To increase the intracellular concentration of DCA, thereby potentiating its effect, DCA-loaded poly(lactic-co-glycolic acid) nanoparticles were produced. At higher DCA concentrations, encapsulation was found to increase its toxicity. These results may help find a new treatment strategy through combined therapy, which could open doors to new treatment approaches.Antiglycolytic agents inhibit cell metabolism and modify the tumor’s microenvironment, affecting chemotherapy resistance mechanisms. In this work, we studied the effect of the glycolytic inhibitors 3-bromopyruvate (3BP), dichloroacetate (DCA) and 2-deoxyglucose (2DG) on cancer cell properties and on the multidrug resistance phenotype, using lung cancer cells as a model. All compounds led to the loss of cell viability, with different effects on the cell metabolism, migration and proliferation, depending on the drug and cell line assayed. DCA was the most promising compound, presenting the highest inhibitory effect on cell metabolism and proliferation. DCA treatment led to decreased glucose consumption and ATP and lactate production in both A549 and NCI-H460 cell lines. Furthermore, the DCA pretreatment sensitized the cancer cells to Paclitaxel (PTX), a conventional chemotherapeutic drug, with a 2.7-fold and a 10-fold decrease in PTX IC Non-small cell lung cancer is one of the most common malignant tumors in men and women and a leading cause of mortality worldwide ,2. The h6H12O5, dichloroacetate (DCA)—C2HCl2O2—and 3-bromopyruvate (3BP)—C3H3BrO3—have been assayed as putative antitumor agents that target glycolysis, hampering this metabolic pathway or, in the case of DCA, redirecting pyruvate to acetyl-CoA synthesis  × 100. The DL (drug loading) was calculated taking into account the total dry weight of PLGA NPs using the following equation: DL (%) = [/Total dry weight of NPs] × 100.® 100 RP-18 (5 mm) (250 mm × 4.6 mm) (Merck). Chromatographic analysis was performed in an isocratic mode where the mobile phase consisted of 5% acetonitrile and 95% of 2% phosphoric acid in ultrapure water. The eluent flow rate was 1.0 mL/min. The column was maintained at room temperature, and the injection volume was 20 μL. Detection was performed by UV at 214 nm. All samples were run in triplicate, and the total area of the peak was used to quantify DCA.Free DCA in the supernatant was quantified by high-performance liquid chromatography (HPLC) in a Shimadzu UFLC Prominence System equipped with two LC-20AD pumps, a SIL-20AC autosampler, a CTO-20AC oven, a DGU-20A degasser, a CBM-20A system controller and a LC solution version 1.25 SP2. The UV detector was a Shimadzu SPD-20A, and the column used was a LiCrospherThe cells were seeded in 96-well plates as described and, after adhesion, incubated for 24 h with medium containing DCA-loaded PLGA NPs or free DCA at different concentrations . The influence of empty PLGA NPs on cell viability was also tested and adjusted according to the concentrations of DCA-loaded PLGA NPs. At least three independent assays were performed in triplicate, and cell viability was determined by the SRB assay, assuming 100% viability for untreated cells in each case.p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.The results presented correspond to the average of triplicates of at least three independent experiments. Results were expressed as means ± SD. For the statistical analysis, GraphPad Prism 8.3.1 software was used. All the assays were analyzed using one-way ANOVA, considering significant values to be * MDR is one of the major causes of treatment failure in cancer. This phenotype can be associated with several causes, including the energetic metabolism of cancer cells, which mainly relies on glycolysis, either in aerobic or anaerobic conditions. In this study, we aimed to exploit the effect of GIs on cancer cell properties, namely by investigating how they can overcome such a phenomenon of resistance to conventional drugs, opening doors for new therapeutic strategies. Thus, our overall objectives were: (i) to analyze the effect of the GIs 3BP, DCA and 2DG on lung tumor cell line properties; (ii) to verify their ability to reverse the MDR phenotype, when used in combination with PTX, a conventional drug used in lung cancer therapy; and (iii) to increase the efficiency of DCA delivery to tumor cells after its encapsulation into polymeric NPs, which may overcome limitations regarding the maximum dose of GIs that can be used.50, using the SRB assay , and determined the respective ICRB assay .50 values for these GIs but a higher IC50 value for DCA , HKII . Further50 values of each GI. Flow cytometry analysis showed that the mechanism of cell death depended on the cell line and on the compound used.To assess the cell death mechanism induced by the GIs 3BP, DCA and 2DG, Annexin V/PI and caspase-3 assays were performed. Concerning the A549 cell line, untreated cells (control) showed a basal level of around 0.5% and 10% of apoptotic and necrotic cell death, respectively. Both DCA and 2DG, but not 3BP, induced an increase in apoptotic levels, whereas necrosis was stimulated by DCA and 3BP but not by 2DG. DCA induced the greatest effect, resulting in 40% of cell death, mainly by necrosis (approximately 36 ± 4.31% of necrosis and 3% of apoptosis). The treatment with 2DG induced an increase in apoptotic cells only (around 3%) and 3BP in necrotic cells only (around 20%).An increase in caspase-3 activity was not observed in A549 cells treated with 3BP, DCA or 2DG , which mConcerning the other cell line, NCI-H460, untreated cells (control) showed a basal level of around 10% and 4% of apoptotic and necrotic cell death, respectively. In this case, and differently from A549 cells, DCA induced apoptosis only, whereas 3BP induced both mechanisms of cell death. No effect, both in apoptosis and necrosis, was observed in 2DG-treated cells. In this cell line, it was 3BP that produced the greatest effect, resulting in 70% of total cell death (45% of apoptosis and 25% of necrosis), whereas DCA significantly induced an average of 38% of total cell death (about 35% of apoptosis and 3% of necrosis). In agreement with these results, treatment with 3BP revealed an increase in caspase-3 activity. However, the increase in apoptotic cells, determined by the annexin assay for DCA-treated cells, was not confirmed, suggesting that the annexin assay is more sensitive than the caspase-3 assay. For 2DG, no increase in apoptotic rate was observed, neither through the annexin assay nor through the caspase-3 activity assay. As such, these results may suggest that 2DG induces cell death by another mechanism—likely autophagy. In fact, some authors have reported that, in vitro, 2DG induces autophagy in different tumor cell types ,56.In summary, for both cell lines, an increase was observed for both apoptosis and necrosis, depending on the cell line, mainly upon 3BP and DCA treatment. Our results are in agreement with other reports that also showed that 3BP induces apoptosis and necrosis and that DCA induces mainly apoptosis ,57. In fIn order to understand if the effect of GIs on cell viability was due to metabolic disturbance in cancer cells, glucose consumption and lactate production, as well as ATP levels, were assessed in A549 and NCI-H460 cell lines .50 values of 3BP, DCA and 2DG. After treatment, extracellular glucose and lactate and intracellular ATP were quantified and normalized against total biomass or protein, respectively  and, in the most sensitive cell line, NCI-H460, this reduction was even more evident (from around 55 mg/dL to 10 mg/dL). DCA treatment also led to the depletion of cellular ATP in both lung cancer cell lines, with a decrease to less than half in A549 and a decrease of approximately 40% in NCI-H460. Accordingly, DCA treatment lowered lactate levels in both cell lines: in A549 cells, lactate produced was reduced from 30 mg/dL (control) to approximately 13 mg/dL (treated cells) and, in NCI-H460 cells, from 35 mg/dL (control) to 10 mg/dL (treated cells). These results indicate that glucose oxidation switched from fermentative glycolysis toward oxidative mitochondrial metabolism. Since DCA can reverse the Warburg effect through PDH activation, DCA-induced stimulation of oxidative metabolism interrupts the metabolic advantage of tumor cells. Due to the frequent occurrence of mutations in their mitochondrial DNA, tumor cells often present dysfunction of the respiratory chain. As a result, they become unable to sustain energy demand . FurtherDifferent results were obtained when the cell lines were treated with the GI 3BP. As previously noted, the A549 cell line was less sensitive to this compound, with a non-significant reduction of the glucose consumed and ATP cell content. However, a significant decrease in lactate production was observed, similarly to the DCA treatment. Consistently with the results that indicate that the A549 cell line has higher rates of oxidative metabolism, the 3BP treatment did not affect the energetic yield of this cell line. In fact, there can be cases where cancer cells also rely on oxidative metabolism. Moreno-Sanchez described the contribution of OXPHOS in a model of lung cancer, where the majority of ATP was produced during OXPHOS ,62. ThisIn NCI-H460, the glucose consumed  was decreased by half after treatment with 3BP, while ATP production was significantly reduced to less than half. However, this alteration was not accompanied by a decrease in lactate production. Due to the metabolic plasticity exhibited by tumor cells, it is not unexpected that these cells could develop resistance to inhibition of a specific pathway through the upregulation of alternative pathways . It is kWhen 2DG was used, the ATP content was not reduced in both cell lines. However, this GI was shown to be able to decrease lactate production, both in A549 and NCI-H460 cells, which is in agreement with its inhibitory effect on the glycolytic pathway.These results show that lung cancer cell lines treated with GIs, namely with DCA, suffer a disruption in their metabolism, with a significant decrease of energy, particularly the NCI-H460 cell line, which is also the most sensitive to the drugs. Furthermore, DCA was the only GI capable of disturbing ATP production in the most resistant cancer cell line, A549.50 value of 3BP, DCA (24 h) or 2DG (48 h)  . The treAs shown in In both cell lines, the highest effect was observed for DCA, where 50% of cell proliferation was inhibited. In effect, the use of glucose supplies cells with intermediates used in other pathways, like lipid, nucleotide and amino acid biosynthesis ,67. As sMigration is a process that offers valid targets for