HSF20 (YZ1)-overexpressed fruits were sensitive to cold and heat conditions but CBF/NF-Y (YZ9)-overexpressed fruits promoted coloring under cold treatment. This study clarified the effect of postharvest cooling and heat treatments on quality and transcriptional mechanism of strawberries fruits. Moreover, these results provided an experimental basis for further research on improving the quality of strawberry berries during postharvest periods.Temperature is one of the most important factors regarding fruit postharvest, however its effects in the strawberry fruits quality in postharvest remains to be evaluated. In this study, the effects of cold and heat storage temperature on fruit quality of ‘Benihoppe’ strawberry were performed. The results showed that different temperatures could affect the metabolism of hormone, anthocyanin, reactive oxygen species (ROS), and transcription level of responsive factors. The synthesis of terpenoids, amino acids, and phenylpropanoids in strawberries were also changed under different temperatures, which finally changed the quality characteristics of the fruit. We found  Fragaria ananassa Duch.) as a fruit appreciated in the food and drug industry is noteworthy for its attractive color, unique flavor, and nutritional benefit  were collected from the vineyard of Nanjing Agricultural University  at April in Nanjing, Jiangsu Province, China. All selected fruits were mature with uniform fruit size and intact shape, grown in a greenhouse under standard cultivation conditions  during spring seasons. The harvested strawberries (28 days after anthesis) were placed in a transparent airtight box and treated with different temperatures including room temperature , low temperature , and high temperature , with a relative humidity of 90% in a constant temperature incubator. There are three repetitions for each treatment, and twelve fruits for each repetition. The samples were randomly collected after 7 days, and separated into two groups including seeds (at room temperature storage (AS), high temperature storage (BS), and low temperature storage (CS)) and fruits (berries under room temperature storage (DF), high temperature storage (EF), and low temperature storage (FF)) , the plasmids were subsequently transfected with Agrobacterium (strain EHA105) and used for the infection of the strawberries during their large green fruit period. For control (CK), samples were infected with the pCAMBIA1302 vector alone. All treatments were performed with three biological replicates, and ten fruits for each repetition. The transformed strawberries were placed in incubators with 4°C, 25°C, 37°C. The samples were collected after 5 days, while the skin and seeds were removed, and the remaining fruit were used for subsequent analysis.Using screening of transcriptome data, we selected six genes in response to temperature changes including: Hsp20/alpha crystallin family , Universal stress protein family , Senescence regulator , EF-hand domain pair , Histone-like transcription factor (CBF/NF-Y) and archaeal histone , and Protein phosphatase 2C  Table S1The anthocyanin content was determined using spectrophotometry according to the method described by −1; mass spectrometry detection range, 29–540 m·z−1; carrier gas, helium; flow rate, 1.0 mL min−1. The column temperature was programmed as follows: the initial temperature was set at 50°C for 6 min and then increased to 250°C at 6°C min−1, which was held for 3 min. The chromatographic and spectral data were evaluated using the TraceFinder software .The aroma components were determined using gas chromatography-mass spectrometry (GC-MS) . Samples® 1st Strand cDNA Synthesis SuperMix for qPCR . The qRT-PCR reactions consisted of 5 μL SYBR Premix Ex Taq™ , 0.3 μL of each primer (10 μM), 2 μL cDNA, and 2.4 μL RNase-free water in a total volume of 10 μL. The reaction was performed using a LightCycler 1.5 instrument , with the preliminary step at 95°C for 30 s followed by 35 cycles at 95°C for 5 s and 58°C for 35 s. The relative gene expression was calculated using the 2–△Ct method  were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexing. The samples were incubated on ice for 5 min and then were centrifuged at 15000 rpm, 4°C for 5 min. A some of supernatant was diluted to final concentration containing 60% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube with 0.22 μm filter and then were centrifuged at 15000 g, 4°C for 10 min. Finally, the filtrate was injected into the LC-MS/MS system analysis.LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto an Hyperil Gold column  using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A  and eluent B (Methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; 2% B, 16 min. Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 35 arb and aux gas flow rate of 10 arb .https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) database to obtained the accurate qualitative results. The peak area was used for quantitative analysis. The quality control (QC) sample was set to evaluate the system stability during the experiment, and the blank sample was used to remove background ions. The statistical software R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6) were used for statistical analysis. The screening of the differentially accumulated metabolites between different samples mainly dependent on VIP (Variable Importance in the Projection), FC (Fold Change) and P-value. In this experiment, the threshold was set as VIP > 1.0, FC > 1.5 or FC< 0.667 and P value< 0.05  for the processing of the raw data files generated by UHPLC-MS/MS and performed peak alignment, peak selection, and quantification for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 minutes; actual mass tolerance, 5ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity,100000. After that, peak intensities were normalized to the total spectral intensity. The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud  SPSS statistics 17.0 , TBtools v1.072 , and OriTo determine the molecular mechanism of temperature on strawberry, we performed RNA-Seq on seeds (S) and fruits (F) under three different temperatures  Table S3According to the PCA analysis of transcriptome data, we observed a strong correlation between cold (4°C) and RT (25°C) treatments in seeds (AS/CS) and fruits (DF/FF) than heat (37°C) and RT treatments in both . These genes were significantly enriched in biological processes (BP), cellular components (CC), and molecular functions (MF). The three most abundant of BP are included protein localization, establishment of protein localization, and cellular catabolic process. Among the 30 GOs that were significantly enriched, the number of genes enriched in each GO in DF was more than that in EF except for phosphorelay sensor kinase activity. Compared to RT-treated fruits, the cold-treated fruits (FFvsDF) had 10,025 and 10,136 up- and down-regulated genes, respectively. These genes were significantly enriched in the BP and MF, which means cold does not bring significant changes in CC. The BP with the largest number of enriched genes was the single-organism carbohydrate metabolic process, and the MF is oxidoreductase activity, acting on CH-OH group of donors. Similarly, the number of enriched genes in each GO belonged to DF was more than that in FF S2.Compared to RT treatment, heat-treated seeds (BSvsAS) had 8,517 and 9,789 up- and down-regulated genes, respectively. These genes were significantly enriched in BP, CC, and MF. Single-organism carbohydrate metabolic process and nucleobase-containing small molecule metabolic process were the two most significant GOs related to BP, while active transmembrane transporter activity and GTPase activity were the two most significant GOs related to MF. GOs related to BP and CC showed a higher expression level in heat than that in RT. Only few CC-related genes, such as proton-transporting ATP synthase complex, had higher expression levels in heat-treated seeds than that in RT. While the cold-treated seeds (CSvsAS) had 5,410 and 6,632 up- and down-regulated genes, showing a completely different pattern from heat-treated seeds. The GOs related to CC were only enriched in chromatin, nucleosome, lipid particle, monolayer-surrounded lipid storage body, protein-DNA complex, and DNA packaging complex, and were up-regulated under cold conditions. BP and MF were mainly enriched in the cellular carbohydrate metabolic process, cellular carbohydrate metabolic process, and oxidoreductase activity, acting on CH-OH group of donors S2.Using KEGG pathway analysis, we counted the number of paths for DEGs for ASvsBS, ASvsCS, BSvsCS, DFvsEF, DFvsFF, and EFvsFF, which were 13, 14, 13, 10, 20, 8 pathways , respectively  and cold to RT storage condition (FFvsDF) were enriched to 12 and 13 paths in negative ion modes and 7 and 14 paths in positive ion modes, respectively Table S6To evaluate the relationship between transcriptome and metabolome, we integrated the KEGG pathway enrichment results for DEGs and metabolome Table S72O2. Furthermore, CAT and POD enzymes act as ROS scavenging and expressed in DF vs FF while were down-regulated in DF vs EF.Cell wall modification is one of the important factors in quality control of fruit and seed. On the whole, the up-regulation of cell wall metabolism-related genes under heat condition was higher than cold Table S8Among responsive genes, most HSPs were up-regulated under heat treatment, although HSP20, HSP70, and HSP90 were identified as differentially expressed genes Table S8The above results showed that defensive mechanisms could be different in seeds and fruits of strawberry when were treated with different storage temperatures like cold and heat treatments. In total, according to transcriptome data, heat treatment had a greater impact on the entire defense system of strawberries than cold treatment.YZ4, YZ5, YZ9, and YZ10 promoted coloring of fruits at RT but inhibited it under cold and heat treatments. Meanwhile, transformation of fruits with YZ8 led to the promotion of strawberry fruit coloring at cold treatment but inhibited it under RT and heat conditions. In fruit transformed with YZ1, we observed the inhibition of anthocyanin content under RT, cold, and heat conditions , which calculates the gene expression levels by the number of fragments per kilo base of transcript sequence per million base pairs sequenced Table S1YZs-overexpressing strawberry. In total, most aroma components were detected after heat treatment (YZ5 increased than non-transforming fruits (CK) but under RT condition, aroma components were more in the transformed fruits with YZ1, YZ4, YZ5, YZ8, and YZ9 than non-transforming fruits. Meanwhile, the aroma components in transformed fruits with YZ1, YZ4, YZ9, and YZ10 were significantly reduced under heat condition than non-transforming fruits , chilling (0-15°C), room temperature (25-27°C), and heat (10-15°C above ambient) , which hAnthocyanins are the main polyphenols that produce fruit pigmentation. The antioxidant activity of strawberries is positively correlated with the content of anthocyanin . FurtherThe nutritional value of strawberry fruits is associated with the content of specialized metabolites, organic acids, soluble sugars, and amino acids . In addiCBF genes were rapidly and dramatically induced by cold shock and orchestrate cold tolerance in plants  genes. The expression of CBF gene is controlled by upstream transcription factors including bHLH transcription factor ICE1 . CBF genn plants . In thisn plants Figure 9FaQR significantly decreased under cold treatment, while FaQR and FaOMT were up-regulated and FaAAT, FaNES, and FaPAL1 down-regulated with increasing of temperature  function in thermotolerance (A sudden increase in temperature initiates the activation of heat shock response (HSR) mechanisms and induce the expression of molecular chaperones to combat the negative effects on proteins caused by stressors . As partolerance . Our resolerance Table S8olerance , therefoolerance . The accolerance . Ascorbaolerance . In thisHSF20 (YZ1)-overexpressed fruits indicated to be very sensitive to both cold and heat treatments, inhibited the color change, and increased the aroma components of strawberry. Cold treatment promoted coloring in Histone-like transcription factor (CBF/NF-Y) and archaeal histone (YZ9)-overexpressed fruits. Therefore, we concluded that the temperature fluctuation affects postharvest responses of strawberries and links with color, flavor, and the nutritional value of fruit.We found the effect of postharvest heat storage on the color of strawberry fruits was developmental stage-dependent. In the big green fruit period, high temperature inhibits the accumulation of anthocyanin but deepens the color of strawberries after harvest. In our study, heat treatment increased the metabolic levels the content of ABA, MeJA, up-regulated HSF, SET-domain, GRAS transcription factors, and down-regulated Histone transcription factor. Cold treatment increased the content of MeJA and decreased the content of ABA and genes expression involved in biotic and heat stress. Three major metabolism pathways including terpenoid biosynthesis, amino acid biosynthesis and metabolism, and pyruvate metabolism responded to cold treatment, while phenylalanine metabolism, phenylpropanoid biosynthesis, glutathione metabolism, and terpenoid biosynthesis were activated in postharvest heat storage of fruits. The original contributions presented in the study are publicly available. This data can be found here: NCBI, PRJNA783909.TZ, conceptualization, data curation, investigation, writing - original draft, writing - review and editing. JL, investigation, software, and writing - original draft. ES, writing - original draft, writing - review and editing. JC, conceptualization, methodology, funding acquisition. HJ, funding acquisition, methodology, supervision, writing - review and editing. All authors contributed to the article and approved the submitted version.This work was supported by grants from Key Project for New Agricultural Cultivar Breeding in Zhejiang Province (2021C02066-6), Jiangsu Excellent Youth Fund Project (BK20180076), National Natural Science Foundation of China (31872047), Jiangsu Independent Innovation of Agricultural Science and Technology (CX(19)3088), Fundamental Research Funds for Central Universities (KYXJ202003).We thank Ruiping Tian from the State Key Laboratory of Crop Genetics and Germplasm Enhancement for helping analyze GC-MS data.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.


