obtained from the brains of chronically infected mice (35 – 45 dpi). Briefly, C57Bl/6 mice were euthanized, their brains were removed and washed with sterile 1× PBS, macerated, and homogenized with a needle and syringe in sterile 1× PBS (1 mL). This suspension (10 µL) was placed on a glass microscope slide and the number of cysts was determined microscopically. Next, C57Bl/6 female mice, 9–10 weeks old, were intraperitoneally infected with 20 cysts/animal. Treatment with Pep1, Pep2a, or Pep2b  started 8 h after infection and lasted until 7 dpi, with a 24/24 h interval. The animals were monitored throughout the experiment  at 5, 7, and 30 dpi. Blood samples were collected for cytokine analysis in serum (as described above). Additionally, the presence of liver enzymes  was quantified . At 30 dpi, the animals were euthanized and the brains cysts were quantified as described above.For p ≤ 0.05.Statistical analyses were performed using Prism v. 8.2.1  by comparing the means ± SEM from controls to the treated samples using the Student’s t-test and one- or two-way ANOVA with the Bonferroni test. Differences were considered significant when T. gondii-infected MOs stimulated with TsV inhibited the production of NO (T. gondii), at 24 and 48 h after stimulation  and IL-6 (24 h) compared to the uninfected F6-stimulated cells. However, the levels of IL-12 (24 h), TNF (24 and 48 h), and IL-6 (24 and 48h) remained higher than that of the unstimulated infected cells (T. gondii decreased the levels of IL-12 (48h), but not TNF or IL-6 production triggered by F7. Notably, F6 was a better inducer of cytokine production than F7. In summary, our results indicate that F6 and F7, mainly F6, retain the molecules that might participate in the control of T. gondii replication, inducing a pro-inflammatory profile in MOs. Thus, the F6 fraction was fractionated to isolate and characterize the molecules with possible anti-T. gondii activity.Recently, our group isolated and characterized the key protein fractions present in TsV that were involved in the induction of NO in MOs , here named F1 – F7, according to the order of elution from the column . AccordiT. gondii activity in MOs was confirmed by the reduced number of parasites in the stimulated cells compared to the unstimulated cells  with a gradient of 0.01 M sodium acetated buffer (% B), resulting in 17 new chromatography peaks that were listed according to the order of elution (peaks 1–17)  and Pep2a (3COCH-FIRPAGFKYSWP-2NH), whereas an extra third compound Pep2b (FIRPAGFKYSWP-2NH) was also proposed by the N-terminal deacetylation of Pep2a. These three peptides were obtained using chemical synthesis, purified by reversed-phase HPLC, and their purity was determined using mass spectrometry. To characterize the molecule/peptide present in Sub6-B, peak 15 of this subfraction was sequenced and its partial amino acid sequence was elucidated: KEGYAMDHEGCCFSCFIGPAG FCDGYCCC(…). This amino acid sequence was searched against the NCBI nr protein databases using BLASTp. The highest BLASTp hit was Ts2 toxin (found in TsV – UniProt ID: P68410 [SCX2_TITSE]), with 93% similarity     , ion cha and K+) , and Mit and K+)  which cu and K+) . Of greagranules .Hitherto, our data led us to hypothesize that TsV and F6 were able to reduce tachyzoite replication in MOs, activating a pro-inflammatory response. Reinforcing this hypothesis, pre-stimulation of the tachyzoites with TsV or F6 before infection did not alter the replicative capacity of the parasite, excluding a direct action on the parasite. Unlike TsV, the F6 fraction was able to stimulate tachyzoite invasion in MOs. We speculate that this was due to the modifications on the membrane of the host cell, facilitating the parasite invasion and/or by activation of the host receptors that sense parasite factors, favoring parasite internalization .+ channels   , and pre/NaV1.6) . Ts2 was. As a result, some structural or biochemical features of the original peptide might be affected, including (but not restricted to) folding, hydrolysis prevention, protein interactions, and cell permeation or subcellular location  and in vivo (ME49 strain) should also be considered  approved all experiments and procedures (Permit Number 449/2015).DA, ML, AP, MB, and FM conceived experiments. DA, MT, ML, AP, RV, JR, LF, CO, and FM wrote the manuscript. DA, PP, PR, RR, LF, JR, MB, and MC performed experiments. LF, CO, RV, MB, ML, AP, MT, ML, AP, and FM supervised and provided expertise and funding. All authors contributed to the article and approved the submitted version.This work was supported by Pró-Reitoria de Pesquisa/UFMG, Conselho Nacional de Desenvolvimento Científico e Tecnológico  and Fundação de Amparo a Pesquisa de Minas Gerais , Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES: CAPES/COFECUB 57914) (Brazil) and the National Institute for Science and Technology in Dengue and Host-microbial interactions (465425/2014-3). DA, PP, PR, RR, RV, LF, CO, JR, MT, AP, ML, and FM acknowledge grants from CNPq.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.


The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin- Osteosarcoma (OS) is the most frequent bone tumour in children and adolescents, in addition to being the most common aggressive malignancy originating from mesenchymal cells , 2. Alth2O2 through Fenton reaction (Fe2+ + H2O2→Fe3+ + HO·+ OH−) to generate many reactive oxygen species (ROS) and trigger lipid peroxidation (LP) to induce ferroptosis [−, which is composed of two subunits, SLC7A11 (light chain) and SLC3A2 (heavy chain), is closely linked to ferroptosis [− [Ferroptosis is a new form of regulated cell death (RCD) that is mediated by intracellular iron and is quite different from other forms of cell death, such as apoptosis, autophagy, or necrosis . Intraceroptosis . Aberranroptosis , 7. Morproptosis . Additioroptosis . The cysroptosis . SLC7A11roptosis , ferroptroptosis , 16. Somroptosis , via inhtosis [− , 19. TheSignal transducer and activator of transcription 3 (STAT3) belongs to the STAT family and is a vital transcription factor involved in inflammation and tumour progression . ClassicPsoralea corylifolia and displays various functions including anti-inflammatory, lipid-lowering, and cholesterol-reducing effects [Flavonoids have multiple biological functions, especially anticancer effects induced by the prooxidant activity . Amentof effects . A previ effects . Recentl effects . It has  effects . Notably effects . AlthougWe hypothesized that bavachin increases intracellular ferrous iron, ROS, and malondialdehyde (MDA) levels and induces ferroptosis by the downregulation of SLC7A11 through inactivating STAT3 to upregulate P53. We hope that our research will provide promising pharmaceutical targets for the treatment of OS.