intervention in important physiological and pathological phenomena, such as wound healing and cancer metastases . To stud50 of 3BP. Therefore, the results suggest that 3BP seems to influence the migratory capacity of cells, and such ability may contribute to its anticancer effect. In the A549 cell lines, again, an increase in cell migration was unexpectedly observed with 2DG treatment, consistently with its effect on cell proliferation.The NCI-H460 cell line exhibited a slightly higher migratory capacity . HoweverPTX is one of the most commonly used anticancer drugs in therapies against solid tumors, although the disease relapses frequently due to the development of resistance to the drug . Such reCells expressing MDR proteins, such as Pgp, are known to require ATP as the energy source to pump out drug substrates . Thus, i50 value decreased when the cells were pre-incubated with DCA, showing that this GI can sensitize cells to PTX. However, this effect is less evident in the nontumor cell line HPAEpic. In fact, although this cell line is more sensitive than the A549 cell line to two of the GIs studied, the effect of potentiation appears to be more specific in tumor cell lines. In contrast, for both cancer cell lines, such effect was very evident, even with a lower concentration of DCA (½ IC50), but much more pronounced in the NCI-H460 cell line. In A549, the most resistant cell line to PTX and to GIs, the IC50 value decreased 2.7-fold, whereas, in the NCI-H460 cell line, the IC50 value decreased 10.1-fold. ATP depletion and exported lactate should affect proteins putatively involved in chemoresistance, which can be present in cancer cells. In fact, the cancer cell line treatment with DCA had almost the same effect on metabolism in both cell lines (as assessed through cellular ATP levels and lactate production), for which a similar effect could be expected for the decrease in PTX IC50 in both cell lines after DCA pretreatment. However, the decrease in PTX IC50 did not parallel the effects on metabolic parameters, being more pronounced in NCI-H460 cells. These were shown to be intrinsically more sensitive to GIs, which suggests that other metabolic parameters and/or membrane transporters and proteins involved in drug resistance may contribute to cell line sensitivity to PTX.The three untreated cell lines presented similar sensitivity to PTX and, in all of them, the ICOur results demonstrate that the biological activity of DCA is mainly due to its ability to decrease tumor cell metabolism. However, there are disadvantages to a metabolism-based approach in cancer therapy, since the metabolic pathways required for cell survival are also present in normal cells. Thus, metabolism-based treatment can face a major hurdle of non-specific toxicity . TherefoThe average size is within the 125–130 nm range. The encapsulation of DCA into PLGA NPs did not change the particle size. However, there was an increase in the polydispersity index  and a decrease in the zeta potential (from −4.31 to −8.99). The increase in PdI and the decrease in the zeta potential may be indicative of some aggregation and fusion of nanoparticles . HoweverThe formulations, represented as DCA and DCA-loaded PLGA NPs, showed a concentration-dependent effect on the viability of lung cells. After 24 h, no significant differences were found in all samples at concentrations between 10 and 100 mg/mL. Viability results showed that PLGA NPs did not lead to significant cytotoxicity in lung cells, which is in agreement with the literature . Howeverp < 0.01 for A549 and NCI-H460 cell lines and p < 0.001 for the HPAEpic cell line), which might be interpreted as a result of increased intracellular deposition of the drug. The decrease in viability might be associated with the fact that DCA has been internalized by lung cancer cells, binding to the intracellular components, namely PDK. By blocking PDK, DCA shifts pyruvate metabolism from glycolysis to OXPHOS, allowing a decrease in cell viability. Indeed, our study demonstrated that encapsulation was successfully achieved, enabling an observable biological effect on lung cancer cells. Although not explored here, it may be expectable that the surface modification of NPs with an active targeting fraction would increase target specificity [Even so, this concentration is much lower than those used in the previous assays. The results showed that DCA-loaded NPs allowed a decrease in cell survival , treateux pumps . This, tOur results with DCA were quite promising, given that the decrease in cell viability upon DCA pretreatment was higher for the tumor cell lines than for the normal cell line. However, the fact that the effect on cell viability was not absent for HPAEpic underlines the need to enhance drug targeting to tumor cells. Since the inhibition of the metabolism of healthy cells and the significant drug accumulation outside the tumor cells could lead to serious adverse effects , we aimeTumor cell biology is extremely complex, and an array of factors can be involved in the MDR phenotype, thus compromising chemotherapy outcomes. Many other components , complementary to those assayed in this work, putatively represent valuable targets of anticancer therapies and are being explored as part of new therapeutic approaches. In this work, the effect of a standard drug already in use (PTX) has been intensified by exploring the reprogrammed metabolism as the ‘Achilles heel’ of cancer cells through the use of a GI (DCA). The effect was further potentiated by NPs encapsulating the DCA. Thus, the results herein presented demonstrate the potential of “all in one” therapeutic approaches, combining multiple strategies  as the key to efficiently and selectively targeting tumor cells.


Flow cytometry is the gold-standard laser-based technique to measure and analyze fluorescence levels of immunostaining and DNA content in individual cells. It provides a valuable tool to assess cells in the G0/G1, S, and G2/M phases, and those with polyploidy, which holds prognostic significance. Frozen section analysis is the standard intraoperative assessment for tumor margin evaluation and tumor resection. Here, we present flow cytometry as a promising technique for intraoperative tumor analysis in different pathologies, including brain tumors, leptomeningeal dissemination, breast cancer, head and neck cancer, pancreatic tumor, and hepatic cancer. Flow cytometry is a valuable tool that can provide substantial information on tumor analysis and, consequently, maximize cancer treatment and expedite patients’ survival. Flow cytometry (FC) is the gold-standard laser-based technique to measure and quantify cell features. It is an indispensable method in basic, translational and clinical research. The utility of cytometry is based on the use of a flow cytometer and specific reagents, including fluorochromes such as fluorescein, and data analysis software . Such a Flow cytometry has been widely utilized in the detection and analysis of several diseases, including hematological malignancies, where it may advise diagnosis, guide therapy and be used to follow-up on disease progression ,2,3. FC Cancer is a leading cause of human mortality. Early cancer detection improves treatment and optimizes patient outcomes and survival. Intraoperative flow cytometry (iFC) has recently emerged as a novel method for cancer diagnosis and treatment, originally being applied in central nervous system tumor surgery and, subsequently, in a number of other malignancies. In the present literature review, we present intraoperative flow cytometry as a novel and adjunct method for standard histopathological and frozen diagnosis to analyze tumor samples and tumor margins. The findings from current reports support the further use of iFC for the accurate characterization of cancer biology and tumor margins.The life cycle of a cell, from the beginning of its existence till its division into two daughter cells, is most commonly called the cell cycle. The cell cycle includes a harmonized sequence of events that leads to two cells that share the same amount of DNA. To better understand and study the cell cycle, we divide it into different stages or phases, including the G1, S, G2, and M phases. The G1 earlier phase is the stage where a cell “decides” whether to divide or not. If not, it enters a state of low metabolic consumption, called G0, and if yes, it enters the latter phase of G1, where it prepares to duplicate its DNA . The nexFlow cytometry provides a valuable tool to assess the DNA quantity of individual cells, which is proportional for the cells in the G0/G1, S, and G2/M phases, following staining of DNA with a fluorescence dye such as propidium iodide. In addition, following comparison with a normal diploid cell, FC may indicate aneuploidy, which holds diagnostic and prognostic significance.Alexiou GA et al. described the tumor index (TI) as an important parameter investigated through the DNA histogram that correlates with the proportion of cells in the S and G2/M phases and the degree of tumor malignancy ,6,7. TheCentral nervous system tumors are the leading cause of cancer death among females younger than 19 years old and among males younger than 39 years old . IntracrMaximal safe resection is the mainstay treatment of low- and high-grade gliomas . A comprGiven the clinical importance of maximal safe resection, several techniques that can guide tumor resection have been developed over the last years. Among them, intraoperative MRI is very useful to maximize tumor resection; however, it is very expensive and time-consuming ,13,14. FRecently, desorption electrospray ionization-mass spectrometry imaging (DESI-MS) has emerged as a promising tool for the intraoperative analysis of a tumor margin resection ,17. SuchAnother promising tool for tumor diagnosis is intraoperative flow cytometry. In an early experimental report by Mesiwala et al., the feasibility of iFC was supported in the analysis of fresh beef-brain tissue samples. An ultrasound aspiration was utilized for the removal and homogenization of pathological tissue from brain tumors, followed by staining and in situ automatic measurements of DNA content .In a report of iFC in human brain tissue, Alexiou G.A. et al. reported normal brain tissue from epilepsy samples that had a G0/G1-phase fraction of 97.1 ± 0.5%, S-phase fraction of 1.7 ± 0.5%, and G2/M-phase fraction of 1.25 ± 0.5% . The higDifferent intraoperative flow cytometry protocols have been developed recently for brain tumor analysis. Shioyama et al. described a detailed flow cytometry protocol for a tumor DNA analysis within 10 min based on the malignancy index . The IoaThe