FCGBP, BPIFB, F5, CST1, and CFB and their correlation to EMT under SMG, rendering them potential tumor suppressor biomarkers. Together, these findings reveal new opportunities to establish novel therapeutic strategies for lung cancer treatment.Microgravity is a novel strategy that may serve as a complementary tool to develop future cancer therapies. In lung cancer, the influence of microgravity on cellular processes and the migratory capacity of cells is well addressed. However, its effect on the mechanisms that drive lung cancer progression remains in their infancy. In this study, 13 differentially expressed genes were shown to be associated with the prognosis of lung cancer under simulated microgravity (SMG). Using gene set enrichment analysis, these genes are enriched in humoral immunity pathways. In lieu, alveolar basal-epithelial (A549) cells were exposed to SMG via a 2D clinostat system in vitro. In addition to morphology change and decrease in proliferation rate, SMG reverted the epithelial-to-mesenchymal transition (EMT) phenotype of A549, a key mechanism in cancer progression. This was evidenced by increased epithelial E-cadherin expression and decreased mesenchymal N-cadherin expression, hence exhibiting a less metastatic state. Interestingly, we observed increased expression of  Space is known for lacking the gravity vector, which affects the body at organ, tissue, and cellular levels. Microgravity, a condition of apparent weightlessness, is a significant space stressor known to significantly impact human health, such as bone loss, muscle atrophy, and cardiac deconditioning ,2. The iLung cancer, with non-small cell lung cancer (NSCLC) as the most prominent type, is a leading cause of death, accounts for 12% of all cancers, and is the most commonly diagnosed cancer . In studIt is well established that epithelial cells undergo several biochemical changes to establish a mesenchymal phenotype that enhances apoptosis resistance, invasiveness, and migratory capabilities. Such a biological process is known as epithelial-to-mesenchymal transition (EMT), a hallmark of cancer progression . The relFCGBP, BPIFB1, F5, CFB, and CST1) that significantly exhibited increased mRNA expression in A549 cells under the effect of simulated microgravity (SMG). In addition, we have evaluated the contribution of SMG in A549 cancer progression via EMT regulation. FCGBP, BPIFB1, F5, CFB, and CST1 and their correlation to EMT key markers displayed a potentially less metastatic phenotype of lung cancer under SMG. The findings of this study provide a basis for future investigations that are required to identify underlying mechanisms, which will develop our understanding and aid in the development of novel therapeutic strategies for the treatment of lung cancer.Despite the many crucial discoveries made on the ability of microgravity to modulate tumorigenic and metastatic processes of cancer, the exact mechanisms driven by microgravity remain relatively unknown, hence rendering open questions regarding the adaptive changes that occur at the molecular level. In this study we have identified a signature of immune-related genes . Interestingly, these genes are enriched in pathways involving humoral immune response and regulated exocytosis (To identify genes involved in lung cancer under simulated microgravity (SMG), we initially investigated genes that are differentially expressed under SMG compared with ground gravity (GG) for the human lung cancer using the publicly available Gene Expression Omnibus (GEO) database. Two datasets were chosen, GSE78210 and GSE36931, which include gene expression for two different human lung cancer cell lines, A549 and Colo699, cultivated in 2D and 3D cell culture conditions. Differences in genetic expression profiles between SMG and GG samples were presented in volcano plots . The ideocytosis B.http://www.kmplot.com/; accessed on 11 November 2021) was used to compare the overall survival (OS) of lung adenocarcinoma (LUAD) patients with high/intermediate versus low expression of candidate genes (p = 0.0058), GDA , TCN1 , and BPIFA1  were observed to be significantly associated with poor prognosis (p = 0.0026) were associated with better prognosis and higher overall survival in LUAD patients (p = 0.078), CFB , VTCN1 , AGR3 , F5 , and BPIFB1 , as shown in To evaluate the clinical prognostic value of the candidate genes on patients with lung adenocarcinoma, Kaplan–Meier plotter , in vitro application using qRT-PCR was performed. Interestingly, using qRT-PCR, our data displayed a significant upregulation of FCGBP mRNA expression, specifically at 48 and 72 h in A549 cells, post-SMG as compared with GG .To validate the expression of the in silico identified genes  and metalloproteinases MMP-2 and MMP-9 mRNA expression levels were measured by qRT-PCR in A549, under both GG and SMG  mechanism. EMT is a hallmark and an important mechanism that drives cancer progression and metastasis, thus enhancing its cell motility and invasive properties . Key EMT and SMG . Upon SMpost-SMG A,B. A noupon SMG C–E. MMP-post SMG F, indicaTo gain a further mechanistic insight into the potential role of the identified genes in A549 and their possible correlation to EMT under SMG condition, a gene-interaction analysis was conducted using GeneMANIA against EMT gene markers. FCGBP, BPIFB, F5, CST1, and CFB, and their correlation to epithelial-to-mesenchymal transition (EMT) under SMG, rendering them potential biomarkers of lung cancer progression. Herein, we also highlight the effect of induced SMG on EMT regulation, a hallmark of cancer progression [Lung cancer, one of the significant and worldwide threatening diseases, has recently gained very much attention in “Space research”. However, despite the advancement in this field, lung cancer progression and its response to treatment remain controversial due to the lack of definitive strategies of assessment that help in the prevention or treatment of cancer . To our gression ,22, in sAZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, and BPIFA1) to be significantly expressed in lung cancer under simulated microgravity (SMG) as compared with ground gravity in silico. Our data showed that these differentially expressed genes are enriched in pathways related mainly to humoral immunity and regulated exocytosis. Interestingly, we also showed that these genes correlate to the overall survival of lung cancer patients, where some showed significant correlation to poor prognosis and others showed no potential correlation, except for FCGBP, which was significantly associated with better prognosis. FCGBP, or the Fc fragment of IgG binding protein, is a mucin-like glycoprotein found in body fluid and thought to play important roles in immunity and cancer [In this study we displayed thirteen genes , and NASA-developed rotating wall vessel. Each is designed to serve a certain research objective, where some are designed to cultivate adherent or suspension cells, and others are used for online measurement of kinetic responses. Of note, the clinostat is one of the simplest and most adaptable platforms used by different experimental applications. In principle, the 2D clinostat is characterized by a rotation axis that rotates continuously at an adjusted constant speed and in a direction that is perpendicular to the direction of the Earth’s gravity vector, hence creating centrifugal forces that mimic real microgravity ,32,33. Iesponses . The BPIesponses . In respesponses ,36. One esponses .BPIFB1 promote the risk of lung cancer, and its downregulation leads to poor prognosis in lung cancer patients [CFB was associated with increased patient overall and disease-free survival in lung cancer patients [Given that, it was found that mutations occurring in patients ,39. In apatients . Furtherpatients . As for patients .FCGBP, BPIFB1, F5, CFB, and CST1, our data interestingly displayed two cellular-related phenotypes of A549 cells under SMG—a morphology change into aggregates and clump-shaped cells and a significant decrease in the proliferation rate post-SMG. This is in parallel with some other studies, where cells that were exposed to SMG exhibited small clumps or multi-layered cell aggregates [E-cadherin (CDH1) and the downregulation of the mesenchymal N-cadherin (CDH2) mRNA levels, key markers of EMT [MMP-2, which is a major component of the basement membrane that normally segregates the epithelial layer from the surrounding mesenchyme, thus leading to the loss of the basement membrane [FCGBP, BPIFB, F5, CST1, and CFB with EMT gene markers as well as cell-migration-related genes MMP2 and MMP9. Interestingly, Xiong et al. have shown FCGBP to be a main regulator of EMT in the gall bladder [In addition to the increased expression of gregates ,44. Thesgregates ,22. In pgregates ,46. Suchgregates . For insgregates ,48,49. Hmembrane . This ismembrane . In genemembrane ,51,52. H bladder . This siCollectively, our findings classify the identified genes as key regulators of EMT, promoting a potential enhancement of a less metastatic phenotype via a mesenchymal-to-epithelial transition (MET) of lung cancer cells.https://www.ncbi.nlm.nih.gov/geo/; accessed on 11 November 2021).Relevant datasets from publicly available Gene Expression Omnibus (GEO) database were extracted for initial analysis. This database is used as a publicly available functional genomics analysis of high throughout gene expression data and microarrays. The datasets selected fulfilled the following criteria: datasets using human lung cancer cell lines only, studies that include matching controls in ground gravity (GG) conditions, datasets with defined classification of lung cancer cell lines, and datasets with human lung cancer cell gene expression using microarray. Two datasets fit this criteria, GSE78210 and GSE36931. In total, 25 samples were included in the studies, and 14 lung cancer cell line samples in simulated microgravity (SMG) were compared with 11 GG controls, as shown in https://www.ncbi.nlm.nih.gov/geo/geo2r; accessed on 11 November 2021), an interactive online tool used to