α (PFT-α), deferoxamine (DFO), ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1) were purchased from Topscience (China). Vitamin E was purchased from Beyotime . Rabbit polyclonal anti-transferrin receptor antibody , rabbit polyclonal anti-divalent metal transporter-1 antibody , rabbit polyclonal anti-ferritin light chain antibody , rabbit polyclonal anti-ferritin heavy chain antibody , rabbit polyclonal anti-SLC7A11 antibody , rabbit polyclonal anti-P53 antibody , rabbit polyclonal anti-STAT3 antibody , rabbit polyclonal anti-p-STAT3 (705) antibody , and rabbit polyclonal anti-glutathione peroxidase-4 antibody  were purchased from Affinity Bioscience (China). HRP goat anti-rabbit IgG was purchased from Earthox (USA).Bavachin was purchased from MCE (China), and pifithrin-2 incubator at 37°C and 5% CO2 and grown in DMEM/high-glucose  supplemented with 10% foetal bovine serum .The human OS cell lines MG63 and HOS were purchased from Procell (China). The cells were cultured in a humidified COAsample − Ablank)/(Acontrol − Ablank) × 100.Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) . According to the protocol, the absorbance values of the samples were read at 450 nm using a fluorescence microplate reader . Subsequently, the cell death ratio was calculated using the formula: cell death ratio (%) =  at room temperature for 24 h. Then, the cells were fixed in 2% osmium tetroxide in PBS at room temperature for 1 h. Next, the samples were dehydrated with a graded series of ethanol solutions (50% to 100%). Subsequently, the samples were embedded in epoxy resin and sectioned into ultrathin sections (60 nm). Finally, the ultrathin sections were steeped with uranyl acetate (1%) and lead citrate (0.1%) and observed and photographed using an H-7500 TEM .MMP was observed using JC-1 assay kit . According to the protocol, the cells were stained with the JC-1 probe and observed under a fluorescence microscope . Green fluorescence indicates a low MMP, whereas red fluorescence indicates a high MMP.Cellular ferrous iron levels were detected using FerroOrange . According to the protocol, the cells were incubated with FerroOrange for 0.5 h. Fluorescence intensity  was assessed using a fluorescence microplate reader . The ferrous iron levels were finally expressed as a ratio to the fluorescence intensity value of the control.μM). The cells were then incubated at 37°C for 20 min. Finally, the fluorescence intensity  was assessed using a fluorescence microplate reader .Cellular ROS levels were measured using a commercial ROS assay kit . According to the protocol, the cells were loaded with a 2,7-dichlorodihydrofluorescein diacetate probe . The harvested cells were crushed by sonication to obtain the supernatant used for measuring the GSH levels in a fluorescence microplate reader . The levels of GSH were expressed as a ratio to the absorbance value of the control cells assessed at 405 nm.MDA levels were assayed using the MDA Assay Kit . First, the cells were lysed using the RIPA Lysis Buffer  with a protease inhibitor and centrifugated at 100,00 g for 10 min at 4°C to obtain the supernatant. The protein concentration of the samples was measured using the BCA Protein Assay Kit . Subsequently, the collected supernatant (0.1 ml) was added to thiobarbituric acid (0.2 ml) and incubated at 100°C for 15 min. Finally, the samples were read at 532 nm using a fluorescence microplate reader . The MDA levels were expressed as a ratio to the absorbance value of the control.μg) was loaded onto an SDS-PAGE (10%–12%) gel and transferred to PVDF membranes. The membranes were blocked with NcmBlot Blocking Buffer  and incubated with the primary antibodies at 4°C overnight. The membranes were subsequently incubated with the secondary antibody at room temperature for 1.5 h. Finally, the bands were developed using a HyperSignal ECL kit .The cells were lysed and homogenized using RIPA Lysis Buffer  with a protease inhibitor. The protein concentration of the samples was measured using the BCA Protein Assay Kit . The proteins were added to the loading buffer  and denatured in boiling water for 5 min. Each protein sample  plasmid was purchased from Vigene Biosciences (China). Briefly, cells were transfected with the plasmids using Lipofectamine 2000 . After 48 h, the cells were subjected to subsequent experiments.n = 3). One-way ANOVA and Tukey's multiple comparisons test were used to analyze the data. All statistical analyses were performed using GraphPad Prism 7 (https://www.graphpad.com/). Statistical significance was set at p < 0.05.Data are presented as means ± SD  for 24 h, 48 h, and 72 h, and then cell viability was measured using the CCK8 assay. As shown in 50) of bavachin at 24 h was determined. The IC50 values for MG63 and HOS cells were 42.32 μM and 34.2 μM, respectively. Therefore, 40 μM of bavachin concentration was selected for the following experiments. Morphological changes in MG63 and HOS cells following bavachin treatment were observed. Light microscopy images showed that the shape of the cells became round, and there was an increase in cell shrinkage and cell death with increasing concentration of bavachin  for 24 h. Compared to the control group, bavachin increased the ferrous iron concentration in a concentration-dependent manner  for 1 h recovered from bavachin-induced  cell death .To evaluate whether bavachin affects ferrous iron accumulation, we assessed the expression of ferrous iron in OS cells treated with bavachin  group and red fluorescence in the control group (μM), Lip-1 (2 μM), and Vit E (150 μM) for 1 h. As shown in To demonstrate bavachin-induced ferroptosis in OS cells, we looked into the morphology of the treated cells. TEM showed that MG63 and HOS cells treated with 40 roptosis . Moreoveol group . BavachiμM, 20 μM, and 40 μM) increased over 24 h . SLC7A11 overexpression attenuates the bavachin-induced accumulation of ROS and MDA  and ferroptosis inhibitors . Moreover, bavachin reduced the MMP and led to mitochondrial shrinkage, increased mitochondrial membrane density, and reduced mitochondrial cristae in OS cells. Furthermore, bavachin elevated intracellular ferrous iron levels by increasing TFRC and DMT1 expression and decreasing FTH and FTL expressions. Bavachin also reduced SLC7A11 and GPX4 expression and promoted ROS and MDA accumulation by downregulating p-STAT3 to upregulate P53 expression .As a new form of RCD, ferroptosis significantly differs from other types of cell death, which is due to characteristics of iron-dependence, GPX4 inhibition, and abnormal LP . Moreove2+ that is involved in ferroptosis progression [Aberrant intracellular iron metabolism, especially ferrous iron overloading, is an initiating factor for ferroptosis. TFRC and DMT1 mediate ferrous iron formation and transport . Previougression .Triggering LP, which is described as a process by which ROS attacks polyunsaturated fatty acids to cause dehydrogenation, is an essential event that drives ferroptosis . Both ROThe SLC7A11/GPX4 axis efficiently protects cells from ferroptoss . SLC7A11α, a P53 inhibitor, these effects were reversed. These findings suggest that bavachin triggers ferroptosis in OS cells by activating P53 to inactivate SLC7A11.P53 is known as a tumour suppressor protein and can inhibit tumour cells by triggering ferroptosis . Jiang eSTAT3 inactivation is also associated with ferroptosis. It has been reported that STAT3 inhibits P53 to facilitate tumour progression by binding to the promoter of p53, which is an important regulator of ferroptoss , 48. QiaTaken together, our study displayed that bavachin triggers ferroptosis in OS cells by increasing intracellular ferrous iron levels and inhibiting the STAT3/P53/SLC7A11 axis. It suggests that bavachin could be a promising pharmaceutical for the treatment of OS.