utility of iFC has also been proved to go beyond diagnosis. Alexiou et al. described the association between the G0/G1 and S phases and tumor prognosis. Patients with a G0/G1 phase lower than 70% and an S phase greater than 6% presented a worse prognosis. Interestingly, such findings were also observed in meningiomas of grades I and II/III in the G0/G1-phase, S-phase, and G2/M-phase fractions [In pediatric brain tumors, including ependymomas, medulloblastomas, atypical teratoid/rhabdoid tumors, astrocytoma, and PNETs, a higher G0/G1-phase fraction and significantly lower G2/M-phase fraction were observed in low-grade lesions ,6,29,30.Shioyama et al. reported the malignancy index (MI) using flow cytometry in patients with high-grade gliomas surgically treated and followed by radiotherapy and temozolomide . The MI In intracranial meningiomas, flow cytometry analysis is a promising tool in prognosis and diagnosis. The malignancy of these lesions has been associated with proliferative potential and aneuploidy, as well as with cerebral edema .Since FC analysis can quantify the expression of phenotypic markers, such information can assist in tumor diagnosis. For example, the CD45 marker, a glycoprotein expressed in lymphohematopoietic cells, is diagnostic of primary central nervous system lymphoma (PCNSL). In addition, the expression of an additional marker, CD20, in PCNSL is considered to have a prognostic and therapeutic value, in which treatment with rituximab, a monoclonal antibody against the CD20 antigen, leads to improved survival. Therefore, flow cytometry may have an additional role in the therapeutic strategy in PCNSL patients . AnotherFlow cytometry has also proved useful in the analysis of cerebrospinal fluid (CSF) tumor dissemination. Leptomeningeal metastases (LM), known as meningeal carcinomatosis, is a diffuse dissemination of tumor cells into the CSF and leptomeninges . Almost The most common tumors that develop LM occur in patients with melanoma, breast cancer, small-cell lung carcinoma, and non-small-cell lung carcinoma ,34,35,36Current methods for LM screening include Gadolinium-enhanced MRI and CSF cytology. The sensitivity of MRI and specificity is 75% and 20–91%, respectively . In patiPatients with undiagnosed LM for solid tumors may benefit from flow cytometry methods. Such a method may reduce the diagnostic uncertainty of LM and unnecessary lumbar punctures can be prevented. Flow cytometry provides information on diagnosis and prognosis in patients with LM, optimizing treatment initiation and improving patient survival and quality of life .In invasive breast cancer, the prognosis is directly related to patient age, histological grade and subtype, tumor size, lymph node status, and mitotic index . In addiIn patients with invasive breast cancer, DNA ploidy has been shown to be determined in the early stages of carcinogenesis . ChromosFlow cytometry analysis provides information on nuclear DNA quantification, including on the DNA index, % of S-phase fraction, and ploidy status. DNA ploidy is associated with survival, and presence of aneuploidy is correlated with poor prognosis . In addiLymph node involvement presents often in patients with a higher incidence of aneuploidy than in patients with no lymph node involvement. Furthermore, it has been observed that there is a correlation between DNA diploidy and node-negative tumors.Breast cancer tumors presenting with a Ki-67 labeling index of 50% or above are highly proliferative or aneuploid . AdditioIntraoperative flow cytometry analysis of the DNA status in breast carcinoma from tumor samples is a powerful tool to inform prognosis, which provides information on the percentage of cells in the S phase, a hallmark of tumor aggressiveness ,50. In aSquamous cell carcinoma of the head and neck is a very aggressive disease with high mortality and morbidity ,52. By tTumor margin resection is a prognostic factor for patients with squamous cell carcinoma, thus improving survival rates and progression-free disease rates . Frozen Recently, fluorescent tumor tracers have proven effective to visualize tumor margins intraoperatively ,55. The Intraoperative flow cytometry analysis is a promising tool in the diagnosis of head and neck tumors ,58,59,60A cut-off value of 88% for the G0/G1 phase had a 97.4% sensitivity and 90% specificity for the diagnosis of neoplastic lesions. For the G2/M-phase fraction, the cut-off value was higher or equal to 5% (80% sensitivity and 86.7% specificity) . The cutDespite the recent advancements in intraoperative tracers and frozen section analysis, flow cytometry is a novel tool for the diagnosis of squamous cell carcinoma in head and neck regions. Such a technique has shown high sensitivity and specificity in the diagnosis of these intraoperative neoplasms.Lung cancer has the highest incidence of all tumors, in which non-small-cell lung cancer comprises 80% of new cases of pulmonary tumors ,62. CompApproximately two-thirds of the patients with lung cancer with a potentially curative tumor resection will present tumor recurrence and/or disseminated disease . AccordiNovel methods have been developed to identify cancer cells disseminated in the circulation at early stages . Among tFlow cytometric analysis provides information on up to four different types of molecules simultaneously on a single-cell basis, thus minimizing the false-positive results . The spePancreatic cancer is considered one of the most aggressive tumors, presenting high morbimortality rates . OverallPositive margins and metastasis in pancreatic cancer are associated with reduced overall and recurrence-free survival. Currently, frozen analysis is the gold-standard technique for the detection of positive margins .Intraoperative flow cytometry has shown that it can evaluate the percentage of proliferating cells and is an effective method for detecting cancer cells and the status of tumor margins . Such a The most common primary liver tumor is carcinoma hepatocellular (HCC), the fifth most common cancer, which is also considered one of the most common causes of cancer mortality in men. Complete surgical resection is the mainstay treatment for overall survival in patients with liver cancer or hepatic metastatic disease ,72.The status of resection tumor margins is used to assess the prognosis and risk of recurrence. Novel studies using intraoperative flow cytometry have the potential to improve surgical resection and, therefore, overall survival.Intraoperative flow cytometry tumor analysis is based on the quantification of DNA content, also known as ploidy, and cell-cycle distribution for tumor cell analysis and margin evaluation. Using flow cytometry analysis software, one may determine the G0/G1 geomeans, according to the fluorescence peak .As mentioned before, such techniques may identify areas corresponding to proliferating cells (S phase and G2/M cell-cycle phases) and/or cells with altered DNA content.In addition, the tumor index reflects the proliferative potential of cell analysis in the S and G2/M phases. A tumor index value over 5% is suggestive of cell proliferation .Markopoulos et al., using intraoperative flow cytometry in patients with hepatic carcinoma margin samples, presented similar results as frozen analysis, demonstrating specificity and sensibility of the flow cytometry method . Such a Circulating tumor cells from tumor tissues are an independent risk factor associated with the prognosis of solid tumors, including prostate cancer, breast cancer, colon cancer, and hepatocellular carcinoma. These cells are a source of tumor recurrence or metastasis. Therefore, patients presenting elevated CTC counts often have a high recurrence rate, poor outcomes, and decreased overall survival after surgery .The number of CTCs is also associated with the presence of microvascular invasion, particularly in patients diagnosed with HCC. The detection of circulating CTCs may provide a therapeutic window before tumor metastasis. Therefore, the early detection of circulating CTCs may be an additional prognostic indicator to conventional TNM staging and may be a marker of early tumor vascular invasion .Liu et al. have described a strong association between CTC counts and the karyoplasmic ratio, the presence of microvascular invasion, and HCC prognosis using imaging flow cytometry . AccordiRecent studies of the intraoperative use of flow cytometry contribute to the characterization of tumor margins, the prediction of CTCs, and the potential for complete tumor removal. Intraoperative knowledge of tumor metastasis and complete surgical resection could identify the patients who are at a high risk for local recurrence, assist with follow-up, and help improve overall survival .Colorectal cancer is the third most common cancer and represents 10% of all cancer diagnoses. Intraoperative flow cytometry has been utilized for the detection of colorectal cancer cells and in potential resection margin evaluations. In a study that included 106 colorectal cancer patients, samples from malignant and normal colon epithelium were taken and analyzed. The results showed that a threshold of 10.5% for the tumor index (S and G2/M cell-cycle phases) predicted the presence of cancer cells with a 91% accuracy (82.2% sensitivity and 99.9% specificity). For the margin assessment in the subpopulation of rectal cancer patients with or without neoadjuvant therapy, the accuracy of intraoperative flow cytometry was 79% and 88%, respectively .Intraoperative flow cytometry has been also utilized for cancer cell identification during surgery for gynecological malignancies. In a recent study, 21 aneuploid cancers were detected following the DNA index calculation. Tumor samples were characterized by a significantly lower percentage of cells in G0/G1, as well as by an increased tumor index .Advances in intraoperative molecular analysis using flow cytometry have the potential to improve real-time tumor diagnosis. Such a technique is an attractive adjunct to frozen analysis, especially in time-sensitive scenarios such as the intraoperative assessment of oncological procedures, particularly in brain tumor biopsies and tumor margin resections. Flow cytometry analysis is feasible, cost-effective, and, most importantly, highly sensitive in detecting tumor cells, including CSF dissemination. It is a promising technique that provides substantial information on tumor biology, enabling oncologists and surgeons to maximize cancer treatment and expedite patients´ survival. The data from a plethora of malignancies support the further development of iFC and its utility in the operation theater as a valuable tool in surgical oncology.