compare GEO series, was used in each dataset to group and identify upregulated and downregulated genes in SMG conditions compared with GG conditions. The genes with p-values less than 0.05 and fold change greater than 2 were selected as differentially expressed genes DEGs in SMG conditions. Consequently, the DEGs of each dataset were intersected with each other, and common genes were identified.GEO2R , an online public database evaluating the effect of selected genes on patient clinical outcomes. Overall survival (OS) was defined as the duration from the time of diagnosis (in months) until death. Gene expression data and the survival information are derived from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and European Genome-phenome Atlas (EGA). Patients were categorized into two groups: (1) high expression  and (2) low/medium expression . Kaplan–Meier plots were used to compare OS of lung adenocarcinoma (LUAD) patients with high/intermediate versus low expression of candidate genes.Next, the resulting short-listed genes were validated using Kaplan–Meier Plotter  for comprehensive gene list annotation and analysis resource alongside GEO2R.To explore whether the identified genes share common pathways, Gene Ontology (GO) pathway enrichment analysis for the identified genes was performed using Metascape web tool  cell line, widely used as a model for lung adenocarcinoma, were grown in Roswell Park Memorial Institute Medium (RPMI)-1640 medium supplemented with 10% fetal bovine serum  and 100 units/mL of penicillin/streptomycin . The cell line was cultured in a 37 °C humidified incubator in an atmosphere of 5% CO® Inc—Houston, TX, USA) is a bioreactor equipped with cell culture vessel incorporating a microporous perfusion core and an in-line oxygenator system, which provides nutrient and external gassing of the media, hence insuring proper gas exchange and a low-shear cell culture environment. The chamber was packed with the culture media containing the cells. This chamber horizontally rotated around one axis perpendicular to the force of gravity at a speed of 10 rpm. The 2D clinostat was placed inside the 37 °C humidified incubator in an atmosphere of 5% CO2. A549 cells were harvested post-simulated microgravity (SMG) at 24, 48, and 72 h.Lung cancer cells (A549) were cultured in a 2D clinostat system mimicking the near-zero gravity environment (microgravity). This Rotary Culture Max . These cells are referred to as the control group and were also harvested at 24, 48, and 72 h.4 cells per 500 µL. Cells were harvested by trypsinization at 24, 48, and 72 h following seeding, then centrifuged at 200× g for 5 min. Obtained cell pellets were reconstituted in culture media.For the GG conditions, A549 cells were seeded in 24-well cell culture plates at a density of 3.0 × 104 cells per 1 mL. Cells were harvested at 24, 48, and 72 h following SMG exposure, then centrifuged at 200× g for 5 min. Obtained cell pellets were reconstituted in culture media.For the SMG conditions, A549 cells were seeded in the 2D clinostat rotating system at a density of 6.0 × 10Trypan blue  dye exclusion assay was performed. Cells count was determined using the CellDrop™ Automated Cell Counter.AZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, and BPIFA1) along with EMT gene markers (Gene expression levels (mRNA levels) of the in silico identified genes , a network algorithm for predicting gene functions using a large set of functional association data. This was conducted to further evaluate interactions between/among the selected candidate genes; FCGBP, CST1, F5, CFB, and BPIFB1 with EMT genes; CDH1, CDH2, TJP1, CTNNB1, and SNAI1, as well as cell-migration-related genes, MMP2 and MMP9.Data from in vitro qRT-PCR was implemented in gene–gene interaction analysis using in silico webtool GeneMANIA (t-test was used to compare various groups. Difference between groups were assessed by one-way anova of variance (ANOVA). p-values were determined and values of p < 0.05, p < 0.01, p < 0.001  were considered as significant. All experiments were performed in triplicate (n = 3).GraphPad Prism software was used to perform statistical analysis. Results are expressed as individual data or as mean ± standard deviation (SD). Student’s


PIK3R1, HRAS, and AR genes. Alterations in the PIK3R1 gene have been previously reported in cases of SPA; however, this study is the first to report two novel clinically significant genomic alterations in the HRAS and AR genes. AR protein expression by immunohistochemistry was strongly and diffusely positive in the neoplastic epithelial cells compared to the adjacent normal salivary gland tissue, which was dead negative for AR. This molecular profile will enhance our understanding of the molecular pathways underlying the development of this tumor. Although this entity was initially thought to be a reactive process, evidence from our case and similar cases strongly support the notion that it is neoplastic due to the presence of specific genetic alterations linked to it.Sclerosing polycystic adenoma (SPA) is a rare neoplasm occurring in the salivary glands, mainly the parotid gland. Although it was originally thought to represent a non-neoplastic process, recent genetic data have proven its monoclonality, supporting its neoplastic origin. We report a case of a 73-year-old woman who presented with left neck swelling and pain. A 3 cm hypoechoic, heterogeneous, solid mass was identified on neck ultrasonography within the left parotid gland. Fine needle aspiration revealed benign acinar cells and lymphocytes. Left partial superficial parotidectomy was performed and a diagnosis of SPA was made. Targeted next-generation sequencing (NGS) revealed three clinically significant alterations in the  Sclerosing polycystic adenoma (SPA) is an uncommon entity occurring in the salivary glands, with the majority of cases 70%) reported in the parotid gland and around 60 cases reported worldwide . SPA was0% reportPTEN loss, and PIK3CA and PIK3R1 mutations [The gross appearance consists of one or more well-delineated, pale, firm, rubbery nodules embedded within a normal salivary gland. Microscopically, the nodules consist of irregularly distributed, bilayered ducts and acini within a sclerotic stroma. Ductal cells may have vacuolated, foamy, apocrine, and mucous appearances. Acini contain numerous, coarse eosinophilic, periodic acid Schiff (PAS)-positive, intracytoplasmic granules. Genetic studies on SPA have reported X-chromosome inactivation, utations  and onlyutations .PIK3R1, HRAS, and AR genes. This surgical case report was conducted and reported in accordance with Surgical CAse REports (SCARE) guidelines for reporting case reports.In this report, we present a case of a 73-year-old woman who presented to our institution due to left neck swelling and pain. A 3 cm hypoechoic, heterogeneous, circumscribed, solid mass was identified on neck ultrasonography within the left parotid gland. Left partial superficial parotidectomy was performed and a diagnosis of SPA was made based on histopathologic examination. Targeted next-generation sequencing (NGS) was performed showing three clinically significant alterations in the A 73-year-old woman with a medical history of hypertension and thyroid disease presented to our institution with left neck swelling and pain. The patient never smoked and had no known allergies. Family history was not pertinent. Physical examination revealed a 2 cm firm smooth palpable left neck mass. A neck ultrasonography showed a hypoechoic heterogeneous solid mass within the left parotid gland measuring 2.9 × 2.9 × 2.2 cm. A computed tomography (CT) scan of the neck showed a 2.7 cm solid-appearing mass in the left parotid gland. A neoplastic process was favored. Fine needle aspiration revealed clusters of benign acinar cells in a background of scattered lymphocytes, favoring a benign pathologic process.Left partial superficial parotidectomy with facial nerve dissection and nerve integrity monitoring was performed and the specimen was submitted to Pathology. The surgical specimen weighed 14 g and measured 5.3 cm from superior to inferior, 3.2 cm from anterior to posterior, and 1.6 cm from superficial to deep. It consisted of a 2.8 × 2.3 × 2 cm circumscribed and lobulated mass with a tan, white, focally yellow cut surface and multiple cystic areas . The mas® 550Dx System with 101bp paired-end reads. The DNA and RNA data were analyzed using the Illumina Software TSO500 v2.0.0, a bioinformatics pipeline, and a customized analysis pipeline from PierianDx. The DNA sequence was aligned and compared to the human reference genome GRCh37p.13 (GRCh37/h19). The Clinical Genomics Workspace software platform from PierianDx was used to call, filter, and analyze variants found in the patient sample. TSO500 can detect multiple classes of variants, including single-nucleotide variants, multi-nucleotide variants (<3bp), small insertions (1-18bp) and deletions (1-27bp), copy-number variants, fusions, and splice variants. The assay quantitatively detects microsatellite instability (MSI) and tumor mutational burden (TMB).Targeted NGS was performed, and the results are summarized in HLA-A, KMT2B, KMT2C, and KMT2D genes are filtered out due to potential mis-mapping as a result of sequence homology with other genomic regions. It is possible that pathogenic variants may not be reported by one or more of the tools because of the parameters used. However, tool parameters were optimized to maximize specificity and sensitivity.Among its limitations, certain types of genetic alterations including large deletions and duplications, complex rearrangements, and repeat expansions may not be detected by the current analysis. A pathogenic change may not be detected if it is located outside of the targeted regions. The following untargeted exons have been identified: HIST2H3A NM_001005464.2 exon 1, HIST2H3C NM_021059.2 exon 1, MYB NM_001130173.1 exon 1, PAX8 NM_003466.3 exon 8, DPK1 NM_002613.4 exon 10, PDPK1 NM_002613.4 exon 3, PDPK1 NM_002613.4 exon 8, PDPK1 NM_002613.4 exon 6, PDPK1 NM_002613.4 exon 4, PDPK1 