To describe the epidemiological characteristics of the first 69,382 patients with COVID-19 infection in Oman.A retrospective case series study of patients diagnosed with SARS-CoV-2 infection in Oman from 24 February to 23 July 2020. The data were obtained from the National surveillance COVID-19 network.n = 51,391) were males, 40,859 (59%) were Omani citizens, and 28,523 (41%) were foreign-born individuals. The most common symptoms at presentation were fever 50% , cough 46% , sore throat 46%  and shortness of breath (SOB) 35% . Overall, 8,960 (12.9%) patients required hospitalization with 1189 (13.3%) individuals requiring admission to the intensive care unit (ICU) and mechanical ventilation (MV). Patients hospitalized with COVID-19 infection were mostly Omani nationals and males between 30 and 39 years old (p < 0.001). The mortality rate was 7.7 per 100,000 population (n = 359) with rates of 9.4 (n = 278) and 4.8 (n = 81) deaths per 100,000 population in Omani nationals and foreign-born individuals, respectively. Females hospitalized with COVID-19 had a mean age of 64 (± 18) years versus a mean age of 55 (± 17) years in males. Mean age of patients with COVID-19-related mortality was 58 (± 18) years with significant differences in mean age between females and males 64 (± 18) versus 55 (± 17) years, respectively.The overall mean age of patients was 34 (± 14) years, 74% . Identifying the epidemiological characteristics and outcomes of COVID-19-infected patients is essential for developing targeted intervention strategies and preparing for the resurgence of anticipated second and third waves of this pandemic. Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has spread worldwide infecting more than 129 million individuals and causing more than 2,778,619 deaths  and hospitalized patients [44.4 (± 23.8)] years , Bangladeshi 12% (n = 8905), Pakistani 2% (n = 1191) with 9% from other nationalities , cough 46% , sore throat 46%  and shortness of breath 35%   patients were treated as outpatients, and 8950 (13%) were hospitalized.The most common reported symptoms on notification were fever 50%  of patients had completely recovered, 0.5% (n = 359) had died, while 30.5%  were still active cases (Table p < 0.001)  were between 30 and 79 years of age, and only 4.8% (n = 43) were under 19 years old.Out of the total number of diagnosed patients, 8960  required hospitalization, among whom 1189 13.3%) required admission to the ICU and subsequently required mechanical ventilation (MV) (Table .3% requin = 5961) comprised the largest proportion of admissions, with the majority of admitted patients  being Omani nationals.The overall mean age of hospitalized patients was 44.4 ± 23.8) years of age, while those admitted to ICU were slightly older (52 ± 19.2 years of age) (Table .8 years n = 1336) compared to 165 per 100,000 population (n = 7624) for adults aged 19 years and above. About 83% (n = 7430) were admitted for < 3 days (p < 0.001) , North Al Batinah  and Dhofar  were the three leading governorates with respect to hospitalization of COVID-19 patients.There were substantial variations in hospitalization rates across the country, with some governorates reporting an admission rate as high as 260 to as low as 119 cases per 100,000 population in Dhofar and Musandam Governorates, respectively Table . Muscat n = 359). The COVID-19 mortality rates were 9.4 (n = 278) and 4.8 (n = 81) deaths per 100,000 population in Omani nationals and foreign-born individuals, respectively. The death rate increased by 15%, in March representing a rate of 0.02 deaths per 100,000 population, and this increased to 0.2 deaths per 100,000 population by the end of July. However, the death notification rate remained below 2 cases per million population from March to July 2020.The mortality rate up to 23 July was 7.7 per 100,000 population (p < 0.001). Mortality from COVID-19 was higher among Omani nationals 58% (n = 206), with the Governorate of Muscat accounting for 51% (n = 185) of the total deaths  years, with significant differences in the mean age between females and males, 64 (± 18) versus 55 (± 17) years, respectively   years. This has been similar to the average age of 36 years that was reported in several studies –16. The Similar to other reports , 14, 18,While the initial reports from Oman indicated a predominance of COVID-19 in foreign-born individuals, this study showed that majority (59%) of the confirmed COVID-19 cases were among Omani nationals. The possible explanation for the lower proportion of cases among the foreign-born population (expatriates) could be the demographic make-up, with most expatriates being younger in age. Additionally, expatriates may be less inclined to go for a test or to hospitals due to fear of victimization or loss of employment or due to lack of health insurance or inadequate information and other cultural barriers [In comparison with all other governates, Muscat Governorate reported the highest rates of COVID-19. The governorate has unique demographic, socioeconomic and community features as it has the largest population with the highest density and many foreign-born individuals, mostly single labourer men living in very crowded houses with poor living conditions .p < 0.001) [Our study supported the finding that men are more likely to become infected by COVID-19, potentially due to sex-based immunological differences , gender < 0.001) .In this case series, 13% of the patients required hospitalization with 13.3% of our hospitalized patients required ICU admission and MV; compared with 4.7% in Saudi Arabia , 14% in Similar to other global studies, 49% of our patients who required MV were above the age of 59. A review conducted among confirmed COVID-19 patients admitted to a hospital in Germany between 26 February and 19 April 2020 showed that 24% of patients between 60 and 69 years of age, 25% of patients between age 70 and 79 years and 12% of patients of age 80 years and above required ICU admission . In anotDuring the study period, Oman experienced a sharp rise in COVID-19-associated deaths . The number of excess deaths reported could reflect an increase in rates of death directly caused by the disease. The mortality rates of COVID-19 were higher than China  , the EU/In this study, we noted significant gender difference in the risk of acquisition of COVID-19 infection among males and females. For reasons that remain unclear, the novel coronavirus appears to present a significantly higher mortality risk in men than in women , 33, 35.In our study, the mean age of patients with COVID-19-related mortality was 58 (± 18) years, with females mean age of 64 (± 18) years and males mean age of 55 (± 17) years. This is similar to findings from a longitudinal cohort from the USA that included 88,747 COVID-19 cases confirmed by PCR between 28 February and 14 May 2020, where patients older than 50 years had a progressively higher mortality compared to patients less than 50 years .To date, this study is the largest study to report the clinical and epidemiological characteristics of laboratory-confirmed COVID-19 patients from Eastern Mediterranean Region (EMR) region. However, the findings are subject to a few limitations. First, because this is a retrospective study, there may be biases related to patient recall, transcription error or missing information that were introduced during a patient encounter. Second, some data on symptoms, comorbidities and risk factors were missing in records from both non-hospitalized and hospitalized patients. Third, the impact of an infection depends not only on the number of individuals infected but also the infection’s transmissibility and the spectrum of clinical severity, and these variables were not collected in this study. Fourth, information on the treatment given to hospitalized patients was not available, which could affect the patient outcomes. Finally, hospitals in the governates had different admission criteria, which limited the number of patients enrolled.To our best of knowledge, this is the largest retrospective case series study describing the epidemiological characteristics of COVID-19 patients in the EMR region. The findings of this study in terms of demographic profile, hospitalization and mortality rates are in keeping with data published from other countries. We found that patients hospitalized with COVID-19 were significantly more likely to be Omani citizens, male and between 30 and 39 years of age. These findings underscore the importance of public health interventions that prevent transmission, mitigate hospital surges and reduce the impact of the second and subsequent waves of COVID-19 infection in this pandemic.