Prostate adenocarcinoma (PRAD) is the most frequent malignancy, and is the second leading cause of death due to cancer in men. Thus, new prognostic biomarkers and drug targets for PRAD are urgently needed. As we know, nuclear receptor Nur77 is important in cancer development and changes in the tumor microenvironment; whereas, the function of Nur77 in PRAD remains to be elucidated. The TCGA database was used to explore the Nur77 expression and its role in the prognosis of PRAD. It was shown that Nur77 was down regulated in PRAD, and low Nur77 expression was correlated with advanced clinical pathologic characteristics  and poor prognosis. Furthermore, key genes screening was examined by univariate Cox analysis and Kaplan-Meier survival. Additionally, Nur77 was closely related to immune infiltration and some anti-tumor immune functions. The differentially expressed genes (DEGs) were presented by protein-protein interaction (PPI) network analysis. Therefore, the expression level of Nur77 might help predict the survival of PRAD cases, which presents a new insight and a new target for the treatment of PRAD. In vitro experiments verified that natural product malayoside targeting Nur77 exhibited significant therapeutic effects on PRAD and largely induced cell apoptosis by up-regulating the expression of Nur77 and its mitochondrial localization. Taken together, Nur77 is a prognostic biomarker for patients with PRAD, which may refresh the profound understanding of PRAD individualized treatment.  Prostate adenocarcinoma (PRAD) is the most frequent malignancy in male adults  and the Nurr77 , belonging to the nuclear hormone receptor superfamily, the NR4A subfamily , plays aAntiaris toxicaria Lesch  [Malayoside is a kind of cardenolide glycoside which is extracted from the Chinese herb oraceae) ,21. Our To uncover the association between Nur77 expression and cancer, we surveyed Nur77 expression levels in 34 kinds of tumors, which indicated a significantly downregulated expression in the major kinds of tumors, including PRAD A,B. The + T cells, regulatory T cells (Treg), macrophages, and mast cells resting , 14 (for 48 h), and 26 (for 72 h) nM, respectively; along with an IC90 value greater than 1 μM (for 24 h), around 300 nM (for 48 h), and about 200 nM (for 72 h), respectively.We found that malayoside A modulatp < 0.001), and apoptosis induction was also in a time-dependent manner.FCM and nuclear morphology analyses were performed to detect the apoptosis effect. The number of apoptotic cells (sub-G1) increased in a time-dependent manner  in a time-dependent E and dosThe malayoside was then imported into the Discovery Studio 2017 to search for targets in two modes. In the shap mode, 34 targets were obtained; and in the no shap mode, 309 targets were acquired; in addition, 100 targets were gained in SwissTarget. After removing the duplicate values, 383 targets for the regulation of malayoside were achieved. The obtained PRAD-related targets were 2946, 683, 851, 107, and 19 by the GeneCards, DisGeNET, OMIM, TTD, and Drug bank databases, respectively. After deleting the duplicate, 3634 PRAD-related targets were gained. Finally, 230 common targets were obtained between malayoside and PRAD. To achieve a better understanding of the biological functions of these 230 common targets, Metascape software was utilized A,B. Figup < 0.05). Among them, the biological process (BP) entry was 228, the cell composition (CC) entry was 34, and the molecular function (MF) entry was 78, accounting for 67.06%, 10%, and 22.94%, respectively. The top 30 entries are shown in The GO function enrichment analysis of the common target of malayoside and PRAD was carried out through the DAVID database A–C. As ap < 0.05) were obtained through the KEGG enrichment screening. We found that these pathways, including MAPK, PI3K-Akt, ErbB, and HIF-1 signaling pathways, were mainly associated with PRAD  simulation with 100 ns was run based on the initial structure of the docked complex of Nur77 protein with malayoside to investigate the conformation sampling of the ligand into the binding pocket and further analyze the dynamics of the ligand-protein interaction. In addition, the root-mean-square deviation (RMSD) A of the The ligand moved considerably closer to the amino acids and binds extensively into the binding cavity as an outcome of these interactions, showing that the ligand matches well with the receptor’s binding site and that the complex could become permanent over a lengthy period of MD simulations. The root-mean-square-fluctuation (RMSF), a frequently used approach for evaluating the variation and movement of the protein backbone and side-chain residues that appear in MD simulations, was computed according to a reference structure that illustrates the mobility of residues in the binding protein. The hydrophobic channel residues exhibited the lowest RMSF value, indicating the reduction of structural rearrangements, deviation, and variance, while less internal mobility of the complex and the adaptability of these residues were found inside the catalytic sites F.Nonetheless, the RMSF scores of backbone residues in the terminal and loop areas were in the range of 0.3–0.6 nm, indicating that these residues have substantial variations. The protein-ligand complex had robust structures based on the MD simulation, as almost all of the residues remained with an RMSF value at 0.1 nm in most of the trajectories, showing the rationality and validation of this docking model.The binding free energy of chemical malayoside with protein Nur77 was determined using the G_MMPBSA tool to evaluate the activity of the therapeutic molecule . As was Furthermore, the exploration of H-bonding revealed that residues Gln57 and Gln73 play critical roles in the molecular recognition of this complex. Additionally, We have reported that malayoside-induced apoptosis in NSCLC was associated with the inducible expression of Nur77 . In PRADA Nur77 homodimer binding site, NurRE, was activated during T-cell apoptosis ,28. To cPrevious studies have indicated that several orphan receptors, such as Nur77 ,26 and CIt has been reported that the redistribution of transcription factors and kinases is important for the regulation of their activities and the execution of their functions; furthermore, phosphorylation of these proteins is necessary for such redistribution ,32,33. Bp < 0.01), but the redistribution was eliminated or weakened by antisense Nur77 receptor transfection  is the second most frequently diagnosed malignancy . Its dia+ or CD8+ T cells) [+ T cells, macrophages, neutrophils, and dendritic cells were positively correlated with Nur77 expression, while B cells and CD4+ T cells were negatively correlated (−/− CD8+ T cells’ adoptive cell therapy (ACT) is better than wild-type CD8+ T cells [4+ single-positive thymocytes differentiation into Treg cells. The lack of Nur77 in Treg cells shows resistance to tumor growth. In CD8+ cytotoxic T cells, the suppression of Nur77 expression can release the effector activities. In TIME, lacking Nur77 stimulated effective immune responses. Nur77 functions as a potent sensor of the cellular microenvironment, with the activities of controlling metabolism, vascular homeostasis, inflammation, and cancer. Numerous studies have shown that Nur77′s functional roles are associated with its expression levels and subcellular localization ,39. HoweT cells)  or B celT cells)  predicterrelated A,B. The  T cells . Thus, NAntiaris toxicaria Lesch  was used for the target search. The obtained drug targets were standardized by the UniProt database .The Ligand Profile in Discovery Studio 2017 was used for reverse targeting. It was conducted in two modes: shap and no shap. The parameters had defaulted and the website of SwissTarget , TTD  and DisGeNET  databases. The disease targets and the compound targets were mapped to take the intersection and then the Venn diagram was drawn. We imported the common targets to Cytoscape 3.6.1 to construct the “Malayoside-Common Target-Related Pathway-Prostate Cancer” network.Using “prostate cancer” as the keyword, we searched and screened disease-related disease targets through the OMIM . The conditions that were set as species (homo sapiens), minimum interaction score , and free protein spots were removed. We imported the obtained the protein interaction network into Cytoscape 3.6.1 and used the Network Analyzer module for visual processing and analysis. In addition, to obtain the function of the intersection gene and its role in the signaling pathway, we used the DAVID 6.8 platform  to process the intersection target obtained by the screening. We performed a GO enrichment analysis by entering the name list and limiting the species to “homo sapiens” , and selecting the top five entries. The GO analysis and PPI network construction were also conducted by Metascape [To construct a protein interaction network, the obtained key targets were imported into the Metascape database . Additionally, the Autodock Vina (Version 1.1.2) [The Protein Data Bank database (n 1.1.2)  was perf+ counter-ions were introduced by substituting water molecules. The solute was subjected to a position-restrained dynamics simulation at 300 K for 200 ps to equilibrate the system. The entire system was put through its paces in an MD production run at 300 K and 4.5 × 10−5 bar−1 pressure using the Parrinello-Rahman scheme [The GROMACS 2019 software validation of this complex utilizes the ambersb99 force field . The Autn scheme . FinallyEMM stands for the energy of the drug-protein interaction. Additionally, ΔGsol is the sum of the polar and non-polar contributions of ΔGPB (electrostatic solvent free energy) and ΔGSA (non-electrostatic solvent component) to the solvent energy. Since several earlier studies have shown that entropy does not contribute much to the binding free energy to the same protein, the entropy contribution was not calculated in this work [The binding free energy for the complexes of Nur77 protein and malayoside chemical was estimated with the G-MMPBSA method based on the Molecular Mechanics/Poisson-Boltzmann Surface Area approach (MM-PBSA) . The govhis work ,92. 2 at 37 °C. Stable transfected pRC1 CMV-Nur77 antisense LNCaP cells were selected by G418 . Simultaneously, the vector was stably transfected in the same way [Both the human prostate adenocarcinoma LNCaP cell line and the African green monkey kidney cell line CV-1, were acquired from the American Type Culture Collection . Cells were cultured in RPMI1640 medium or Dulbecco’s Modified Eagle’s Medium (DMEM), respectively, and supplemented with 10% FBS, antibiotics, and 5% COsame way . LNCaP cells were inoculated in a 96-well plate at 3000 cells/well. As described previously , after tThe process was the same as described previously . BrieflyThe process was almost the same as the previous protocol . In brieTransient transfection was performed using a modified calcium phosphate precipitation procedure ,94. In tg. The heavy membrane (HM) and cytosolic fractions were obtained by the centrifugation of the supernatants. Both nuclear and HM fractions were lysed by a lysis buffer.As in our previous report, the cells were suspended in a hypotonic buffer , containCells were incubated overnight and then transfected with the plasmid pCMV-Myc-Nur77 or GFP-Nur77 ,72 by LiCells were plated onto coverslips before the treatment with malayoside, then fixed at room temperature using 4% paraformaldehyde. After being permeabilized with 0.1% Triton X-100, the cells were blocked and reacted with the primary antibody, then continued byCy3 labeled anti-mouse IgG or anti-goat IgG conjugated with Alexa fluor 488. The nuclear localization was visualized by DAPI staining.t-test using Prism 5 . The data are shown as mean ± SEM. Differences between groups were compared by a Student’s In this study, we used a bioinformatics analysis to explore whether Nur77 might be a useful prognostic marker for PRAD patients and if it could be a marker for the change of TIME. Moreover, Nur77 could be a target for PRAD. The molecular mechanism of malayoside in PRAD cells has been mapped, which included the NRs and MAPKs signaling pathways. Malayoside may target Nur77 to induce PRAD cell apoptosis and to affect immune infiltration. However, further studies are necessary to elucidate Nur77 expression levels and subcellular localization with tumor-infiltrating cells in PRAD patients. The concise mechanism, especially the exact signaling pathway, will be determined in our future study.