NM_002613.4 exon 5, PDPK1 NM_002613.4 exon 9, RANBP2 NM_006267.4 exon 13, RANBP2 NM_006267.4 exon 8, REL NM_002908.2 exon 9, RICTOR NM_152756.3 exon 19, and SUZ12 NM_015355.2 exon 3. Additionally, all small variant calls in the PIK3R1, HRAS, and AR genes were identified. No loss of PTEN was found. Specifically, the lesion harbored a missense hotspot alteration, c.182A>G (p.Q61R), in the HRAS gene, a missense alteration, c.1690A>G (p.N564D), in the PIK3R1 gene, and a splice site alteration, AR-V7, in the AR gene. In addition, six genomic variants of uncertain significance were identified, including DIS3 , ETV4 , HGF , IRS2 , PREX2 , and SPEN . Microsatellite instability (MSI) and tumor mutational burden (TMB) scores were also tested, showing a stable MSI score of 4.1% (≤20% is considered stable) and a low TMB score of 4.8 muts/Mb (a high TMB score is defined as ≥10 Mutb/Mb).As a result, three clinically significant alterations in the We performed immunohistochemical (IHC) staining to assess the protein expression of AR using an automated Benchmark ULTRA staining platform . Interestingly, there was strong and diffuse AR expression in the neoplastic epithelial cells, whereas the adjacent normal salivary gland tissue was dead negative for AR and served as an internal negative control .Post-operatively, the patient recovered well with no clinical signs or symptoms of hypocalcemia  or hematoma formation. The patient was discharged the next day. On follow up after 1 year, the patient appeared to be recovering satisfactorily. The surgical incision was well-healed. Accordingly, no further therapy was warranted at this time and routine follow up was recommended.Sclerosing polycystic adenoma (SPA) consists of a circumscribed, lobular proliferation of acini and ducts with granular, vacuolated, or apocrine cellular features. Acini contain coarse red zymogen granules embedded in a fibrotic stroma, whereas the ductal elements are proliferative, creating a resemblance to low-grade intraductal carcinoma . Although SPA has histologic features that resemble fibrocystic changes of the breast, the admixture of acini, ducts, and sclerotic stroma present in this lesion makes it unique, unlike other well-established salivary gland neoplasms. In the 2017 World Health Organization Classification of Head and Neck Tumors, SPA was classified as a reactive non-neoplastic lesion . NeverthAR) gene. This alteration leads to the loss of the ligand-binding domain of the canonical AR transcript [AR [AR is common in prostate cancer (PCa) [AR gene in 35 tumor specimens and cell lines derived from salivary duct carcinoma revealed AR overexpression in 70% of the cases [In our patient, a total of 523 genes were subjected to targeted NGS analysis. Splice site alteration, AR-V7, was identified in the androgen receptor   and is aer (PCa) ,18. For er (PCa) . Intereshe cases . This suhe cases .HRAS proto-oncogene. This alteration results in decreased HRAS GTPase activity [HRAS is a member of the small GTPase family that, upon activation by growth factors, stimulates multiple downstream pathways such as RAF and PI3K to promote cell proliferation and survival [HRAS mutations represent more than 90% of all RAS mutations, and they mainly occur in epithelial-myoepithelial carcinomas and salivary duct carcinomas [In our case, a missense hotspot alteration, c.182A>G (p.Q61R), was also identified in the activity . HRAS issurvival ,23. Actisurvival . In salircinomas .PIK3R1 gene. This alteration confers a loss of function to the PIK3R1 protein, leading to constitutive PI3K signaling, phosphorylation of AKT, and increased tumor growth, as demonstrated in animal models [PTEN, PIKCA, and PIK3R1 [Lastly, a missense alteration, c.1690A>G (p.N564D), was identified in the l models ,27,28,29d PIK3R1 , which id PIK3R1 . Interesd PIK3R1 ,31,32,33d PIK3R1 .PTEN mutations, including pleomorphic adenoma, acinic cell carcinoma, and mucoepidermoid carcinoma. Nonetheless, genomic alterations in HRAS and PIK3R1, which we found in our case, have also been described in salivary duct carcinomas, implying a mutual genetic background among neoplasms occurring in the salivary glands. However, each behaves differently in a benign or malignant fashion. In SPA, recurrence has been reported only in around 10% of cases, and this is attributed to either multifocality or incomplete excision of the lesion [Due to the histologic features of this tumor, the main differential diagnosis of SPA includes chronic sclerosing sialadenitis (which exhibits dense lymphoplasmacytic infiltrate with lymphoid follicles) and intraductal carcinoma (a more complex cystic pattern with nuclear atypia). In fact, since both SPA and apocrine intraductal carcinoma harbor similar genetic aberrations, we hypothesize that those two entities may be closely related, and that SPA may indeed be a precursor lesion to apocrine intraductal carcinoma. Other neoplasms of the salivary glands have not been shown to harbor e lesion . Furthere lesion .PTEN loss, and PIK3CA and PIK3R1 mutations. We report, for the first time, two novel clinically significant genomic alterations in the HRAS and AR genes in a SPA of a 73-year-old woman, not including PIK3R1 gene alteration, which has been already reported. This molecular profile will enhance our understanding of the molecular pathways underlying the development of this benign neoplasm.In conclusion, sclerosing polycystic adenoma (SPA) is a rare neoplasm occurring in the salivary glands, mainly the parotid glands, with around 60 cases reported worldwide. Genetic studies on patients with SPA have reported X-chromosome inactivation,


To explore the correlations between the expression of zinc finger protein 521 (ZNF521) with immune invasion and prognosis of gastric cancer.  Expression of ZNF521 was examined by immunohistochemistry in gastric cancer cases. Kaplan–Meier plotter was used to determine the relationships between ZNF521 and prognosis. TIMER and GEPIA were used to analyze the correlation between ZNF521 expression and gene markers of immune cell infiltration.  The expression of ZNF521 was up-regulated in gastric cancer samples. Kaplan–Meier analysis indicated that higher expression of ZNF521 was associated with poor prognosis. The expression of ZNF521 was correlated with infiltrating levels of CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells in gastric cancer, which also correlated with diverse immune marker sets.  ZNF521 is correlated significantly with immune cell infiltration and is a valuable biomarker for prognosis in gastric cancer. The incidence and mortality rate of gastric cancer ranked 5th and 3rd worldwide, respectively, in 2018, and their respective rates in China were 44.1% and 49.9% . Immune Zinc finger protein 521 (ZNF521) encodes a transcription factor with a zinc finger domain that is widely expressed in many tissues and plays important roles in tumor formation and development , 9. ZNF5In this study, we detected ZNF521 expression in gastric cancer by immunohistochemistry (IHC) and analyzed the correlation between its expression with pathological parameters and prognosis. Kaplan–Meier plotter databases were used to analyze the expression of ZNF521 and its correlation with the prognosis of gastric cancer. Moreover, we analyzed the correlation between ZNF521 expression and infiltrating immune cells in different tumor microenvironments through the tumor immune database TIMER. ZNF521 promotes the malignant characteristics of gastric cancer cells in part by interacting with immune infiltrating cells. The findings of this report elucidate the important role of ZNF521 in stomach adenocarcinoma (STAD) and suggest a potential relationship and mechanistic link between ZNF521 and tumor immune interaction. We focused on ZNF521 expression and its relationship with clinicopathological features and prognosis to reveal the relationship between ZNF521 and tumor immune infiltration.Tissue microarrays were performed as described in our previous study . This stn = 7,830), ovarian , lung , and gastric  cancers. Sources for the databases included the Gene Expression Omnibus, European Genome-Phenome Archive, and The Cancer Genome Atlas (TCGA). The primary purpose of the tool is meta-analysis-based discovery and validation of survival biomarkers. We used this database to assess the relationship between TNF521 expression and patient outcomes  ). ZNF521 was primarily localized within the cytoplasm of cancer cells, and ZNF521 expression in non-tumor mucosa was also recorded . AccordiTo explore the clinical significance of ZNF521 in gastric cancer and to analyze correlations between ZNF521 expression and the clinical characteristics of gastric cancer patients, patients were divided into two groups according to their ZNF521 expression levels: the high and low ZNF521 expression groups.P < 0.01; P > 0.05; ZNF521 was highly expressed in 58.7% of patients, and its expression was significantly correlated with tumor location, tumor size, Lauren classification, histological classification, histological differentiation, depth of invasion, regional lymph nodes, distant metastasis, and tumor node metastasis (TNM) stage : HR = 1.72, 95% CI = 1.35–2.2, P = 1.2 × 10−5; post-progression survival (PPS): HR = 1.94, 95% CI = 1.48–2.56, P = 1.4 × 10−6; Figures The prognostic value of ZNF521 on the basis of Affymetrix microarray data in gastric cancer was evaluated using the Kaplan–Meier Plotter database. High ZNF521 expression was associated with poor prognosis in gastric cancer . Specifically, higher ZNF521 expression was correlated with worse OS in stage II  and stage IV  patients, with PFS in stage II  and stage III  patients, with PPS in stage II, III, and IV patients , but was not associated with OS, PFS, or PPS in stage I patients or OS and PFS in stage N0 patients. In particular, we analyzed the prognostic value of ZNF521 expression in gastric cancer patients with lymphatic metastasis. Higher ZNF521 expression was correlated with worse OS in stage N1  and stage N3  patients, worse PFS in stage N1, N2, and N3 patients , worse PPS in stage N0, N1, and