A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA Glioblastoma (GBM), an astrocytoma grade IV, represents the maximal evolution grade of astrocytomas and it is among the most lethal human malignancies ,2. The m1–6) coupled to the Gα proteins: Gαq/11, Gαi/0, Gα12/13, Gαs; LPA can exert an extensive range of physiological effects such as wound healing, proliferation, neurogenesis, angiogenesis and survival, depending on the cellular context [ENPP2) is a 125 kDa enzyme from the family of ectonucleotide pyrophosphatases/phosphodiesterases. This enzyme is secreted to the extracellular space in a constitutively activated form and is the responsible for the main LPA production in many pathologies [The lysophosphatidic acid (LPA) is a small molecule with a phosphate head group and an acyl  chain at the sn-1 (or sn-2) position of the glycerol backbone . It inte context . LPA is  context . Subsequ context ,9. ATX  [Aberrant LPA signaling has been linked to diverse conditions such as cancer ,11; besi and GBM ,13,14,15C (PKCs) ,17 and tC (PKCs) ,19,20.1 receptor, promotes PKCα nuclear translocation, inducing progesterone receptor (PR) phosphorylation at S400 [The role of PKCs in cancer progression is well-known . In GBM, at S400 .VEGF, cyclin D1 and EGFR [In previous work, we have demonstrated that activation of PKCs phosphorylates PR at S400 and that this phosphorylation induces PR transcriptional activity ,25. The and EGFR ; howeverLPAR1, LPAR3 and ENPP2 genes.Therefore, this study aimed to evaluate if LPA induces PKCα/PR interaction and if this association phosphorylates and activates PR, thereby increasing GBM cell migration. Using the TCGA database, we correlated patient survival with mRNA expression of 1/3 receptor antagonist Ki16425  was added 30 min before the LPA treatment when used. Progesterone (P4)  was used to activate PR; selective PR modulator, RU486 , was added 30 min before the P4 treatment when used.Human glioblastoma-derived cell lines U251 and LN229 ) were grown in 10-cm dishes and maintained in DMEM medium , supplemented with 10% fetal bovine serum at 37 °C under a 95% air, 5% CO2 atmosphere. 1-Oleoyl Lysophosphatidic Acid  was used to activate LPA receptors and subsequently PKCα. LPAThe National Institute of Genomic Medicine in Mexico City did proof of cell validation for the U251 cell line and for LN229, we get the ATCC certificate analysis.® kit  according to the manufacturer’s instructions. A total of 4000 cells were plated in 16 well chambers  in DMEM medium with 10% fetal bovine serum for 24 h. Eighteen hours before treatments, the medium was changed for phenol red-free DMEM without fetal bovine serum and cells were incubated at 37 °C under a 95% air and 5% CO2 atmosphere. Afterward, cells were stimulated for 0, 5 and 15 min with LPA 100 nM, washed twice with ice-cold PBS, fixed for 15 min with PBS/Paraformaldehyde 4% and permeabilized with 0.5% Triton X−100 for 30 min. Then, cells were blocked with 40 μL of blocking solution for 1 h at 37 °C in a humidity chamber and incubated overnight at 4 °C with the primary antibodies: monoclonal mouse antibody against PKCα  and polyclonal rabbit antibody against total Progesterone Receptor . To detect the primary antibodies, secondary proximity probes binding rabbit and mouse immunoglobulin  were diluted 1:15 and 1:5 in blocking solution, respectively. The cells were then incubated with the proximity probe solution for 1 h at 37 °C, washed three times in 50 mM Tris pH 7.6, 150 mM NaCl, 0.05% Tween-20 (TBS-T) and incubated with the hybridization solution containing connector oligonucleotides  for 45 min at 37 °C. Samples were washed with TBS-T and subsequently incubated in the ligation solution for 45 min at 37 °C. The ligation solution contained T4 DNA ligase , allowing the ligation of secondary proximity probes and connector oligonucleotides to form a circular DNA strand. Subsequently, the samples were washed in TBS-T and incubated with the amplification solution, containing phi29 DNA polymerase  for the roller cycle amplification for 90 min at 37 °C and washed three times with TBS-T. Finally, the samples were incubated with the detection mix solution  for 1 h at 37 °C, washed twice in SSC-T buffer  and were coverslipped with a fluorescence mounting medium . Fluorescent signals were detected by laser scanning microscopy  and PLA-positive signals were quantified using MetaMorph software. At least 50 nuclei were measured for each experimental condition.PLA experiments were performed using Duolink5 U251 cells were plated per well in six-well plates. At 24 h before treatment, the growth medium was replaced by phenol red-free DMEM supplemented with 10% charcoal-dextran filtered SFB. Then, cells were treated for 24 h with LPA (100 nM), P4 (10 nM), RU 486 (1 µM) and the conjunct treatments LPA + RU486 or P4 + RU486 (same concentrations). After treatment, cells were washed with PBS and TRIzol® LS Reagent  was added to detach cells from the plate. The RNA extraction was performed according to the manufacturer recommendations by the phenol-guanidine isothiocyanate-chloroform method. After extraction, RNA quantity and purity were measured with the NanoDrop 2000 Spectrophotometer . Additionally, optimal RNA integrity was assessed by the electrophoresis of 1 µg of total RNA in a 1% agarose gel in 0.5× TB buffer. cDNA was synthesized from 1 µg of total RNA with the M-MLV Reverse Transcriptase  and oligonucleotides (dT)12–18 Primer  according to the manufacturer’s protocol. A total of 2 µL of such reaction was used to determine the expression of VEGF, EGFR, or the 18S ribosomal gene as expression control by qPCR with the LightCycler® FastStar DNA Master SYBR Green I and the LightCycler 1.5  according to the manufacturer’s protocol. Oligonucleotide sequences were: 5′-CCACACCATCACCATCGACA-3′ forward VEGF primer, 5′-CCAATTCCAAGAGGGACCGT-3′ reverse VEGF primer (amplified fragment of 153 bp); 5′-GCCTTGACTGAGGACAGGCAT-3′ forward EGFR primer, 5′-TGGTAGTGTGGGTCTCTGCT-3′ reverse EGFR primer (amplified fragment of 152 bp); 5′-AGTGAAACTGCAATGGCTC-3′ forward 18S primer, 5′-CTGACCGGGTTGGTTTTGAT-3′ reverse 18S primer (amplified fragment of 167 bp). A reaction without RT was performed as a negative control. The relative gene expression was calculated with the 2−ΔCt [To evaluate the effect of LPA on the gene expression induced by PR, we quantified VEGF, EGFR, TGFβ1 and cyclin D1 expression 24 h after LPA or P4 stimulation. 2 × 10he 2−ΔCt ,31. One-2 atmosphere. After the treatments, cells were fixed for 20 min in 4% paraformaldehyde solution at 37 °C and permeabilized with 100% methanol for 6 min at −4 °C. Next, fixed cells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature and incubated at 4 °C for 24 h with 1µg/mL of rabbit anti-pS400PR  in 0.5% bovine serum albumin in PBS. The samples were rinsed thrice in PBS for 5 min each and incubated in the dark with 0.5 µg/mL anti-mouse Alexa Fluor 488-labeled secondary antibodies  for 45 min. Nuclei were stained with 1 µg/mL Hoechst 33342 solution . The cells were coverslipped with a fluorescence mounting medium . The samples were visualized in a Nikon A1R + STORM confocal microscope.Eight thousand cells per well were plated in Millicell EZ 4-well glass slides . Eighteen hours before treatments, the medium was changed for phenol red-free DMEM without fetal bovine serum and cells were incubated at 37 °C under a 95% air, 5% COSpecific characteristics of the antibodies described in 1/PR signaling pathway affects migration, the scratch-wound assay was used. 300,000 cells were plated with DMEM medium in six-well plates until 90% confluence and the formation of a uniform monolayer were observed. With a 200 mL pipet tip, a scratch per well was made in a previously marked well to allow the identification of 4 separate segments of scratch. To evaluate if blocking LPAAt0h = total area at time 0; AtΔh = total area after “×” hours.The detached cells were washed by aspiration. Cells were incubated with DMEM medium with DNA synthesis inhibitor cytosine β-D-arabinose-furanoside hydrochloride . One hour later, RU486 and Ki16425 were added and the scratch images were taken with an inverted microscope Olympus IX71 , at 4X magnification at 0 and 24 h. The percentage of wound closure from migrating cells in the scratch area was calculated as the mean of four fields using the ImageJ software  and the formula:LPAR1, LPAR3 and ENPP2 (ATX gene) mRNA determination, we used the California University, Santa Cruz platform: UCSC Xena  and the database from TCGA. After applying the filters and deleting the duplicated ones, we obtained 258 samples for grade II (GII), 271 for grade III (GIII) and 166 for grade IV astrocytomas (GIV or GBM). mRNA levels were obtained and plotted on Graph Pad Prism 5.0.For LPAR1, LPAR1 and ENPP2 (ATX gene) mRNA relationship with patient survival, Kaplan–Meier curves were performed using the platform of the California University, Santa Cruz: UCSC Xena  and the database from TCGA for tumor tissue. We obtained the following data from “TCGA low-grade astrocytoma and glioblastoma” and after applying the filters and deleting the duplicated ones: for LPAR1: Grade II: 65 samples, Grade III: 132 samples and Grade IV: 166 samples. For LPAR3: Grade II: 129 samples, Grade III: 134 samples and Grade IV: 96 samples. For ENPP2: Grade II: 254 samples, Grade III: 265 samples and Grade IV: 153 samples. Statistical analysis between high and low expression was performed with a Log-rank (Mantel–Cox) test. A value p < 0.05 was considered statistically significant, as stated in figure legends. All these analyses were performed in GraphPad Prism 8.0 .For the evaluation of PGR and LPAR1 or ENPP2, the gene expression database called “TCGA Glioblastoma (GBM)” was extracted from which the samples that did not have data for both genes were eliminated; 155 samples were used for each graph and the Spearman coefficient was determined using the