Europe is considered the largest producer of wine worldwide, showing a high market potential. Several wastes are generated at the different stages of the wine production process, namely, vine pruning, stalks, and grape marc. Typically, these residues are not used and are commonly discarded. Portugal generates annually approximately 178 thousand metric tons of wine production waste. In this context, the interest in redirecting the use of these residues has increased due to overproduction, great availability, and low costs. The utilization of these lignocellulosic biomasses derived from the wine industry would economically benefit the producers, while mitigating impacts on the environment. These by-products can be submitted to pre-treatments  for the separation of different compounds with high industrial interest, reducing the waste of agro-industrial activities and increasing industrial profitability. Particularly, vine-pruning residue, besides being a source of sugar, has high nutritional value and may serve as a source of phenolic compounds. These compounds can be obtained by bioconversion, following a concept of biorefinery. In this framework, the current routes of the valorisation of the pruning residues will be addressed and put into a circular economy context. Nowadays, environmental concerns and dependence on fossil resources encourage the scientific community to devote great efforts to the pursuit of renewable sources of energy for the manufacture of products. Strategies and policies for the development of sustainable growth based on the efficient use of natural resources are one of the main targets to achieve a circular bioeconomy in compliance with the sustainable development goals of Agenda 2030 . In factLignocellulosic materials are composed of 0–10% non-structural  and structural compounds  . ResidueAmong food industries, the winemaking sector represents one of the most important in the world. In this context, 7.5 million ha of vineyards were dedicated to grape crops, reaching a production of 73.3 million metric tons in 2017. The European Union has stood out in the world production of grapes (37%) and wine (279 million hL). Among the European countries, Italy (15.9%), France (16.4%), and Spain (12.1%), where the wine industry is an important part of both the economy and culture, had the highest production in the years 2018 and 2017 . The thi4, N2O, CO, and NO2) and heat to the atmosphere, leading to environmental damages [According to the International Organization of Vine and Wine, Portugal appears as the 11th wine producing country and the 9th exporter in the world ,11. Gene damages ,13. Prom damages .The residues of the winemaking industry could be an interesting lignocellulosic biomass to be used in a biorefinery scheme due to their enriched composition in antioxidant compounds (phenolic compounds derived from lignin processing) , xylooliThe aim of this review was the identification of lignocellulosic residues derived from the winemaking industry, as well as their composition and the pre-treatments used for the valorisation of the main fractions into value-added compounds. In addition, different strategies for the integral use of this biomass are addressed within a biorefinery scheme.The harvest of vine and the winemaking process (known as vinification) involves seasonal stages in which a large number of residues (including product enriched with a biodegradable content) are generated . Among tBriefly, grapes are harvested mechanically or manually, stems are removed before fermentation in a process called destemming, and fermentation is carried out by yeasts already present on the grapes (or inoculated). After days of maceration (contact between grape skins and must), the grape must is pressed to separate wine from grape skins, from which grape marc is obtained . These bIn Portugal, the cultivation of vineyards produces about 222,700 ha year, which generates around a range of 2 to 4 t/ha of cultivation of vine-pruning residue ,20,21. FThe grape marc is the main waste of winemaking and accounts for 20 to 30% of the total weight of the grapes . Every 6w/w)   [22,23,2Lactobacillus fermentum CECT 285, Leuconostoc mesenteroides CECT 8146, and Lactobacillus intermedius NRRL B-3693). The bacteria Lactobacillus intermedius NRRL B-3693 reached the production of approximately 69 g/L of mannitol from the red wort and approximately 80 g/L of mannitol from the white wort in 48 h of fermentation, and these results reached yields of 0.888 and 0.895 mol/mol, respectively. The yeast extract was replaced by wine lees in order to reduce fermentation costs, which was possible to reach 59.4 g/L of mannitol for red must and 65.6 g/L for white must in 144 h. The compounds extracted from waste of winemaking are of great importance in several sectors  of the processed material, with a production of approximately 2 to 3 million tons per year [Grape stalks (the grape cluster skeleton) are an important by-product of the wine industry and are present in the bagasse, representing about 3 to 7.5%  and fractionated use (selective separation of lignocellulosic components previously to its use). 2, and hydrocarbons of low molar mass), resulting in liquid (gaseous condensation product) and solid (biochar) fractions. Liquefaction is based on the conversion of a gas into a liquid.The global use includes methods of combustion, gasification, pyrolysis, and liquefaction. Combustion is a chemical reaction between two or more reactants (fuels and oxidants) with a release of energy in the form of heat. Gasification is based on the chemical transformation of solid or liquid fuels into a synthesis gas, capable of being ignited immediately for energy production or serving as raw material. Pyrolysis is based on the thermal decomposition of the lignocellulosic material in the controlled presence of gases . Nevertheless, the main challenge is component separation without chemical degradation. These processes can be classified considering the main fraction obtained: delignification methods (solubilization of lignin), hydrolysis of polysaccharides (solubilization of polysaccharides), and a combination of both.The first step in a biorefinery approach is to alter the recalcitrant structure of the lignocellulosic biomass, also known as pre-treatment. A physical, chemical, physico-chemical, or biological pre-treatment plays a significant role in the fractionation of lignocellulosic materials, to obtain suitable fractions for the production of the desired compounds or value-added products.To follow the concept of biorefinery, a physical treatment must be carried out in advance. This physical treatment consists of reducing the size by milling, or milling drying the biomass, to improve the processing efficiency. Other physical treatments such as ultrasound application, vaporization, extrusion, autoclaving, and voltage electrical discharges are also widely used to alter the structure of the lignocellulosic materials by increasing the surface area of the biomass .2SO4 [Chemical pre-treatment consists of using different catalysts, such as acids, alkali, and oxidizing agents. The most widely used acid for the fractionation of lignocellulosic materials is H2SO4 . It conv2SO4 . These aThe physico-chemical pre-treatment consists of thermal treatments such as hot water, autohydrolysis, and steam explosion. This process solubilizes the components of the lignocellulosic material according to the pH, moisture content, and temperature .Simple and economical operation;sLimited requirements of energy, process water, and chemicals;Limited corrosion;Ability to alter the structure of lignocellulosic material;Selectivity towards polysaccharide losses;High recovery of valuable hemicellulose-derived products;Limited production of undesired degradation products;Production of substrates with high cellulose content and susceptibility towards enzymatic hydrolysis;Generation of high-quality lignin or lignin-derived products; andLimited generation of waste.Biological treatments are based on the use of biological agents, such as fungi, which act as biocatalysts with the function of selectively degrading lignin while preserving cellulose. The enzymatic hydrolysis also has the function of catalysing fermentable sugars from insoluble cellulose or hemicellulose, releasing it into the medium ,49,50. TWith this approach, the residues of wine making have been used as raw materials to produce value-added products as xylitol, ethanol, and antioxidant compounds. Vine-pruning residue (VPR) is a promising lignocellulosic biomass due to its high sugar content, low cost, and abundance. Most free phenolic compounds are present in cell vacuoles. However, it is in the cell wall that lignin, flavonoids, and insoluble polyphenols are conjugated to sugars, carbohydrates, organic acids, proteins, and polysaccharides. However, access to these compounds is required to carry out one or more pre-treatments, generally followed by enzymatic hydrolysis to release the fermentable sugars . Table 2ε-viniferin, trans-resveratrol, and trans-piceid) that have been extensively explored in VPR, flavonoids , and phenolic acids , which are both present in higher concentrations in some vine varieties [Despite representing a small fraction, the extractives have high commercial value due to their pharmacological potential. Furthermore, they are mainly composed of phenolic and volatile compounds, including stilbenes  can also be evaluated using a combination of two pre-treatments  [According to Sánchez-Gómez and co-workers , lignin rolysis) .2SO4 for 15 min at 130 °C, liquid solid ratio 7.64:1 g/g) allows the conversion of the major polysaccharides into monosaccharides. The resulting solid from the acid pre-hydrolysis is subjected to an alkaline hydrolysis  to promote delignification. The phenolic profile analysis carried out shows that the majority of the hydroxycinnamate acids used are ferulic and p-coumaric with high concentrations . The hydroxybenzoic acid that presents the highest concentration is gallic acid (164.4 mg/L) [The acidic pre-hydrolysis (3% H.4 mg/L) .Some authors have used physical treatments such as PEF, HVED, and UAE to extract polyphenol and proteins. There is a significant increase in phenolic compound extraction in both treatments, although at different energy intensities. The treatment that obtains better results with a lower energy expenditure is HVED, with an energy input of 254 kJ/kg, causing greater damage to the cellular structure of the treated VPR. The highest yield of total phenolic content (TPC) is 