N2 patients , CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and DCs  in gastric cancer , M1 and M2 macrophages, neutrophils, NK cells, DCs, Th1 cells, Th2 cells, Tfh cells, Th17 cells, regulatory T cells (Tregs), and exhausted T cells (Tex) in gastric cancer . The resZNF521 expression also had a strong correlation with markers of DCs, such as HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, BDCA-1 (CD1C), BDCA-4 (NRP1), and CD11c ITGAX; . ZNF521 The tumor microenvironment plays an important role in the dynamic regulation of cancer progression. Therapeutic strategies targeting the tumor microenvironment have emerged as promising cancer treatments. Immunotherapy has been approved in clinical trials and has broad application prospects . BroadlyA comprehensive analysis of tumor-infiltrating immune cells will help elucidate the mechanisms of tumor immune escape and provide opportunities for the development of new therapeutic strategies. ZNF521 is a multifunctional transcription cofactor that regulates many biological processes, such as hematopoietic differentiation, cell proliferation, autophagy, inhibiting EBF1, and promoting the development of B-cell leukemia , 25–28. ZNF521 expression was correlated with levels of infiltrating CD4+ and CD8+ T cells, macrophages, neutrophils, and DCs in bladder cancer, lung squamous cell carcinoma, and gastric cancer, and was also correlated with diverse immune markers. Additionally, the correlation between ZNF521 expression and immune cell markers indicates that ZNF521 regulates tumor immunity in bladder cancer, lung squamous cell carcinoma, and gastric cancer. First, markers of M1 macrophages, such as inducible nitric oxide synthase, interferon regulatory factor 5 (IRF5), and prostaglandin-endoperoxide synthase 2 (PTGS2) were not correlated or were only weakly correlated with ZNF521 expression, while markers of M2 macrophages, such as CD163, V-set immunoglobulin-domain-containing 4 (VSIG4), and MS4A4A were strongly correlated with ZNF521 expression . These rDCs are the most powerful full-time antigen-presenting cells of the immune system and play a key role in the initiation and regulation of immune responses . In mostTogether, these findings suggest that ZNF521 is a valuable prognostic biomarker for gastric and other cancers that is significantly correlated with immune infiltration.


Vitis vinifera L. is highly susceptible to the biotrophic pathogen Plasmopara viticola. To control the downy mildew disease, several phytochemicals are applied every season. Recent European Union requirements to reduce the use of chemicals in viticulture have made it crucial to use alternative and more sustainable approaches to control this disease. Our previous studies pinpoint the role of fatty acids and lipid signalling in the establishment of an incompatible interaction between grapevine and P. viticola. To further understand the mechanisms behind lipid involvement in an effective defence response we have analysed the expression of several genes related to lipid metabolism in three grapevine genotypes: Chardonnay (susceptible); Regent (tolerant), harbouring an Rpv3-1 resistance loci; and Sauvignac (resistant) that harbours a pyramid of Rpv12 and Rpv3-1 resistance loci. A highly aggressive P. viticola isolate was used (NW-10/16). Moreover, we have characterised the grapevine phospholipases C and D gene families and monitored fatty acid modulation during infection. Our results indicate that both susceptible and resistant grapevine hosts did not present wide fatty acid or gene expression modulation. The modulation of genes associated with lipid signalling and fatty acids seems to be specific to Regent, which raises the hypothesis of being specifically linked to the Rpv3 loci. In Sauvignac, the Rpv12 may be dominant concerning the defence response, and, thus, this genotype may present the activation of other pathways rather than lipid signalling. Vitis vinifera L. is the most well-known and disease-susceptible grapevine species. In viticulture, one of the major threats is caused by Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni, a biotrophic obligatory oomycete that causes downy mildew disease F, IIGSANINQRS133 and PiP2-binding .PI-PLC proteins were predicted to be in the mitochondria  and in the cytoplasmic membrane  . Regardicis-elements associated with JA response, wounding and pathogen/defence response and on the chromosomal location (within Rpv loci).A total of 12 genes were selected for expression analysis using the following criteria: previously described as being associated with defence against pathogens; VviPI-PLC1, VviPI-PLC2, and VviPI-PLC7, two nonspecific: VviNPC3 and VviNPC4); three belong to the PLD superfamily  and four genes already described by Laureano and colleagues [VviFAD8 and VviFAD2-1 and the soluble desaturase 1 (VviSAD-1)).Five selected genes belong to the PLC superfamily  were analysed by qPCR in V. vinifera cv. Chardonnay (susceptible), Regent (tolerant) and Sauvignac (resistant) inoculated with the P. viticola isolate NW-10/16 , at 4 hpi, VviPI-PLC7, VviPLDζ, VviFAD8 and 2VvisPLA were downregulated and the others were upregulated. At 6 hpi, VviPLDα3 was upregulated and VviPI-PLC7, VviPLDζ, VviFAD8 and 2VvisPLA were down-regulated, whereas the remaining half were at control levels. At 12 hpi, two genes were downregulated (VviPI-PLC7 and VviPLDα3), VviNPC4 had fold-change near one, and the others were all upregulated. Finally, at 24 hpi, VviPI-PLC7, VviNPC3, VviPLDβ, VviPLDζ and 2VvisPLA were upregulated, whereas the other seven were downregulated.Regarding the results observed for the resistant cultivar Sauvignac, VviPLDα3, which was downregulated at 4 hpi, although the higher expression value observed in this analysis was from this same gene at 24 hpi. VviPI-PLC1, VviNPC4 and VviFAD8 along with VviPLDα3 presented the most noteworthy differences comparing 4 hpi and 24 hpi, with a significant increase in expression in this last time point.All genes were upregulated at all time points in the tolerant cultivar Regent, except for V. vinifera cultivars  with different degrees of susceptibility and tolerance to P. viticola, the FA composition of total leaf lipids was determined at 6 h, 12 h and 24 h post-inoculation, by gas chromatography. The most representative fatty acids were α-linolenic (C18:3), linoleic (C18:2) and palmitic (C16:0) acids (To investigate differences in the fatty acid profiles of the three 0) acids .P. viticola, Sauvignac, was the genotype that presented the most constant FA profile along the infection, with no significant changes, with the exception of a slight increase in C18:1 at 24 hpi.At the constitutive level, the three cultivars presented distinct FA profiles. The susceptible one, Chardonnay, showed a higher relative amount of C18:3 compared to the more tolerant cultivars Sauvignac and Regent . Upon paCellular membranes undergo alterations in their lipid and fatty acid compositions in response to a myriad of biotic and abiotic stresses. Additionally, lipids are indispensable components in plants, providing energy for metabolic processes, acting as structural components of membranes, and playing a central role in plant defence through signalling processes (reviewed in ). Lipid Phospholipases are at the genesis of lipid signalling pathways by their ability to catalyse the hydrolysis of phospholipids into phosphatidic acid (PA), diacylglycerol (DAG), free FA and inositol polyphosphates . These iV. vinifera cultivar, Regent, inoculated with a less aggressive P. viticola pathovar (not able to overcome the Rpv3 resistance), a large lipid and FA modulation occurs [P. viticola [We have previously shown that in the tolerant n occurs . These cviticola ,15 in thviticola ,13.P. viticola [P. viticola pathovar (NW-10/16). This pathovar was shown to overcome Rpv3 loci-based resistance (Regent), aggravate disease symptoms in susceptible cultivars (as Chardonnay), and be unable to infect a grapevine genotype that harbours an Rpv3.1/Rpv12 pyramid (Sauvignac) [In our previous studies, we performed a genome-wide characterisation of the PLA gene family and showed the involvement of some members in grapevine resistance against viticola . PLDs weviticola  but a neuvignac) . We haveThe grapevine PLC family harbours two groups, the PI-PLC being the larger sub-group with eight members. These results are in accordance with their orthologues in Arabidopsis ,55, soyb3) and diacylglycerol (DAG) [2+-binding EF-hand of Arabidopsis PLCδ, the so-called “EF-loop” [2+-dependent biological processes, unique to plants. This domain is around 130 residues in length and can bind Ca2+ and other effectors, including phospholipids, inositol phosphates and proteins, as mentioned in [PI-PLC acts on phosphatidylinositol (PI), producing inositol trisphosphate (IPol (DAG) . In grapEF-loop”  or EF-haEF-loop” , that prEF-loop”  predicteEF-loop”  or targeEF-loop” ,61. The EF-loop” . The C2 ioned in ,63,64.3G and TxPNR, and two to be variable, DE(x)2G(x)2DHV and GxRVP(x)5P, as previously described [The grapevine NPC members were predicted to be in the membranes of several cell compartments, where they can generate DAG from the hydrolysis of both PC and phosphatidylethanolamine (PE), as reviewed in . The graescribed ,66.V. vinifera PLD, the largest subgroup was the C2-PLD, containing 13 proteins, whereas the PXPH-PLD and SP-PLD had only one protein each. These results are in agreement with their PLD orthologues in Arabidopsis [V. vinifera PLD sub-groups presented specific domains at the N-terminal, as predicted in previous works [2+-dependent phospholipid-binding C2 domain, requiring Ca2+ for its enzymatic activity [V. vinifera PXPH-PLD and SP-PLD are Ca2+-independent enzymes. The PXPH-PLD showed two specific domains, the Phox homology (PX) and Pleckstrin homology (PH) domains, which are protein regulatory modules involved in cell signalling [2 has a critical role in PLD activation in mammals and plants, being required for the enzyme activity of some PLDs [Regarding the bidopsis , soybeanbidopsis  and ricebidopsis . The