GraphPad Prism 8.0.2 program.To obtain the correlation between the degree of expression of p = 0.05 or less was considered statistically significant, as stated in figure legends.All data were analyzed and plotted using the GraphPad Prism 5.0 software for Windows XP . Statistical analysis of comparable groups was performed using a one-way ANOVA with a Bonferroni or Tukey’s post-test. A value of 1 receptor activation, a Proximity Ligation Assay (PLA) was performed  targets known to contribute to GBM progression.In previous work, we showed that activating the LPAd 15 min . Since td 15 min , we wantTo evaluate whether LPA receptors activation modulates the expression of PR target genes, we measured their respective mRNAs with RT-qPCR after 24 h of stimulation with 10 nM of P4, 100 nM of LPA, 1 μM of RU486 (selective modulator of PR) and the combination of RU486 with P4 or LPA in U251 cell line .VEGF expression, similarly to P4, after 24 h of stimulation. The mRNA expression was downregulated when stimuli were combined with the selective PR modulator, RU486. However, this modulator acted as a receptor agonist when used alone. EGFR mRNA expression increased after P4 treatment and RU486 blocked this effect; however, no regulation by LPA was observed  caused a 55% closure in U251 and a 30% in LN229, suggesting a slightly additive effect of both inhibitors.1/3 receptors. We have previously demonstrated that U251 cells express the LPA1 receptor at a protein level and LN229 cells express LPA1/LPA3 receptors [Ki16425 is an antagonist for LPA1/PR pathway induced PR targets transcription and migration of GBM cells; therefore, it is probable that this signaling pathway enhances tumor progression. Thus, we questioned whether LPA receptors  and PGR mRNA expression. In this case, there was a positive correlation between these genes’ expression , we investigated if ENPP2 had possible progesterone response elements (PRE) in its promoter  for PR binding that could, further, explain their correlation. We found seven possible binding sites in the ENPP2 promoter with over 90% probability and one with over 95%  [Additionally, we wanted to evaluate whether the LPAe (PIBF) , a knowne (PIBF) .EGFR and VEGF, which are regulated by PR [VEGF expression was upregulated after LPA treatment similarly as P4 did it. In contrast, EGFR expression was only increased when the cells were treated with P4.Other genes involved in GBM cell proliferation and migration are ed by PR . TherefoEGFR and VEGF are differentially regulated. VEGF has PRE consensus sites in addition to AP1 and SP1 sites in its promoter [1 receptor), that in turns phosphorylates PR, modulating VEGF expression by PRE.promoter ,36. In ppromoter ; thus, LEGFR has SP1 sites but does not have canonical PREs. It is known that PR can activate MAPK or c-Src after its stimulation with P4 and that these signaling cascades promote receptor phosphorylations in different sites. Phosphorylations can promote PR binding to SP1 sites , inducing the transcription of genes without PRE in their promoters [EGFR expression.Conversely, romoters ,38,39, wVEFG expression, we observed an increase in this gene transcription when used alone. This may be due to the role of RU486 as a partial agonist [Although the selective modulator RU486 combined with P4 and LPA inhibits  agonist ,41.VEGF transcription [In addition, RU486 is a modulator of the glucocorticoid receptor, which could also induce cription .1/PR reduces cell motility. Interestingly, the inhibitors’ combined effect had a slight additive impact suggesting that other pathways such as MAPK could contribute to cell migration through LPA1 receptor signaling cascade or non-canonical PR pathways. It is worth noting that despite using a complete medium as a source of LPA, which includes growth factors, the effects of inhibiting LPA1/PR or both were marked. Thus, due to the invasiveness nature of GBM, these results suggest that targeting both proteins is potentially beneficial at a therapeutical level. Moreover, the signaling from both proteins impacts more than just migration; although further studies are needed to corroborate our findings, they could also affect invasion, proliferation, survival and other important tumor progression features. It would be essential to point out that U251 cells express the LPA1 receptor at a protein level and LN229 cells express LPA1/LPA3 receptors [1 receptor would be the main receptor involved in PR phosphorylation by PKC and in promoting cell migration.Additionally, the wound healing assay showed that inhibition of LPALPAR1 increases with tumor malignancy. However, it was only linked to survival in astrocytomas grade III. LPAR3 mRNA expression did not vary among grades and ENPP2 (ATX gene) decreased with malignancy; neither mRNAs had a link to patient prognosis. It is worth noting that the TCGA database does not harbor data of mutations of LPA receptor or ATX; therefore, we cannot account for mutants derived from LPAR1, LPAR3 and ENPP2 that could impact patient survival.We were also interested in analyzing if the mRNA of these LPA receptors and autotaxin could function as a predictor of patient survival among astrocytomas. Remarkably, LPAR3 and ENPP2 gene expression, we cannot disregard their protein role in GBM progression. In contrast, it would seem that LPAR1 mRNA levels could be correlated with high protein levels, as demonstrated previously [Additionally, GBM differences between the transcriptome and proteome have been found, suggesting that mRNA levels do not always reflect protein content. Lemeé et al. found a poor correlation between mRNA and protein content of the neurofilament light polypeptide and synapsin 1 in GMB patient samples . Moreoveeviously  and it hFurthermore, in a recent work from our lab, we have shown that PR protein expression increases with tumor malignancy, although mRNA levels seemed to decrease with tumor grade . MoreoveENPP2 regulation by PGR, which could be through a positive feedback loop since we found probable PRE in the ENPP2 promoter; thus, PRpS400 through LPA1 receptor activation may upregulate ATX in GBM. However, the evidence so far is insufficient and requires further investigation.Despite the latter, our data suggested a possible correlation of 1/PR signaling cascade upregulates VEGF expression and induces cell migration contributing to GBM progression.LPA stimulates PKCα-PR interaction inducing receptor phosphorylation at S400, mainly in the cell nucleus. LPALPAR3 and ENPP2 mRNAs is not a biomarker for prognosis in patients’ survival.The expression of


Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water  was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL−1 after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity. Influenza viruses are one of the most relevant etiological agents of acute respiratory infections. Influenza A viruses are highly mutagenic and can infect both animals and humans, causing seasonal epidemics and pandemics, for instance the influenza AH1N1) 2009 pandemic, which led to significant morbidity, mortality rates, and burden to public health N1 2009 p. TherefoSiparuna are used in Brazilian folk medicine −, common for dihydrochalcones + corresponding to stepholidine 9 -pentoside and quercetin-3-O--β-galactopyranoside . In. Inm/z 3entoside  39]. In. Inm/z 352-132]−  and m/z 52-162]− , indicatranoside ,41.® Brazil  were used for the preparation of extracts. CPC fractionation was performed with ultrapure water (18.2 MΩ-cm) prepared with a PURELAB® Classic system , and analytical grade solvents from Tedia® Brazil . LC-HRMS/MS analyses were carried out using analytical grade solvents from Tedia® Brazil , ammonium formate (CAS number: 540-69-2) purchased from Sigma-Aldrich®, formic acid from Vetec Química Fina®, LTDA , and ultrapure water (18.2 MΩ-cm).Analytical grade solvents from TediaSiparuna glycycarpa (Ducke) S.S. Renner & Hausner were collected at Reserva Adolpho Ducke, Manaus, Brazil, in August 2015 and deposited at Instituto Nacional de Pesquisas da Amazônia (INPA) herbarium  under the registration INPA 269732. This research was authorized by the Directing Council of Genetic Heritage  under the authorization A3C04CB. The leaves were dried and ground (101.97 g) and were extracted by percolation with ethanol 96° GL. Part of the crude ethanol extract  was fractionated by liquid–liquid extraction between water–methanol 7:3 (v/v) and organic solvents affording n-hexane (2.80 g), dichloromethane (2.20 g), ethyl acetate (2.33 g), and n-butanol (2.18 g) extracts.Leaves of n-butanol extract, solvent system selection tests, and CPC fractions were performed on silica gel TLC plates . The TLC plates were eluted with the organic phase of n-butanol–acetic acid–water (BAW) , and the results were visualized under ultraviolet light (UV) (Spectroline CL-80 model) at 254 nm and 365 nm.The preliminary analyses of the −1, and a maximum rotation speed of 3000 rpm. The PLC features a fraction collector and a built-in detector that measures the absorbance and sends the chromatogram to the onboard control software, Gilson Glider Prep (GGP) software. The dissolved sample was injected through the sample loop of 10 mL.The fractionation step was performed on a CPC Lab System  that combines a PLC purification system (model 2050) with a CPC 