34.5 mg of gallic acid equivalent (GAE) per g of VPR, with 89% purity and 4.4 mg/g proteins .p-coumaric acid (140 μg/g VPR). These results show the efficiency of the use of HVED combined with enzymatic hydrolysis, which favours the extraction of phenolic compounds in the delignification process [The physical treatment by HVED is combined in another work with alkaline (delignification) and biological treatments (enzymatic hydrolysis) in VPR to obtain different products, such as polyphenols, reducing sugars, and soluble lignin. The TPC (3.7 mg GAE/g VPR), reducing sugars (110 mg/g VPR), and soluble lignin (1.5%) present higher yields when high voltage electric shocks are applied before enzymatic hydrolysis, which also improves the subsequent delignification process, reducing the lignin content by 10%. This combination of treatments also positively influences the concentration of individual phenolic compounds such as ferulic acid (186 μg/g VPR), resveratrol (26 μg/g VPR), and  process .O-rutinoside (125 mg/100 g).In addition, some studies  suggest trans-resveratrol, 156 mg/100 g of trans-ε-viniferin, and 69 mg/100 g of r2-viniferin. The stilbene yield obtained by PFE  is lower than the yield obtained by PLE and Soxhlet. These differences are attributed to the molecular dimensions where the solubility is reduced according to the dimensions of the solute molecules. The extracts obtained by PLE yield 240–258 mg/100 g of trans-resveratrol, which are higher when ethanol is added at 10 MPA and 80–100 °C, with extraction times and solvent consumptions lower than those used in extraction with Soxhlet. However, r2-viniferine 10–70 mg/100 g has an equal or greater yield than that obtained in Soxhlet when using temperatures of 100 °C or less. Nevertheless, a decline is observed when the extraction is performed above 100 °C, probably due to thermal degradation. However, when combined with PSE and PFE treatments, it is possible to obtain a yield of 10–172 mg/100 g trans-resveratrol, 7–99 mg/100 g trans-ε-viniferin, and 0–40 mg/100 g r2-viniferine. These values are lower than those obtained by Soxhlet and PLE. Therefore, although high pressure extraction techniques have been shown to be more economical and efficient for the extraction of phenolic compounds of VPR, further studies are necessary to optimize the combination parameters of the methods [Other high-pressure techniques were also applied to the extraction of VPR stilbenes, such as SFE and PLE. These extraction methods were compared with the ethanolic extraction in Soxhlet, which reaches 231 mg/100 g of  methods .p-coumaric acid (10.3 mg/L extract), gallic acid (8.4 mg/L extract), cinnamic acid (7.2 mg/L extract), and ferulic acid (5.0 mg/L extract). Based on these results, it can be stated that the severity of the treatments may influence the TPC concentration and the phenolic profile of the VPR extracts [It is described in the literature that the hydrothermal treatments can be an environmentally friendly alternative to obtain extracts with a high content of high value-added phenolic compounds. From the extracts obtained by hydrothermal treatments and purified by the ethyl-acetate stage, the yield obtained is between 0.9 and 3.8 g extract/100 g VPR, with a maximum TFC yield of 0.9 g, and routine equivalents (RE)/100 g of VPR and TPC of 1.6 g of GAE/100 g of VPR at a temperature of 215 °C. In the study by Gullón et al. , the comextracts .In the search to optimize the parameters  for the extraction of bioactive compounds of VPR using environmental friendly methodologies and using non-toxic solvents as hydroalcohol mixtures, a study of the extraction was carried out using the CHE method . For CHERecently, ohmic heating was used for the extraction of phenolic compounds in vine prunings. The extracts obtained by OH in two different intensities of electric fields (LEF refers to low electric field and IEF to intermediate electric field) showed 12 different types of polyphenolic compounds, where the ones with the highest concentrations were apigenin (384.2–157.5 mg/100 g VPR), quercetin (287.2–286.8 mg/100 g VPR), ellagic acid (222.9–77.7 mg/100 g VPR), and hesperidin (180.3–149.0 mg/100 g VPR) in IEF and LEF, respectively . TherefoAs previously shown, VPRs contain a large number of phenolic compounds which are of great interest for industrial and pharmacological applications. The results reported in the literature show that the extracts obtained from VPR stand out over other lignocellulosic biomasses  ,83. AmonAlthough apigenin has been little described in VPR, there are some studies that have detected it in high concentrations. This flavanol has great antioxidant activities with analgesic potential, as well as anti-cancer, anti-inflammatory, and healing properties . Furtherβ-damascenone), vanillin derivatives (vanillin and acetovanillone), and volatile phenols (guaiacol and syringol) [Due to their great potential, the bioactive compounds of the VPR were explored in search of different and innovative applications . Some auyringol) .v/v) and 5 g/L of tartaric acid, adjusted to pH 3.5 with 1 m NaOH, where the concentrations of vanillin or guaiacol were higher than the wine odour limits, depending on the variables applied, as well as high concentrations of trans-resveratrol and ellagic acid. Due to the high transfer rate of compounds, it can be said that the VPR has oenological capacity that can increase the sensorial and functional quality of the wines.Cebrián-Tarancón and co-workers  evaluatetrans-resveratrol had higher concentrations, and an increased concentration of 4 mg/L. As for the red wine “Cencibel”, the compound that presented the highest concentration was vanillin, which was four times higher than its odour threshold in all grapevine varieties at all times of addition, independent of the grape variety, shape, and moment of addition. β-ionol was detected only in wines in which VPR was added after the fermentation process. In this sense, the use of VPR as an innovative alternative to modulate the chemical composition of wines, elaborating different wines, enables the connection of viticulture and oenology in a new concept of circular viticulture [Another study  evaluateiculture .Spodoptera littoralis, Leptinotarsa decemlineata, and Myzus persicae. The study proved that the extracts presented significant antifeedant activity using Lolium perenne seeds and stimulated the root lengthening of Lactuca sativa.The antifeedant activity of the aqueous extract of VPR was investigated  in the iListeria innocua, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Salmonella spp.). From extracts obtained by hydrothermal treatments and extraction with ethyl acetate with extract yield between 0.95 and 3.80 g of extract/100 g of VPR, the identified compounds were vanillin, acetovanillone, guaracyl acetone, syringaldehyde, and acetosyringone. The antimicrobial activity of the extracts presented a minimal inhibitory concentration and minimal bactericidal concentration (between 5–20 mg/mL). Thus, the results obtained showed that VPR extracts obtained by autohydrolysis and extraction by ethyl acetate showed great potential of bioactive compounds with antimicrobial and antioxidant activity [Over the years, the emergence of drug-resistant microorganisms in different environments has been increasing, which has reduced the efficiency of antibiotics. Due to the significant presence of high-value compounds of VPR extracts, some authors  have evaactivity .Escherichia coli and Escherichia coli ESA37, resistant to cephalosporins), Gram-positive bacteria (Streptococcus mitis and Streptococcus mitis ESA65 from lactam resistance), and the yeasts Candida albicans ATCC 10231 and C. albicans from amphotericin resistance. The “Touriga Nacional” variety was associated with higher antioxidant activities, with FRAP values of 24.3 mg of ascorbic acid equivalent (AAE)/g of VPR and of DPPH of 35.3 mg of Trolox (TE)/g VPR, both for the extracts obtained by SWE. However, for the protective effect on haemolysis induced by AAPH2,2′-azobis-2-amidinopropane and the antimicrobial activity, the extracts of the variety “Touriga Nacional” obtained by MAE showed better results. These ranged from 0.6–4.0 mg of extract/mL for minimal inhibitory concentration, 1.5–6.0 mg extract/mL for minimal lethal concentration, and IC50 of 9.59 μg/mL for inhibition of oxidative haemolysis [Other authors  have evaemolysis .Thus, the results showed that the VPR extracts present great potential as bioactive compounds with antimicrobial, antioxidant, and inhibitory activity against the α-amylase and acetylcholinesterase enzymes, and they could be used as adjuvants in the treatments of diseases such as Alzheimer’s and diabetes. In addition, the extracts obtained through MAE extraction present better results in comparison with autohydrolysis, although this treatment showed higher concentrations of total phenolic compounds. This difference is probably due to the phenolic composition present in each extract as a result of the severity of the treatment. The extracts obtained by autohydrolysis presented higher concentrations of lignin-derived compounds, whereas in the extracts obtained by MAE extraction, the majority of the compounds were derived from the cellular wall. Another factor that may have had influence is the synergy of the complex mixture of the bioactive compounds.