V. us works ,44,49,50activity . In contgnalling . Finallygnalling ,44,49,50gnalling . Except gnalling . The IGSgnalling . PiP2 haome PLDs . The graome PLDs .V. vinifera genotype, the susceptible genotypes are composed of higher levels of plastidial lipids and PUFAs, and the tolerant/resistant genotypes have higher contents of PA and neutral lipids [P. viticola, the grapevine genotypes act differently. Only the genotypes presenting a resistant genetic background (Rpv3.1) demonstrate lipid and FA modulation as well as an upregulation of the coding genes of enzymes involved in lipid signalling [VviPI-PLC1, VviPI-PLC2, VviPI-PLC7, VviNPC3, VviNPC4, VviPLDα3, VviPLDβ, VviPLDζ, VviFAD8, VviFAD2-1, VviSAD-1 and 2VvisPLA) as well as characterised the modulation of FA during the interaction of the three grapevine genotypes  and resistant (Rpv3.1 and Rpv12)) with P. viticola.Both lipid and FA composition vary according to the l lipids . Additiognalling ,13. To eRpv3.1/Rpv12) genotypes, the global modulation pattern is very similar, with several genes being downregulated at 24 hpi.When comparing the global modulation of the three grapevine genotypes, there is a clear difference in the modulation pattern. In Regent, there is an overall increase in gene expression, particularly at 12 and 24 hpi, whereas, in the susceptible and resistant  analysed had their expression increased mainly at the later time point (24 hpi). In response to pathogens, these grapevine PLCs may be acting in the release of DAG and IP3 from membrane lipids, whereas PI-PLCs act on PI and NPCs act on PC and PE [OsPI-PLC1 is activated in response to the fungus Magnaporthe grisea, being reported as essential in the activation of the resistance mechanisms against this pathogen [SlPLC2 and SlPLC5 had their expression increased in response to fungal elicitors [Cladosporium fulvum [P. viticola was previously reported [P. viticola [VviFAD2-1 could be related to an increase in C18:2 production in extraplastidial lipids such as PC. VviFAD8 showed upregulation at 12 h and 24 hpi, being the higher fold-change identified at 12 hpi. Corroborating this evidence, an increase in C18:3 content is observed in Regent at the same time point. The accumulation of plastidial C18:3 as well as the activation of the FAD8 gene have been reported in the first hours of the interaction of Regent with P. viticola [VviPLA has been related to the JA production that uses C18:3 as a substrate upon its release from membrane lipids by the action of PLAs [VvisPLA2 upregulation occurs in Regent, mainly at 24 hpi. Secretory PLAs are involved in the response to pathogens by triggering JA synthesis, stomata opening regulation and H+-ATPase activation [VviPLA suggests that this enzyme may be acting as a defence mechanism response to downy mildew by being involved in JA production and the production of ROS by activating H+-ATPase and closing the stomata aperture, which is the P. viticola infection starting point.Considering the PLD genes, the main modulation occurred in Regent, where lycerols  and in llycerols . The actlycerols . The ROSlycerols . Additiolycerols . In planlycerols . In turnlycerols . All theC and PE . InositoC and PE . MoreoveC and PE . The actC and PE . In ricepathogen . In tomalicitors  and inocm fulvum  leading m fulvum . A high viticola ,14. The viticola . The incviticola . The higtivation ,80. The VviFAD2-1 and VviSAD-1) may be related to the production of FA conducted by the pathogen demand to uptake nutrients from the host [Plasmopara viticola is an obligatory oomycete and keeps the host alive to complete its life cycle. After infection, P. viticola is able to switch the grapevine metabolism in order to uptake nutrients from its host [The susceptible cultivar, Chardonnay, presented some similarities to the resistant hybrid, Sauvignac, in the expression analysis results. In general, the majority of the analysed genes did not have their expression increased. In Chardonnay, the gene upregulation occurred mostly at 6 hpi. A slight increase in FA desaturases’ coding genes , and for those, the expression is significantly higher than in Chardonnay. This genotype also did not present alterations in the FA profile upon infection, possibly due to its resistance mechanisms, conferred by the presence of two resistance loci, rp3.1 and Rpv12.Sauvignac only presents the upregulation of a few genes (P. viticola isolates is changing the grapevine resistance paradigm as these isolates are able to overcome crossing hybrids harbouring resistance loci. The isolate (NW-10/16) is more aggressive and is already able to partially overcome Rpv3.1 (some sporulation is seen in Regent) resistance but not Rpv3.1/Rpv12 pyramid resistance (no sporulation occurred in Sauvignac). Lipid modulation and signalling were previously shown to be specifically activated in the Rpv3.1 background when inoculated with a less aggressive pathovar, with no modulation occurring in a susceptible genotype. In the interaction with NW-10/16, despite overcoming the Rpv3.1-associated resistance, lipid modulation was also shown to occur. In this case, host lipid modulation and signalling events do not occur as fast and to the extent that they occurred with a less aggressive pathovar, but FAD, lipid signalling-associated phospholipase activation and C18:3 accumulation was shown.The emergence of new Rpv3.1/Rpv12 pyramid resistance genotypes and the susceptible genotypes were very similar, suggesting that other mechanisms, rather than lipid modulation and signalling, would be activated. This way, we propose that there could be a link between the genetic background of the host and the activation of a lipid signalling pathway as a defence strategy against P. viticola. To further prove this hypothesis, more genotypes with different Rpv backgrounds should be analysed.The patterns of fatty acid and lipid modulation and the gene expression of both


PpHSP20 genes were detected in the peach genome and were randomly distributed onto the eight chromosomes. The primary modes of gene duplication of the PpHSP20s were dispersed gene duplication (DSD) and tandem duplication (TD). PpHSP20s in the same class shared similar motifs. Based on phylogenetic analysis of HSP20s in peach, Arabidopsis thaliana, Glycine max, and Oryza sativa, the PpHSP20s were classified into 11 subclasses, except for two unclassified PpHSP20s. cis-elements related to stress and hormone responses were detected in the promoter regions of most PpHSP20s. Gene expression analysis of 42 PpHSP20 genes revealed that the expression pattern of PpHSP20-32 was highly consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’, which is a temperature-sensitive semi-dwarf. PpHSP20-32 was selected for further functional analysis. The plant heights of three transgenic Arabidopsis lines overexpressing PpHSP20-32 were significantly higher than WT, although there was no significant difference in the number of nodes. In addition, the seeds of three over-expressing lines of PpHSP20-32 treated with high temperature showed enhanced thermotolerance. These results provide a foundation for the functional characterization of PpHSP20 genes and their potential use in the growth and development of peach.The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42  During these times of global environmental change, temperature is arguably the most important factor affecting plant growth and geographical distribution ,2. PlantHSP100s, HSP90s, HSP70s, HSP60s, and HSP20s [Arabidopsis thaliana) [Oryza sativa) [Capsicum annuum) [Solanum lycopersicum) [Citrullus lanatus) [Vitis vinifera) [Sorghum bicolor [Malus domestica) [Tremendous amounts of research have shown that multiple heat shock proteins (HSPs) emerge as central players in the temperature response, including the growth/development and stress responses ,12,13,14d HSP20s ,16. HSP2haliana) , rice (O sativa) , pepper  annuum) , tomato lanatus) , grape (inifera) , Sorghum bicolor , and appHSP20s participate in a wide range of developmental processes and abiotic stresses, such as heat, salt, and drought [SlHSP20s are constitutively expressed in almost all tissues, indicating that they might play specific housekeeping functions under normal growth conditions [SlHSP20s were up-regulated, implying their potential roles in regulating the response to stresses [CaHSP20s were highly induced by heat stress [CaHSP16.4 enhanced the tolerance to heat and drought stresses by stabilizing the ROS-scavenging system [HSP20s were induced by cold stress in soybean [VvHSP20s responded to H2O2 treatment [AtHSP22 participated in auxin-regulated hypocotyl elongation under high temperatures in Arabidopsis thaliana [ drought ,24,25,26t stress . Overexpg system . Unlike  soybean . In grapreatment . In addiPrunus persica L.), an economically important crop and a popular fruit with consumers, is widespread in both temperate and subtropical regions, although peach trees can also be found in high altitude and severe cold regions [TSSD) peach cultivar, showed extremely shortened internodes below 30 °C and normal internode length above 30 °C [TSSD categorize them as semi-dwarf [PpHSP20 participated in the temperature sensitivity of ‘Zhongyoutao 14’ is unknown. In this study, the expression patterns of PpHSP20s were analyzed during shoot elongation of ‘Zhongyoutao 14’ at four critical growth stages. Furthermore, the function of PpHSP20-32 was analyzed via ectopic expression in transgenic Arabidopsis plants. The thermotolerant characteristics of transgenic Arabidopsis carrying PpHSP20-32 were also analyzed. This study provides valuable information as a step toward the further investigation of the functions and regulatory mechanisms of potentially important PpHSP20 genes that may be crucial in temperature tolerance and plant height in peach trees.Peach  contained second structures in the ranked quantities of random coils > alpha helix ≥ extended strand > beta turn, while the quantity ranking of the structures of the other members (21.4% of 42 PpHSP20s) was random coil > extended chain structure > alpha helix > beta turn .PpHSP20 genes were