250. The column has a total volume of 250 mL and can be used with a pressure up to 100 bar, flow rate up to 15 mL·minn-butanol–methanol–water was selected, and different volume ratios , , and  were screened. The n-butanol extract (2 mg) was dissolved in test tubes containing 2 mL of each phase of the solvent systems and were shaken to allow compounds to partition between the two phases  was thoroughly equilibrated and the two separated phases were degassed by ultra-sonication for 10 min. The sample solution was prepared dissolving 500 mg of the n-butanol extract in 10 mL of the solvent system .The selected solvent system −1 flow rate in the descending mode until hydrostatic equilibrium, when the sample was injected into the CPC column. Three fractionations of the n-butanol extract were performed with different rotation speeds: 1000 and 1300 rpm and the UV monitoring at different wavelength . In the first and second fractionations 47 tubes of 12 mL were collected (22 tubes with classic elution step and 25 tubes with extrusion step). For the last fractionation, the local minimum function was applied for the fraction collection step. A total of 167 fractions were collected: 94 fractions in the elution step, corresponding to 250 mL mobile phase (1.0 column-volume) and 73 fractions in the extrusion step, corresponding to 302.2 mL (1.2 column-volume) of pumped upper phase.The fractionations were performed with the lower aqueous phase as mobile phase and upper organic phase as stationary phase (descending mode). The CPC column was first entirely filled with the upper organic stationary phase and then the lower aqueous mobile phase was pumped through the column at 6 mL·minS. glycycarpa n-butanol extract were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg·mL−1 (stock solution) for the in vitro tests. Cell culture medium was used to dilute fractions, and n-butanol extract and oseltamivir carboxylate (OST-car) were used to obtain the assays concentrations. For all assays, DMSO did not exceed 1% (v/v).Dried fractions and 2 atmosphere.Madin-Darby Canine Kidney (MDCK) cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin  and maintained at 37 °C in a 5% COCell infection assays were performed with influenza A (IAV) strain (A/Michigan/45/2015), an influenza A(H1N1)pdm09 virus, propagated in MDCK cells. IAV was grown and titrated according to the World Health Organization (WHO) manual for the laboratory diagnosis and virological surveillance of influenza . Viral sThe cell and virus were kindly provided by the Centers for Disease Control and Prevention  and Influenza Reagent Resources (IRR).4 MDCK cells in 96-well plates were treated with 100 µg/mL of n-butanol extract from S. glycycarpa and fractions for 3 days. OST-car at 0.05 µM was used as control. The culture medium was removed and 5 mg/mL of  (MTT) in phosphate-buffered saline (PBS) 1x was added. Cells were incubated for 2 h at 37 °C. A 10% w/v sodium dodecyl sulfate (SDS) solution was added followed by another 2 h-incubation period. The plate was read in a spectrophotometer at 570 nm.Monolayers of 2 × 104 cells/well) were infected with influenza A/H1N1 virus at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C. The inoculum was removed, and cells were treated with 25 or 100 µg/mL of n-butanol extract from S. glycycarpa and fractions for 24 h at 37 °C. OST-car at 0.0125 or 0.05 was used as control. These concentrations are equivalent to EC50 and 4 times the EC50 values for S. glycycarpa n-butanol extract and OST-car [Monolayers of MDCK cells in 96-well plates  was measured in the viruses present in the supernatants harvested from the influenza replication inhibition assays using the NA-Star kit , according to the manufacturer’s instructions. Briefly, culture supernatants were incubated for 30 min with a chemiluminescent neuraminidase substrate at room temperature, and then an accelerator solution was added triggering light emission from the cleaved substrate. The chemiluminescent signal was read in the luminometer.All the antiviral and cytotoxicity tests were performed at least three times, and the results are displayed as the mean ± standard error, calculated using Excel 2010 for Windows software (Microsoft).® BEH C18 ; 1.7 µm particle size; Waters, Dublin, Ireland) heated at 40 °C. The mobile phase consisted of water–5 mM ammonium formate–0.1% formic acid (A) and methanol (B). The gradient conditions were set as B = 50% at 0 min, B = 99% at 10 min, B = 100% at 30 min and remains at 100% until 32 min, B = 50% at 35 min and remains at 50% until 45 min at a flow rate of 0.2 mL·min−1. The source temperature was set at 200 °C, 8 L·min−1 dry gas (nitrogen) flow rate and 4 bar nebulizer gas pressure (nitrogen). Data were acquired in positive and negative ionization modes in the mass range of m/z 50–1200, and collision-induced dissociation (CID) cell energy was set to 20 eV. The chromatograms and mass spectra were processed in Bruker Daltonics ESI Compass Data Analysis software version 5.0 SR1 .Analysis of the CPC fractions were performed on an ultra-fast liquid chromatography (UFLC) apparatus (Shimadzu), coupled to a MicrOTOF-QIII-MS  mass spectrometer with ESI ionization source. The samples (0.5 mg) were dissolved in 2 mL of methanol, and 3 µL was injected on a column Acquity UPLCS. glycycarpa n-butanol extract and its bioactive fractions were analyzed and organized in molecular networks by using the GNPS platform  [https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=daf5b5b0355e4862b3f163d3d62052cc, accessed on 21 October 2021, and https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=9dc29a10767344f5902b709fdb440722, accessed on 21 October 2021.Product ion spectra arising from the LC-HRMS/MS analysis of the er 2021) . The MS/er 2021)  and thenS. glycycarpa n-butanol extract by using a solvent system that failed stationary phase retention on HSCCC, therefore acquiring bioactive fractions. With the use of a local minimum function tool, it was possible to improve resolution during chromatographic separation. Different compounds were annotated through dereplication tools, while changing the mobile phase nature made it possible to obtain a distinct chemical profile of bioactive fractions composition. Furthermore, different compounds such as flavonoids with galloyl units and O-triglycosylated flavonoid were annotated in fractions with cell viability values below 50%. Benzylisoquinolinic and aporphine alkaloids were annotated in the most bioactive fractions, whereas tetrahydroprotoberberines were at the least active ones. Moreover, glycosylated flavonoids derived from quercetin and kaempferol and dihydrochalcones were also annotated in one of the most active fractions, suggesting this group of compounds, along with the described alkaloids, were responsible for antiviral activity. Compound isolation is the aim of future research in order to assay their individual antiviral potential.The search for new anti-influenza agents is essential for public health. In this study, the application of CPC enabled the bioassay-guided fractionation of


In total, 25 aroma compounds were identified and used to test 33 flavoured commercial products to evaluate the usefulness of the IsoVoc database for fruit flavour authenticity studies. The results revealed the possible falsification for several fruit aroma compounds.Flavour is an important quality trait of food and beverages. As the demand for natural aromas increases and the cost of raw materials go up, so does the potential for economically motivated adulteration. In this study, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis of volatile fruit compounds, sampled using headspace-solid phase microextraction (HS-SPME), is used as a tool to differentiate between synthetic and naturally produced volatile aroma compounds (VOCs). The result is an extensive stable isotope database (IsoVoc—Isotope Volatile organic compounds) consisting of 39 authentic flavour compounds with well-defined origin: apple (148), strawberry (33), raspberry (12), pear (9), blueberry (7), and sour cherry (4) samples. Synthetically derived VOCs (48) were also characterised. Comparing isotope ratios of volatile compounds between distillates and fresh apples and strawberries proved the suitability of using fresh samples to create a database covering the natural variability in δ Fruits play a significant role in human nutrition. While their consumption depends on several factors, including colour, texture, appearance, and nutritional value, the flavour, which is a combination of aroma and taste, remains the primary selection criteria . The sam2H/1H, 13C/12C), which is increasingly being applied in food quality control and in determining the authenticity of natural flavourings. Much effort has also gone into developing isotopic methods to detect adulteration of natural products with synthetic compounds. Among them, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) seems to be the most specific and sophisticated method that can discriminate between natural and synthetic aromas based on the isotopic values of selected volatile organic compounds, VOCs [New methodologies are being developed to detect food fraud since the analysis of chiral separation of enantiomers, single components, and total aroma spectra have