(E)-ε-viniferine, (E)-resveratrol, (E)-piceatannol, ampelopsin A, vitisin B, pallidol, (E)-δ-(E)-ω-viniferine, (E)-trans-cis-benzol C, isorhapontigenin, scirpusin A, and a new isomer called isoscirpusin A. The results obtained showed that the oligostilbenoids isolated from VPR have potential benefits to health [A recent study  tested to health .Alternaria sp., Cladosporium cladosporioides, Phoma violacea, Penicillium italicum, and Penicillium expansum. The results obtained for the antifungal activity varied according to the concentrations and extracts; however, none of the concentrations showed 100% inhibition. The antiproliferative potential was also evaluated in four different cancer cell lines  and one non-cancer cell line (CCD 841 CoN) using the MTT method [50 values after 48 h of exposure of 62.8, 54.7, 89, 7, 49.7, and 71.0 μg/mL, respectively. These results can be attributed to the great potential of the compounds  present in the extracts, which provided the selective reduction of viability on cancer cells.Jesus et al.  reportedT method . SimilarSubsequently, Jesus and collaborators  evaluate2SO4 in an autoclave at 130 °C) of vine pruning in the production of adsorbent compounds for the treatment of agro-industrial effluents. In these reports, it was verified that the non-encapsulated VPR hydrolysate removed 27.8% of the dyes, while the in the case of calcium alginate, the spheres removed 77.3%. In view of these results, it can be stated that VPR are a potential biocomposite in the treatment of industrial effluents.There are reports in the literature  proposinLactobacillus paracasei from the hemicellulosic fraction obtained by acid pre-treatment , followed by alkaline delignification , where they obtained about 70.7% cellulose, 1.7% hemicellulose, and 25.5% lignin. After the enzymatic hydrolysis, the glucose yield was 47% and there was an ability to reduce the surface tension of 25.1 mN/m and a minimum concentration to maintain the lower surface tension of an aqueous solution of 1.35 mg/mL of biosurfactant. These values represent an advantage from an industrial point of view due to their greater efficiency.Other authors  have stuThe fraction of VPR cellulose previously fractionated by alkali treatments (4 wt% NaOH solution at 80 °C for 2 h) and the fibers obtained in this process by bleaching three times in acetate buffer  and aqueous sodium chlorite (1.7%), followed by an acid hydrolysis (NaOH) process, were also experimented upon for the production of cellulose nanocrystals regarding the development of nanocomposite materials. The obtained cellulose nanocrystals presented a needle shape with an average length of 456 nm and an average diameter of 14 nm, and had a high crystallinity index , which allowed the removal of 69% of hemicellulose and obtained a recovery of 96% of cellulose, followed by alkaline treatment with 3% NaOH at 120 °C without stirring for 60 min, which recovered 75% of cellulose and 25% of lignin. However, for the improvement of enzymatic hydrolysis efficiency, it was necessary to carry out a milder pre-treatment of the biomass with 2% NaOH at 100 °C to achieve a glucose yield of 98.72% with 35.06 g L and 31.9% of lignin [Other authors have evaluated the acid and alkaline treatments for the best recovery of cellulose from the VPR, allowing an efficient enzymatic hydrolysis. The pre-treatment conditions used were acidic Hf lignin .2SO4) in a microwave technology as an alternative treatment to solubilize the lignin and hemicellulose of VPR in order to obtain a solid rich in cellulose. In this study, it was possible to solubilize 85% of lignin of good purity, resulting in a solid of 75% cellulose. After the treatment, the lignin was separated from the medium and the hemicellulose derivatives of the aqueous phase were converted into 64.6% furfural.Recent studies  used a c2SO4, 134 °C for 17 min with 10% w/w VPR, followed by enzymatic hydrolysis. These values are within the range described by other authors for VPR [Bacillus coagulans DSM 2314, Geobacillus stearothermophilus DSM 2313, and G. stearothermophilus DSM 494 were tested for lactic acid production under aerobic and non-sterile conditions. The strain that showed the best results in fermentation was B. coagulans DSM 2314, which was able to produce approximately 29 g/L of lactic acid in 24 h, reaching a sugar consumption of 99 and a yield of 96% when supplemented with the lees of red wine, without the need for detoxification steps.In another study, Garita-Cambronero and co-workers  applied  for VPR . After sBifidobacterium population increased by 14%. These results demonstrated efficiencies in the use of VPR oligosaccharides treated as a prebiotic constituent with functional properties important for human health. Finally, some of the potential applications of oligosaccharides derived from vine-pruning hemicellulose are shown in Recent studies  have invPrevious studies have shown that VPR was also exploited as an energy source for boilers in the wine industry, replacing pine pellets with VPR chips. These have a high heating value (18 MJ/kg on a dry matter basis) . Combust2 and N2 use at two absolute pressure levels (0.1 and 1.1 MPa) at 600 °C of pyrolysis of VPR. They found that pyrolysis in CO2 and N2 atmospheres yields a similar fixed carbon and biochar mass. In a CO2 atmosphere, the CO2 yield was reduced; however, the CO2 yield increased. Therefore, these results indicate that the recycling of CO2 obtained by the combustion of VP as the pyrolysis atmosphere is a promising substitute for N2, which is a relatively expensive gas.Methodologies such as pyrolysis lead to the significant production of combustible gases and are a good source of graphite carbon, with higher calorific value. Due to the high lignin content, grape pruning in nature is an excellent raw material for the production of biochar. Some authors  have stu2 and KOH, where the CO2 absorption capacity at different temperatures (0.25 and 75 °C), apparent CO2 selectivity on N2, and isosteric adsorption heat were tested. The charcoal activated chemically at 25 °C and presented a higher adsorption capacity of CO2, overcoming results obtained in similar works with other lignocellulosic materials. However, the coals prepared with physical activation at 800 °C during 1 h of immersion presented a higher adsorbent capacity of CO2 with high adsorption rates at higher temperature. Based on these results, it can be stated that VPR biochar is a promising material that can also be used as an efficient adsorbent with several applications, such as CO2 after combustion, biogas upgrading, and H2 purification.Other authors  have evaFor the production of biofuels through lignocellulosic biomass, it is necessary to perform different pre-treatments (such as hydrolysis) to break down the recalcitrant lignin structure, aiding the enzymatic and microbial accessibility ,30,47,51w/w), 125 °C, and 110 min) and subsequent enzymatic hydrolysis, a strategy adopted to reduce the concentration of phenolic inhibitors and increase the recovery of fermentable sugars. These conditions allowed obtaining 42.84 g/L of total sugars corresponding to 65.21% recovery and 0.84 g/L of phenolic compounds. The hydrolyzate produced was subjected to fermentation by 11 strains of Clostridium, where the highest concentration of butanol (8.0 g/L) was obtained by the strain C. beijerinckii CECT 508.Garita-Cambronero and collaborators  studied As for the production of bioethanol, some authors used pre-treatments by alkaline hydrolysis of VPR, followed by enzymatic hydrolysis and fermentation for the production of glucose as a source of carbon for the production of bioethanol . The resSome authors  have useTherefore, this work shows that VPR is a promising biomass for the production of biofuels, energy, and chemical products, as it is a renewable and low-cost alternative to petroleum. Previous studies have shown that vine pruning has a higher xylan and glucan content than those found in other residues from the wine industry  and fromCurrently, the biorefinery concept is encouraged for the valorisation of residues obtained from the agricultural and food industries in order to achieve a sustainable development based on a bioeconomy. Considering the above-mentioned chemical composition of VPR, several value-added products can be obtained using different biomass pre-treatments. In this sense, it is necessary to develop biorefinery configurations to promote the efficient separation of their components, as well as their further conversion into chemical compounds and derivatives.This approach has been recently applied by several authors ,69 that Thus, through the studies developed so far for the full recovery of VPR, it is possible to consider an integrated biorefinery approach, based on different processes, to obtain a cascade of multi-products . In thisThe solid resulting from the two autohydrolysis processes can be explored in different ways. They can be subjected to acid hydrolysis to obtain nanocrystalline cellulose, or saccharified through enzymatic hydrolysis. After conversion, the glucose can be fermented, thus giving rise to different products such as ethanol, biobutanol, L(+)-lactic acid, biogas, and biosurfactants. At this point, the solids resulting from the lignin-rich fermentation process can be subjected to pyrolysis and transformed into biochar. On the other hand, other authors have suggested the valorisation of the solid fraction resulting from the fermentation process for the production of wood bioethanol and grape stems as a source of raw material for the production of cellulose nanocrystals ,111.Thus, the biorefinery concept presented is suggestive for the full recovery of vine-pruning residues, making these promising substrates for the economic production of bioethanol, xyoligosaccharides, bioactive compounds, and lignin, among others. These studies show that it is possible to make the recovery of waste from the wine industry, leading to a reduction in production costs and waste avoidance, in accordance with the principles of the circular economy.The composition of lignocellulosic biomass may vary depending on geographic location, climate, process, and variety of vine studied. There are several lignocellulosic biomass conversion studies of derived wine residues, and these are here being reviewed. Still, there are some fractionation studies of wine residues related to the extraction and production processes of phenolic extracts and antioxidants. Nevertheless, a biorefinery approach could be addressed to obtain added-value products from the different fractions of residues generated in the winemaking industry. The main limitation on the use of lignocellulosic materials obtained from the winemaking industry is to know their chemical composition and to use a suitable pre-treatment for the proposed objective.This review addresses the most recent studies that use the valorisation of VPR as a raw material for the production of products of industrial interest, as well as their perspectives, their potential, and future challenges. The main limitations encountered were the diversity of processes and products found and the lack of standardization of the units of measurement, which increased the complexity of the comparisons. Based on the analyses, it was possible to observe that there is a need to find an effective, clean, and economically viable methodology for the effective fractionation of VPR. VPR is a high value-added by-product, and the development of an effective fractionation methodology promises to make possible the full use of all the existing fractions within an integrated biorefinery. This would allow directing the refined products to specific industrial sectors, and thus contributing to the circular economy.