unevenly distributed among the eight chromosomes of peach  and tandem duplication , , . Eight g 30.9%), .PpHSP20 genes were also predicted. Fourteen PpHSP20s were intronless (33.3%), 17 (40.5%) had one intron, and seven (16.7%) had two introns  contained Motif 1, 20 (47.6%) contained Motif 2, 34 (81.0%) contained Motif 3, and 32 (76.2%) contained Motif 6. Motif analysis showed that the PpHSP20s containing similar motifs were grouped in the same class A,C. For Arabidopsis thaliana sequences, 45 Glycine max sequences, 22 Oryza sativa sequences, and 42 peach sequences , light-responsive, MeJA-responsive, and anaerobic-induction elements , including abscisic acid-responsive), MeJA-responsive, salicylic acid-responsive (SA), auxin-responsive and gibberellin-responsive (GA). The abiotic and biotic stress-responsive elements included anaerobic induction and low temperature-responsive elements. In the plant development-related category, seed specific regulation, cell cycle regulation, and circadian control were identified. These results suggested that PpHSP20s were not only related to stress response, but also related to other physiological responses.The promoters in the upstream 2000 bp region of 42 elements A,B. The PpHSP20s were compared at four critical stages  of shoot elongation in the temperature-sensitive semi-dwarf peach cultivar ‘Zhongyoutao 14’, grown in the field under regular management with natural ambient temperature. According to their expression patterns, the 42 PpHSP20s could be classified into four groups , compared to RGP and the SGP. Group IV, the second largest group containing 12 PpHSP20s, showed higher expression levels in IP or RGP, compared to IEP and RGP. The internodes length of IP (1.21 mm) and IEP (2.57 mm) were significantly less than that of RGP (11.27 mm) and SGP (12.54 mm) [PpHSP20s in Group III (marked in red). Among the Group III genes, the expression level of PpHSP20-32 was most consistent with the terminal internode length, as shown in PpHSP20-32 might participate in temperature-induced shoot growth in this temperature-sensitive peach cultivar.The expression patterns of the r groups A. Group 2.54 mm) . It showPpHSP20-32 overexpression vector and transformed it into Arabidopsis thaliana using an Agrobacterium-mediated method. The phenotypes of three transgenic lines  and WT were recorded , except for five genes that did not fall into any class [Arabidopsis thaliana, the CVII subgroup gene AtHsp14.7 was constitutively expressed, and its transcript level did not change under different stresses [The 42 PpHSP20s were divided into 11 classes , except for 2 unclassified PpHSP20s, based on the phylogenetic tree which was constructed using the amino acid sequences of peach, rice, ny class . Afterwany class . Most ofny class ,28. In tny class , switchgny class , apple [ny class , and thrny class . In Arabstresses . It was stresses .PpHSP20-13, PpHSP20-14, PpHSP20-35, and especially PpHSP20-32, showed a correlation with the length of the terminal internodes in the shoots of temperature-sensitive semi-dwarf peach. Populus trichocarpa, a transgenic line overexpressing PtHSP17.8, showed enhanced tolerance to heat and salt stresses [CaHSP16.4 participated in heat and drought stress by enhancing the scavenging of reactive oxygen species [PpHSP20s might play important role in the regulation of shoot elongation at non-stressful temperatures. In addition, HSP20 responded to the phytohormone ABA and modulated polar auxin transport [Plant HSPs are molecular chaperones that protect the functions of their target proteins under various stress conditions to help maintain growth and development ,16. In tstresses . In pepp species . These rransport .AtHSP70 is expressed at a level proportionate to the ambient temperature [AtHSP90 integrates environmental temperature and auxin signaling to regulate temperature-dependent plant growth by stabilizing the auxin co-receptor F-box protein TIR1 [AtHSP22 promoted hypocotyl elongation at high temperatures by regulating polar auxin transport, which required the ABI1 protein phosphatase [PpHSP20-32-overexpressing transgenic lines produced larger rosette leaves and taller plants than WT. The plant height of the transgenic lines was higher than that of WT. There was no significant difference in the length of the internodes between the transgenic lines and WT, indicating that the increase in plant height of the transgenic lines may be caused by the increase in the number of internodes. The PpHSP20-32-overexpressing lines also showed enhanced heat tolerance. Similar results were observed in rice, pepper, and poplar, which together demonstrate that HSP20 genes enhance thermotolerance [In the ambient temperature-sensing pathway, perature . AtHSP90ein TIR1 ,43. A resphatase . In thisolerance ,25,27.PpHSP20-32 contained four types of phytohormone-responsive elements, namely ABRE, MeJA-responsive, salicylic acid-responsive, and gibberellin-responsive elements  Heynh Columbia 0 (Col-0) was used for transformation with PpHSP20-32.The peach cultivar ‘Zhongyoutao14’, a temperature-sensitive semi-dwarf, is planted in the experimental station of the Horticulture College, Henan Agricultural University . The shoot internode length was temperature-dependent. Shoot tips were collected at four critical growth stages, namely the initial period (IP), initial elongation period (IEP), rapid growth period (RGP), and stable growth period (SGP) . The inthttp://pfam.xfam.org, accessed on 10 May 2022) and used to identify HSP20 genes in peach. An hmmsearch was performed against the peach genome files (v2.1), downloaded from the JGI database . The isoelectric points and other physical properties were approximated from ExPASy .The hidden Markov model (HMM) profile (PF00011), characteristic of HSP20, was downloaded from the Pfam website .According to the genome location annotation given by Phytozome V12.1, the chromosome location of each  TBtools . AccordiArabidopsis thaliana, Oryza sativa, Glycine max, and peach were saved as FASTA format files. The phylogenetic tree was constructed by the maximum likelihood method using MEGA 7.0 software  [http://alternate.meme-suite.org/tools/meme, accessed on 12 May 2022) was used to analyze the motifs in each protein sequence.The amino acid sequences of the HSP20 genes of , Japan) . The onlPpHSP20 gene (2000 bps upstream of the start codons) was downloaded from the peach genome. The cis-acting elements of the HSP20 promoters were detected using PlantCARE .The promoter sequence of each PpHSP20 genes were obtained from our previous transcriptome data of the four critical stages of shoot growth in the cultivar ‘Zhongyoutao 14’ . After sequence confirmation, the full coding sequence of PpHSP20-32 was amplified with primers including restriction sites Hind III and Xba I . Three biological replicates were taken, for a total of nine seedlings observed. The height, the length of internodes, and the number of internodes and branches (five plants per line) in the different transgenic lines and WT were determined.After screening for kanamycin resistance and PCR verification , the transgenic plants were allowed to flower. Seeds from Tt-test.To detect the function of PpHSP20-32 in heat tolerance, heat stress treatment (46 °C for 30 min) was performed. Before heat stress treatment, seeds of WT and transgenic Arabidopsis lines were sown on MS medium and kept dark at 4 °C for 2 d, and then at 22 °C for 2 d. Then, the seeds were exposed to 46 °C for 30 min, followed by being transferred into a climate chamber . The germination of seeds was counted daily and photographed. More than 60 seeds of each line were used in each plate, with three replicates. Differences in heat stress tolerance were confirmed using Student’s p < 0.05) using IBM SPSS Statistics 20 .Data were analyzed by ANOVA, Tukey HSDa, and Duncan’s multiple range tests  and tandem duplication (TD) were the primary modes of gene duplication of PpHSP20s. Except for two unclassified PpHSP20s, the other 40 PpHSP20s were classified into 11 subclasses. The gene structures, basic classification, conserved motifs, and cis-elements were also analyzed. The expression pattern of PpHSP20-32 was highly consistent with shoot length changes during four critical growth stages of temperature-sensitive semi-dwarf peach ‘Zhongyoutao 14’ in response to increasing temperature. Transgenic Arabidopsis lines overexpressing PpHSP20-32 demonstrated that the gene can increase plant height and enhance thermotolerance. The results in this study supplied general information on the PpHSP20 gene family and further revealed the potential roles of PpHSP20-32 in plant height, in addition to the response to heat stress.In this study, 42