limited applicability ,6,7. Oneds, VOCs ,23,24,25ds, VOCs  and a pads, VOCs  in whichArticles on determining the authenticity of fruit volatile organic compounds based on GC-C-IRMS are summarised in When assessing authenticity, it is essential to have a comprehensive database of authentic samples ,40,41,42δ13C values of multiple VOCs between distillates and fresh fruit samples (apples and strawberries), (2) compare δ13C values of VOCs between different fruit types to define the natural variability in δ13C values of VOCs, (3) establish a database of δ13C values of authentic natural and synthetic aroma compounds, and (4) identify aroma compounds as markers of fruit flavour authenticity.Obtaining all of the necessary distillates of various fruit types to build a database requires extensive resources. However, this task could be significantly reduced by directly analysing the aroma of fresh fruits. Therefore, the present study deals with constructing a stable isotope database (IsoVoc) based on HS-SPME-GC-C-IRMS data to recognise the authenticity of fruit aroma used in food products. Further, the possibility to use fresh fruits instead of distillates for constructing the authentic database on aroma compounds was also investigated. Thus, the specific objectives were to (1) compare Fresh apples (n = 86), strawberries (n = 17), raspberries (n = 4), blueberries (n = 3) and pears (n = 2) harvested in Slovenia were obtained from the Agricultural institute of Slovenia and from local food producers, between 2016 and 2019. Additional fresh apples were obtained from the Czech Republic (n = 4), Italy (n = 2), Japan (n = 2), Poland (n = 2), and Slovakia (n = 3). Samples of aroma volatiles, recovered in the water phase (used in text as distillates) in apples (n = 43) and strawberries (n = 17), were prepared by steam distillation at the Biotechnical Faculty, University of Ljubljana. Additional distillates of apples (n = 6), strawberries (n = 2), blueberries (n = 4), raspberries (n = 8), sour cherries (n = 4) and pears (n = 7) with known origin were obtained from a commercial flavour supplier.1, ethyl acetate (n = 2); 2, ethyl butyrate (n = 2); 3, ethyl 2-methyl butyrate (n = 2); 4, butyl acetate (n = 1); 5, hexanal (n = 3); 6, 2-methyl butyl acetate (n = 2); 7, 1-butanol (n = 1); 8, amyl acetate (n = 1); 9, butyl butyrate (n = 1); 10, (E)-2-hexenal (n = 3); 11, hexyl acetate (n = 1); 12, (E)-2-hexen-1-ol, acetate (n = 2); 13, 1-hexanol (n = 1); 14, (E)-2-hexen-1-ol, (n = 3); 15, benzaldehyde (n = 2); 16, hexyl 2-methyl butyrate (n = 1); 17, methyl butyrate (n = 1); 18, hexyl butyrate (n = 1); 19, propyl acetate (n = 1); 20, methyl acetate (n = 1); 21, 2-heptanone (n = 1); 22, 1-octanol (n = 2); 23, linalool (n = 1); 24, isopropyl butyrate (n = 1); 25, propyl propionate (n = 1); 26, hexanoic acid (n = 1); 27, isoamyl acetate (n = 3); 28, methyl hexanoate (n = 2); 29, acetic acid (n = 1); 30, E-nerolidol (n = 1) and 31, ethyl hexanoate (n = 2).Pure synthetically derived aroma compounds were obtained from Sigma Aldrich and from a commercial flavour supplier and include (number in the brackets present the number of obtained samples): Fruit samples were mashed and filtered to obtain fresh juice immediately before analysis. Volatile organic components were extracted from both juice, and the distillates using a Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) SPME fibre (50/30 µm thickness) purchased from Sigma-Aldrich . The SPME fibre was previously conditioned according to the supplier recommendation. The analysis of the VOCs was performed by sampling the headspace of a 10 mL SPME vial (with silicone/PTFE septa) containing 1 mL of sample. The equilibration time was 10 min at 30 °C, followed by extraction for 20 min at the same temperature. The analytes were then desorbed at 250 °C for 1 min. A working standard solution was prepared by diluting 1 µL of each synthetically derived aroma compound (samples 2 to 15) in 20 mL of water, which was then stored at 4 °C before use. All analyses were performed within three months.13C/12C ratios were determined using a Vario PYRO Cube analyser  couple to an IsoPrime 100 isotope ratio mass spectrometer, IRMS . The accuracy and precision of measurements were controlled using internal working standards: absolute ethanol (δ13C = −27.38 ± 0.09‰) and a rum distillate (δ13C = −13.81 ± 0.09‰) previously calibrated against the certified reference material BCR-656 wine alcohol  from the Institute for Reference Materials and Measurements—IRMM, Belgium).The δ-notation in per mil (‰) relative to the VPDB international standard as follows:13C/12C ratios in the sample and standard, respectively. The precision of the measurements was ± 0.1‰.Carbon isotope ratios are expressed in the −1 (held 1 min), then to 100 °C at 7 °C min−1, to 180 °C at 10 °C min−1 and finally to 200 °C at 15 °C min−1 (held 1 min). The carrier gas was helium at a constant flow of 1.5 mL min−1. The injection was performed at 250 °C in the split mode (1:10), using a Straight Ultra Inert Liner for SPME . The ion source was set to 230 °C and the interface temperature to 250 °C, with a scan range of 30 to 400 m/z. The system was controlled using the ChemStation software . The identification procedure was performed by comparing retention times and mass spectra with the NIST 14 Mass Spectral Library  and pure standards.VOCs were identified using a 7890B & 5977A Series GC/MSD , where chromatographic separation was achieved on a VF-WAXms capillary column . The overall temperature program was as follows: 40 °C (1 min) to 60 °C at 5 °C min−1. Injections were performed at 250 °C in splitless mode. The oxidation reactor (Cu/O) in the 6890N GC/C system was set to 900 °C.Method optimisation and validation are described in detail in Strojnik et al. . Briefly13C/12C ratio was first determined in pure standards and compared with the values obtained by EA-IRMS. Next, the 13C/12C ratios were determined in the distillates and fruit samples. The multiple-point linear method was used for data normalisation [Before each measurement, stability and linearity were checked. Acceptable values were < 0.03‰. Reproducibility and accuracy were determined routinely using the working standard. The lisation . The repMetadata  and isotopic values were imported into Excel. Then, data visualisation and data analysis were performed in RStudio. A Mann-Whitney U test for non-normally distributed data was then used to reveal any statistically significant differences. In this case, principal component analysis (PCA) could not be applied since the dataset included many missing values of individual variables.The primary purpose of creating the IsoVoc stable isotope database for food flavour authentication within the present study is its applicability and usefulness for industrial partners to determine the authenticity of raw ingredients. For this reason, the database must be as broad as possible to cover the widest variety of flavoured fruit products. Therefore, in establishing the IsoVoc database, the following steps were included: selecting authentic reference samples, database creation and authenticity assessment of commercial samples.δ13C values of specific VOCs present in distillates and fresh fruit samples of apple and strawberry. Only aroma compounds that occur in at least five samples per group (fruit samples and distillates), wherein the compound must be present in both groups, were accepted for further data analysis. In this way, we identified 15 characteristic aroma compounds for apple and 11 aroma compounds for strawberry.To address the usefulness of fresh fruits instead of distillates for database creation, we compared the δ13C values ranged between −43.7‰ (acetone in fruit) and −24.1‰ (methyl hexanoate in fruit). A Mann-Whitney U test revealed statistically significant differences in median isotope values for (E)-2-hexanal , amyl acetate , butyl acetate , butyl propionate , hexanal , hexyl 2-methyl butyrate  and hexyl acetate  between fruit samples and distillates respectively.We included 49 recovery aromas and 99 samples of fresh apple juice . For theδ13C values ranged between −47.0‰ ((E)-2-hexenal in fruit) and −26.0‰ (2-methylbutyl acetate in distillate). In this case, the Mann-Whitney U test revealed statistically significant differences for (E)-2-hexanal, , acetone  and ethyl butyrate  between fruit samples and distillates, respectively.For strawberry, 17 distillates and 17 fresh strawberry samples were analysed . For theδ13C values for seven investigated VOCs in apple and three in strawberry. Although Ells et al. [Data analysis also revealed several outliers in apple and strawberry that were not a result of analytical problems or random errors but are likely due to differences in variety, stage of maturity, and geographical origin, although further investigation is required. The outliers were not removed from the database. It is also necessary to consider natural isotopic variation caused by, e.g., geographical location, variety, temporal and seasonal variation, and processing to make the database more robust. Further, before data analysis it is important to understand if various technological processes such as distillation cause any isotope fractionation in isotopic values of aroma compounds in specific sample type. When comparing