Therefore, in our study, the effects of Ag NPs suspension (containing Ag0 at 20 mg/L) and AgNO3 solutions (with the concentration of Ag+ ions at 20 and 50 mg/L) on the seed germination and early seedling growth (4 days) of pea (Pisum sativum L.) were compared. Both Ag NPs and AgNO3 did not decrease seed germination, and even stimulated seedling growth. In seedlings developing in the Ag NPs suspension, an increase in monosaccharides, homoserine and malate was noted. In the next experiment, the effect of short-term seed imbibition (8 h) in AgNO3 at elevated concentrations, ranging from 100 to 1000 mg/L, on the further seed germination, seedling growth (in absence of AgNO3) and their polar metabolic profiles were evaluated. The seed imbibition in AgNO3 solutions at 500 and 1000 mg/L reduced seed germination, inhibited seedlings’ growth and caused morphological deformations (twisting and folding of root). The above phytotoxic effects were accompanied by changes in amino acids and soluble carbohydrates profiles, in both sprouts and cotyledons. In deformed sprouts, the content of homoserine and asparagine (major amino acids) decreased, while alanine, glutamic acid, glutamine, proline, GABA (γ-aminobutyric acid) and sucrose increased. The increase in sucrose coincided with a decrease in glucose and fructose. Sprouts, but not cotyledons, also accumulated malic acid and phosphoric acid. Additionally, cotyledons developed from seeds imbibed with AgNO3 contained raffinose and stachyose, which were not detectable in sprouts and cotyledons of control seedlings. The obtained results suggest the possible disturbances in the mobilization of primary (oligosaccharides) and presumably major storage materials  as well as in the primary metabolism of developing seedlings.The use of silver nanoparticles (Ag NPs) on plants is accompanied by the occurrence of Ag Thus, evaluation of the effects of Ag+ ions should accompany or precede studies of the effects of Ag NPs on plants.The growing interest and use of silver nanoparticles  due to their beneficial properties in various industry sectors, e.g., medicine, textile, cosmetics, home appliance, environment, construction, food, printing, agriculture, sports and fitness, electronics and others, also causes their increasing release to the environment . Nanopar+ ions [+ ions are taken up from the soil by the roots’ epidermal cells, then are translocated between cells through the plasmodesmata  and later on via vascular bundles to the shoot [+ ions can be accumulated in cells, causing oxidative stress [Phaseolus vulgaris and Zea mays [Pisum sativum [Trigonella foenum-graecum [Oryza sativa [Triticum aestivum [Solanum lycopersicum, Raphanus sativus and Brassica oleracea [In recent years, there has been an increasing number of reports indicating the effects of Ag NPs on various plant species ,10, incl+ ions ,18. Ag Nhe shoot ,20. Bothe stress ,22 and de stress ,23, watee stress , mitotice stress ,26. In sZea mays , Pisum s sativum  and Trig-graecum . The effa sativa , Triticuaestivum , Solanumoleracea .3 has been used in in vitro culture of plants as a plant growth modulator in organogenesis, morphogenesis and other physiological processes [Glycyrrhiza glabra L.) seeds after germination in Murashige and Skooge (MS) medium containing AgNO3 had increased root, hypocotyl and shoot growth, and decreased stomata and trichomes in 20-day-old seedlings [3 also increased cell divisions in root and shoot tips, decreasing the number of vascular tissues, the epidermal thickness, and intercellular spaces in the mesophyll [+ ions applied at low doses also enhanced the growth of cowpea seedlings [AgNOrocesses ,33,34,35rocesses . Licoriceedlings . AgNO3 aesophyll . Ag+ ioneedlings .P. sativum L.) is one of the most important commercial legume crops used as food for humans and livestock, mainly due to the high protein content in seeds [+ ions originating from Ag NPs can also affect cell metabolism [Pea  on the further seed germination and seedlings’ growth (in absence of AgNO3) of ten pea cultivars were evaluated to find the toxic concentrations of AgNO3. Finally, the study focused on the effect of Ag+ ions on the primary metabolism in pea seedlings of the Nemo cultivar.Therefore, in the present study, the effects of Ag NPs and AgNO3 at 20 and 50 mg/L concentrations of Ag+ ions did not affect germination of pea seeds (Silver nanoparticles (Ag NPs) at a concentration of 20 mg/L and AgNOea seeds A. Howeveea seeds B, as welea seeds C,D.In seedlings, 42 polar metabolites were identified . There w3, only slight changes occurred in polar metabolites. Regardless of the seed’s treatment, the concentration of TIPMs was higher in the root and epicotyl than that in the cotyledons  led to an increase in TIPMs . On the other hand, in epicotyl, the decrease in TIPMs was due to a drastic reduction in sucrose  and seedling development . However, the content of total amino acids (TAAs) and total organic acids (TOAs) in sprouts was ca. three- and five-fold higher than that in cotyledons, respectively , decreased to 20.13 and 19.92 and to 4.15 and 4.01 mg/g DW. In contrast, these contents decreased to 8.66 and 14.83 and to 3.56 and 3.60 mg/g DW in deformed sprouts, respectively  than in the control (51.24 mg/g DW), while in deformed ones TSCs reached a level of 42.16 mg/g DW  and in cotyledons (0.48 mg/g DW) of deformed seedlings compared to the control .3 increased its content to 10.21 mg/g DW in sprouts and decreased in cotyledons (1.08 mg/g DW).The phosphate was present at a considerably high level in control seedlings , did not negatively affect seed germination. Additionally, they stimulated the early growth of the seedling  had no effect on seed germination, AgNO3 at 500 and 1000 mg/L clearly reduced the process as well as seedlings’ growth. Similar results were also demonstrated in previous studies on the effect of silver nanoparticles on the germination of Cucurbita pepo [A. thaliana [O. sativa [Vicia faba [3, demonstrated in our study  than the concentration of Ag NPs in the mentioned work. Thus, the phytotoxicity of Ag NPs seems to be much higher than that of Ag+ ions. Similar results were obtained previously in studies on A. thaliana [Hordeum vulgare [+, manifested in greater root damage and its inhibited growth. Additionally, previously it was shown that silver nitrate affects callus induction as well as shoot and root formation in wheat [3 also accelerated the growth of licorice seedlings [3 had toxic effects on growing pea seedlings.The changes of root morphology in pea seedlings developed from seeds imbibed with AgNO000 mg/L  were simthaliana ,24 and H vulgare , for whiin wheat . AgNO3 aeedlings  but at leedlings . The resThe much higher contents of quantitatively main polar metabolites  in pea sprouts than in cotyledons  presumabmyo-inositol and traces of monosaccharides (<0.2 mg/g DW). Hydrolysis of RFOs results in the release of galactose and sucrose, which are used to maintain the osmotic potential in the cells of growing sprouts [In dry pea seeds, the major sugars are sucrose and its galactosides  . Their c sprouts  and acts sprouts . Besides sprouts , which e sprouts .3 affected the content of some amino acids in developing seedlings. There were significantly higher decreases in homoserine and asparagine content in deformed seedlings than in those with normal morphology  was constant , thus the osmotic imbalance appears to be questionable. It is considered that malic acid and sucrose are involved in the osmotic regulation of cells [3 treatment, and water/osmotic stresses [The content of malic acid, the major organic acid, was higher in both sprouts and cotyledons of deformed seedlings compared to morphologically normal ones. Previous studies have shown accumulation of malic acid in leaves  and shooof cells , as wellof cells . The malof cells . Thus, tstresses ,70, leadstresses .+ ions can be reduced to Ag NPs, with reducing monosaccharides, ascorbic acid/citric acid, present in cells as it was revealed by Marchiol et al. [+ ions in pea seedlings.On the other hand, organic acids  can chelate metal ions, including heavy metal ions, participating in their accumulation/transport in plants ,78, as wl et al. . Howevermyo-inositol as phytin. During germination, phytin or phytates are a source of myo-inositol, phosphate, K, Mg, Ca, Mn, Fe, and Zn which are released by the action of phytase enzymes and then translocated to developing seedlings [3 associated with seedling deformations. So far, the involvement of phosphoric acid in pea’s response to environmental stresses remains unclear. In addition to the presence in the form of phytic acid, phosphorus compounds play a crucial role in plants’ metabolism as sugar phosphates as well as phospholipids and nucleotides [Another distinct feature found in our study was the high phosphoric acid content in pea sprouts. In the dry seeds, the phosphate is present in a bound form to eedlings . In our leotides .Pisum sativum L.) cv. Tarchalska  were germinated on Petri dishes  in double-distilled water (control) and a suspension of Ag NPs  with a size of 10 nm (at a concentration of 20 mg/L) or in AgNO3  solutions  for 4 days in the darkness . After every 24 h of germination, the number of germinated seeds was scored. After 4 days, the length of root and epicotyl and fresh weight of root, epicotyl and cotyledons of seedlings from 3 replicates were measured, and tissues were frozen in liquid nitrogen and stored in an ultra-refrigerator (at −80 °C) for 2 days. Then, samples were freeze-dried  for 48 h, and dry tissues were pulverized (for 2 min at 22 Hz) in a mixed mill  for extraction of polar metabolites.Seeds of pea  solutions  for 8 h. Then, seeds from each replicate were placed on double-folded sheets of filter paper  wetted with double-distilled water, rolled, and transferred into 250 mL glass cylinders. After 4 days of incubation at 22 °C in the darkness, the length of root and epicotyl and fresh weight of root, epicotyl, and cotyledons from 3 replicates were measured and tissues were dried at 80 °C for 24 h to estimate their dry weight.Seeds of ten pea cultivars , obtained from DANKO and Poznan Plant Breeding Companies (Poland), were imbibed on Petri dishes  in double-distilled water (control) and AgNO3 at concentrations of 500 and 1000 mg/L causing a decrease in sprout (root and epicotyl) growth were used for polar metabolites profiling. Moreover, sprouts developed from seeds imbibed in AgNO3 were separated into two groups—sprouts of typical morphology  and those with visible deformations (deformed). Sprouts and cotyledons were collected separately, frozen in liquid nitrogen, stored in an ultra-refrigerator (at −80 °C) for 7 days, and then freeze-dried  for 48 h.The control seedlings (sprouts and cotyledons) of cv. Nemo, and those developing from seeds imbibed in AgNOv/v, containing 100 μg of ribitol as internal standard) at 70 °C for 30 min. Homogenates were centrifuged  and aliquots of clear supernatant (600 μL) were mixed with 400 μL of cold chloroform to remove non-polar compounds. After centrifugation, the polar fraction was concentrated to dryness in a speed vacuum rotary evaporator . The metabolites were derivatized with 40 μL of O-methoxamine hydrochloride at a concentration of 20 mg/mL in pyridine by heating at 37 °C for 75 min with continuous shaking (500 rpm). Then, 160 μL of a mixture of MSTFA (N-methyl-N-trimethylsilyl-trifluoroacetamide) with pyridine  was added and the mixture was heated at 70 °C for 30 min [m/z. Data were collected and analyzed using GC-MS Solution and LabSolutions software . For the quantitative calculation of the content of identified polar metabolites, only the data obtained for the peak areas from the GC-FID analyses were used.Polar metabolites were extracted from freeze-dried and pulverized roots, epicotyls and cotyledons of seedlings cv. Tarchalska  and spror 30 min . The mix shaking 0 rpm. Thp < 0.05) for multiple comparisons in GraphPad Prism and Statistica. Principal component analysis (PCA) for multivariate statistics was performed using COVAIN [The results are means of 3 independent replicates and they were subjected to analysis of variance using a one-way ANOVA and Tukey’s post hoc test  in the cotyledons, as well as glutamate/glutamic acid, alanine, malic acid and phosphoric acid in the sprouts at an early stage of development indicates a strong disturbance in carbon and nitrogen metabolism, and presumably impaired mobilization of storage compounds. Moreover, AgNO3 can cause oxidative stress resulting in increased accumulation of proline and GABA in pea sprouts. It seems that the effect of Ag+ ions is related to changes in the proteome and transcriptome, which may be the subject of future research. The results also indicate that the phytotoxicity of Ag NPs to pea seeds/seedlings seems not to be determined by the release of Ag+ ions from them. However, further study is needed to verify this hypothesis.Low concentrations of Ag NPs and AgNO