This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature  was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor  cell proliferation and the osteogenic ability of different donors. Mesenchymal stem cells (MSCs) have become the basis of numerous bioengineering studies due to their ability to differentiate into multiple cell lineages  and secrete bioactive factors essential for tissue repair ,2,3. HowInter-donor variability is perhaps one of the major contributions to MSCs heterogeneity, potentially reflecting differences in donor age , gender To the best of our knowledge, only a few metabolomic studies of MSCs heterogeneity have been carried out ,28,29,30In this work, untargeted NMR metabolomics was carried out in hAMSCs, for the first time to our knowledge, to search for osteogenesis-specific metabolic markers, which may be considered independent of donor origin and of underlying cell proliferation metabolism. To achieve this, the detailed polar endometabolome adaptations of hAMSCs harvested from two randomly chosen donors, found to differ in proliferating and differentiation extensions, were measured at several timepoints throughout 21 days, under osteogenic and basal (control) conditions. The detailed and holistic nature of NMR metabolomics ,36,37 enhAMSCs harvested from two randomly chosen healthy donors (1 and 2) were considered in this study. Adipose tissue from donor 1 was collected through abdominoplasty, under a cooperation agreement between the COMPASS Research Group from CICECO-University of Aveiro and Hospital da Luz, Aveiro, Portugal, upon approval of the hospital Ethics Committee. The patient’s informed consent was obtained under anonymous conditions . Samples were processed within 24 h after surgery, and hAMSCs isolation was carried out as previously described . The hAM6 cells/flask . After reaching nearly 100% confluence under the above-mentioned conditions, the culture medium was exchanged and supplemented with osteogenic differentiation factors, namely, 10 mM β-glycerophosphate , 50 μg/mL L-ascorbic acid , and 10 nM dexamethasone . For both donors 1 and 2, parallel control experiments were carried out . The medium was exchanged twice a week for 21 days. On days 0, 1, 4, 7, 14, and 21, cells were trypsinized  and rinsed twice in phosphate-buffered saline (PBS) solution, to avoid medium contamination. For donor 3, day 4 samples could not be collected. At least 1 × 106 cells per pellet were used for metabolomics. The above experiments were carried out in 48-well plates to assess cellular proliferation and osteodifferentiation. At each timepoint, plated cells were scraped, rinsed twice with PBS, lysed by osmotic/thermal shock and stored at −80 °C. Media samples (>3 mL) were also collected.Prior to osteoinduction, hAMSCs were harvested and seeded at a density of 0.5 × 10escribed ) and coln = 6 samples/timepoint) were centrifuged , and supernatants used for double-stranded DNA (dsDNA) quantification . Dye fluorescence was assessed at emission and excitation wavelengths 528 ± 10 nm and 485 ± 10 nm, respectively . dsDNA concentrations were calculated using a calibration curve in the 0.0−2.0 μg/mL concentration range.Cell lysates were thawed at room temperature (RT), exposed to ultrasounds (10 min) and kept at −20 °C overnight. This was repeated twice to improve DNA extraction. Cell lysates (n = 6 samples per timepoint). The mixture was incubated at 37 °C for approximately 1 h and then 80 μL of 3 M NaOH (in MiliQ water) was added. pNP absorbance at 450 nm was measured  and ALP activity was calculated using a pNP calibration curve (0.0−0.5 mM). For calcium quantification and determination of osteocalcin  was assntiation ) protocontiation ,37. ALP g, 20 min, RT), and polar and lipophilic phases were separated. Polar extracts were dried under vacuum and stored at −80 °C until analysis . Importantly, the above conditions constituted an improved protocol in relation to that used for donor 3 cells, reported earlier [2O  containing 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid  for chemical shift referencing. After vortexing, 550 μL were transferred to 5 mm NMR tubes. NMR spectra were recorded on a Bruker Avance III spectrometer operating at 500.13 MHz for 1H (298 K). Standard unidimensional spectra were acquired with the noesypr1d pulse sequence under the following conditions: 7002.801 Hz spectral width, 32 k data points, 2.3 s acquisition time, 4 s relaxation delay (d1) and 512 scans. Each free induction decay was zero-filled to 64 k points, multiplied by a 0.3 Hz exponential line-broadening function and Fourier transformed. Spectra were manually phased and baseline corrected. As described before [1H-1H total correlation (TOCSY) and 1H-13C heteronuclear single quantum correlation (HSQC) spectra, spiking experiments, the literature and spectral databases, such as the Bruker BBIOREFCODE AMIX database, the human metabolome database (HMDB) and Chenomx NMR Suite . Statistical total spectroscopy (STOCSY) [Intracellular metabolites were extracted using a methanol-chloroform-water method described elsewhere . Briefly earlier ,37. Polad before ,37, peak(STOCSY)  was used1H NMR spectra using SIMCA-P 11.5 , upon exclusion of water (5.11−4.69 ppm) and TSP (0.30−0.00 ppm) spectral regions from the matrices. Due to possible contamination, methanol (3.38−3.34 ppm) and ethanol (3.68−3.63 and 1.21−1.17 ppm) regions were also excluded. Spectra were aligned using recursive segment-wise peak alignment [Multivariate analysis was applied to the full resolution lignment , to minilignment . Multivalignment ,37. hAMSCs cultured in basal medium (controls) presented overall similar dsDNA patterns (indicative of cell proliferation) to those observed in osteogenic media until day 14 , except 1H NMR spectra of the polar extracts of hAMSCs from donor 1  +), uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), ethanolamine (Etn), 1-methylnicotinamide (1-MNA) and the unassigned resonance at δ 3.48. We hypothesize that the trajectories followed by these 21 metabolites , with amino acid levels at day 21 approaching those at day 0 (blank rectangles in the right column in δ 3.48 (found to correlate to membrane precursors and adenosine through STOCSY).In order to propose possible osteogenic-specific donor-independent metabolites , selected metabolite trajectories were identified when meeting the following two conditions simultaneously: (i) specificity for osteogenic differentiation , and (ii) apparent donor-independence . We propose that 11 metabolites may serve as candidates for osteogenic-specific and donor-independent markers : (1) taum-inositol feature in both P1 and P2 but lack the symbol †  the 6 robust proliferation markers: alanine, aspartate, proline, tyrosine, ADP and ATP; (ii) the 11 proposed osteogenic-specific metabolites, found in section O1/O2 and in addition to the Cr/PCr, Cho/PCho, Etn/PCho ratios; and (iii) the 5 metabolites found to be osteogenic-specific at least for one of donors 1 and 2: BCAAs, glycine and phenylalanine) were sought for an independently chosen donor 3, in order to attempt some marker validation . This work has compared the detailed metabolic profiling of two randomly chosen donors, during cell proliferation alone and osteogenic differentiation. Donor 1 was found to be more osteogenic  and slightly more proliferating , compared to donor 2. As only two donors were considered, discussion of the reasons for these distinct characteristics is out of scope here, however, the metabolic information found may be correlated to the different donor biochemical parameters.+ levels, increased inosine and decreased 1-MNA), and protein glycosylation (decreased UDP-GlcNAc) [Cell proliferation in donor 1 is accompanied by a well-defined metabolic trajectory, which is not as clear in donor 2. However, amino acids generally tend to accumulate before day 4 in both donors (perhaps in preparation for use for protein synthesis) ,61, is g-GlcNAc) ,62 also -GlcNAc)  to vary Upon osteogenic cues, the most osteogenic donor (donor 1) exhibits a clearer metabolic switch from day 7 to day 14, the overall process being accompanied by generally more persisting changes in several amino acids. This indicated that a higher osteogenic differentiation extension is clearly reflected in amino acid metabolism, at least including their involvement in the biosynthesis of osteogenic protein markers, but also probably as substrates to feed into the TCA cycle. More extensive osteogenesis also leads to lower levels of creatine  and of the cell membrane precursors Cho and Etn. Indeed, the increases in Cr/PCr, Cho/PCho and Etn/PCho ratios identified before as markers of osteogenesis are not as pronounced for the more extensively osteogenic donor. In relation to Cho/PCho and Etn/PCho ratios, it is unclear at this stage if their diminished levels result from differentiation and/or proliferation mechanisms, as the more osteogenic donor 1 also shows slightly enhanced cell proliferation. Furthermore, choline levels are also known to depend on intra/extracellular cross-talk mechanisms , which mIn spite of the above-discussed metabolic differences between the two donors, some osteogenic-specific metabolite changes could be advanced as donor-independent characteristics of osteogenic differentiation. These comprise increased GSH (oxidative protection), increased Cho and Etn and corresponding ratios to PCho , This paper describes, for the first time to our knowledge, an untargeted NMR metabolomics study to measure hAMSCs polar endometabolome as a function of proliferation and osteogenic differentiation, to identify donor-dependent and donor-independent metabolic features of each process, firstly considering two donors. A set of 18 metabolites could be identified as a donor-independent marker of cell proliferation, including particularly robust variations in alanine, aspartate, proline, tyrosine, ATP and ADP, which are not affected by osteogenic differentiation. Regarding osteogenesis, a set of 11 metabolites were proposed as composing an osteogenic-specific donor-independent signature. This signature includes altered levels of taurine, PCr (and Cr/PCr ratios), GSH, methylguanidine, adenosine, inosine, uridine, Cho, Etn, PCho (and Cho/PCho and Etn/PCho ratios) and a still unassigned resonance at 3.48 ppm. In particular, some metabolites previously suggested to relate to osteogenesis were now linked preferably to the underlying cell proliferation process. Although the fact that only two donors were considered in detail is a clear limitation of this work, their distinct proliferation and osteogenic abilities enabled putative correlation between these properties and the metabolic adaptations to be advanced. Interestingly, many of the above signatures could be confirmed for a third donor, although the results reported here necessarily require validation in a larger cohort of donors, then also enabling a more robust correlation between the metabolic profile and (i) biochemical markers of proliferation and osteogenesis and (ii) donor characteristics. Future developments may entail the use of a validated metabolic signature of successful osteogenic differentiation to help monitor and guide the process into a pure lineage outcome. We further propose that such metabolic features may also potentially be used to predict the osteogenic ability of different donors/tissues at an early stage of the process, and even help evaluate in vivo post-implantation MSC performance.