distillates with the freshly prepared fruit juice, we observed a difference in s et al.  did not δ13C values are within the min and max value of the distillates, this is possible, as in the case of 1-hexanol, (E)-2-hexen-1-ol, 2-methylbutyl acetate, ethyl butyrate, and propyl acetate for apples and 1-hexanol, ethyl butyrate, hexyl acetate, methyl butyrate, and methyl hexanoate for strawberries. That means that an authenticity assessment will not be influenced by this effect. However, if we want to cover more fruit VOCs, we need to include both distillates and fruit samples in the database.Importantly, the following question arises: can fresh fruit samples substitute distillates in the database? For all aroma compounds where the δ13C values of 1-hexanol, (E)-2-hexen-1-ol, (E)-2-hexenal, benzaldehyde, butyl acetate, ethyl acetate, ethyl butyrate, ethyl hexanoate, hexanal, and hexyl acetate. We also found that apples and strawberries account for most of the most variability in the natural range of δ13C values and contain the highest number of VOCs, which makes them the most appropriate fruits for database creation.Next, for apple and strawberry, distillates and fruits were first combined into a single group . Finallyδ13C values for authentic natural samples and not for the synthetic samples, we used the Mann-Whitney U test rather than principal component analysis, PCA. We also excluded butyl propionate, isobutyl acetate, estragole, acetone, (Z)-2-hexen-1-ol, acetate, and isopropyl butyrate from δ13C values reported in the literature. However, the δ13C values for authentic compounds determined in our study are presented, since they could be of a general interest to other researchers and are as follows:: butyl propionate , isobutyl acetate , estragole  in apple, and acetone , (Z)-2-hexen-1-ol, acetate  and isopropyl butyrate  in strawberry.The number of unique VOCs present in at least five samples was 39 . Not all13Cδ values. Most data are in good agreement with literature data. For example, Elss et al. [δ13C values for trans-2-hexenal ranging from −39.1 to −31.5‰, 1-hexanol ranging from −42.5 to −38.4‰ and trans-2-hexenol ranging from −42.2 to −36.8‰ in apple aroma.All authentic samples were combined in one range -2-hexen-1-ol, hexanoic acid, hexyl butyrate, isobutyl acetate and linalool) compared with this study. For example, isoamyl acetate δ13C values for bananas are higher, ranging from −29.6 to −27.2‰ [δ13C values in the literature data for γ-decalactone, α-ionone, β-ionone is most likely due to the low number of samples and, consequently, the smaller variation in δ13C values in samples used in our study. The δ13C values for γ-decalactone and δ-decalactone in prunus fruits such as peaches, apricots, and nectarines ranged from −38.4 to −34.0‰ [δ13C values of γ-decalactone ranged from −31 to −28‰ [δ13C values for γ-decalactone in organically produced strawberries range from −29.7 to −28.2‰ and cannot be distinguished from other natural strawberry samples. Raspberry has also been subjected to isotope analysis [δ13C values for α-ionone, β-ionone, and α-ionol in these samples ranged from −36.6 to −30.3‰, which shows that it is possible to differentiate between natural and synthetic derived compounds. Although compounds appearing in peach and raspberry are well researched, many more samples are needed to represent better the actual variation in δ13C values for those compounds.However, for certain VOCs, the authentic range of o −27.2‰  comparedo −34.0‰  and wereδ13C values. In addition, having only natural aroma compounds in the database is likely to result in samples being misclassified. Different market brands should also be analysed to increase the range of δ13C values observed in synthetic samples.A database should not contain only the isotopic values of authentic natural samples. For example, we observed several overlaps in the isotopic values of synthetic samples, which meant that it was not possible to differentiate between natural and synthetic samples, mainly (E)-2-hexanal, 2-methylbutyl acetate, amyl acetate, E-nerolidol, ethyl acetate, ethyl hexanoate, hexyl acetate, and linalool Despite this overlap, the method could successfully separate 25 of the 33 VOCs for which we have data for the synthetic compounds .δ13C values of individual compounds, estimating the average amount of synthetic compound in the sample is possible, as shown by Strojnik et al. [δ13C distribution is described by the min, max, and median value. Thus, the δ13C value of a mixture (mix) of two compounds, both natural (nat) and synthetic (syn), can be expressed with the following relationship:x corresponds to the added synthetic compound in the mixture, and in the case where x = 0.5, both components are present in equal amounts. Assuming an extensive dataset of samples that is representative of the population, we can use 13δC values in natural compounds  to estimate the lowest value x that can be detected for compounds in those regions where natural and synthetic samples are separated. Based on isotopic mass balance using k et al. . Here weδ13C values is higher than the natural range, we use the following expression:When the range of synthetic δ13Cnat value of −31.3‰ and max value δ13Csyn value is −25.7‰, then x = 0.59, meaning that if a mixed sample contains more than 59% of a synthetic fraction, it is likely (with 50% chance) that adulteration will be suspected since the shift in the δ13C values will be significant . If the calculation is made using the minimum measured values of δ13Cnat = δ13Cnat min and δ13Csyn = δ13Csyn min, then x = 0.79, meaning that if a mixed sample contains more than 79% of a synthetic fraction, the measured δ13C value will fall out of natural sample range.For example, we chose benzaldehyde with a median δ13C values is lower than natural ones, the corresponding fraction x of a synthetic sample is calculated by replacing the maximum values of the measured ranges with the minimum values and vice versa. The detectable fractions of synthetic compounds for different VOCs are presented in If the range of synthetic The necessary synthetic fraction of selected aroma compounds lies between 5% and 83% for a 50% detection threshold and between 19% and 100% for a 100% detection threshold. Therefore, the method is the most sensitive for detecting adulteration with β-ionone, acetic acid, α-ionone, and 1-butanol and less sensitive at detecting adulteration of ethyl 2-methyl butyrate and hexanal. However, evaluating their usefulness will require a study on how adding different amounts of synthetic aroma compounds to a sample with a weak intrinsic aroma can affect the odour.δ13C values towards the synthetic range for 2-heptanone, benzaldehyde, butyl acetate, ethyl butyrate, hexanal, hexanoic acid, isoamyl acetate, methyl acetate, methyl hexanoate, and propyl acetate suggest the possible falsification of specific fruit VOCs despite being labelled as natural fruit extracts  and natural range (−28.0‰), we can estimate that the minimum amount of synthetic fraction is 59%, but it can range up to 95%.We can also estimate the amount of synthetic fraction x present in the commercial fruit distillates by using the measured δ13C signature similar to that of a plant [13C and 2H) of VOCs would be needed to prove their natural authenticity.It is impossible to distinguish certain synthetic (petroleum-based) VOCs from natural ones since they can have a  a plant , which wδ13C values for 39 aroma compounds present in fruit and distillate samples  and 31 VOC of synthetic origin were established. This study proves that samples of apple and strawberry are sufficient for database construction regarding the variability in 13Cδ values. The data are comparable with literature data, and despite some overlap between the natural and synthetic range of values, the method allows the successful separation of 25 of the 33 target VOCs. When testing commercial fruit distillates, the results show possible falsification for several fruit aroma compounds. However, extensive testing of flavoured products on the market is necessary to determine the extent of adulteration and evaluate the usefulness of IsoVoc for different flavoured products. Moreover, the development of an HS-SPME-GC-IRMS method for 2H/1H ratio determination of fruit VOCs is required to upgrade the existing database, which could be more successfully used in fruit flavour authenticity studies.The falsification of natural flavours by either dilution, mixing with synthetic compounds, or false declaration of origin of natural ingredients creates a demand to control their authenticity. HS-SPME-GC-C-IRMS analysis of volatile fruit compounds is a valuable tool for differentiating between synthetic and naturally occurring aromas and, together with a database containing flavour compounds with well-defined origins, can be used for authenticity assessment. However, selecting suitable reference samples that need to be analysed to create such a database is critical. When comparing distillates with freshly prepared fruit juice, differences in isotope values in some of the investigated VOCs in apple and strawberry samples is observed. We needed to include both distillates and raw fruit samples to obtain maximal natural stable isotope variability of selected VOCs. A database of