Mechanistically, DIO inhibited the expression of NOX4 and restored mitochondrial respiratory chain (MRC) complex I-V expressions. Further, DIO inhibited mitochondrial apoptosis by ameliorating mitochondrial membrane potential (MtMP) and down-regulating the expressions of CytC, Apaf-1, caspase 3, and caspase 9, while up-regulating Bcl2 expression. Moreover, the ER stress and its associated cell apoptosis were inhibited through decreasing PERK, p-PERK, ATF4, IRE1, p-CHOP, and caspase 12 expressions. Collectively, DIO inhibited ROS production by modulating NOX4 and MRC complexes, which then suppressed apoptosis regulated by mitochondria and ER stress, thereby attenuating DN.Diosgenin (DIO) is a dietary steroid sapogenin possessing multiple biological functions, such as the amelioration of diabetes. However, the remission effect of DIO on diabetic nephropathy (DN) underlying oxidative stress and cell apoptosis remains unclear. Here, the effect of DIO on ROS generation and its induced cell apoptosis was studied in vitro and in vivo. Renal proximal tubular epithelial (HK-2) cells were treated with DIO  under high glucose  conditions. DN rats were induced by a high-fat diet combined with streptozotocin, followed by administration of DIO for 8 weeks. Our data suggested that DIO relieved the decline of HK-2 cell viability and renal pathological damage in DN rats. DIO also relieved ROS (O Diabetes mellitus (DM) is one of the common chronic metabolic diseases with an increased incidence in recent years. Globally, approximately 451 million adults (20–79 years old) suffer from DM in 2017, and the number of people with diabetes is estimated to reach 700.2 million by 2045 , and has2O2 under renal pathological conditions, such as chronic kidney diseases and DN [Oxidative stress is highly positively correlated with the progression of DN ,6. The os and DN . Inhibits and DN  indicates and DN  showed tN-acetylcysteine, tempol, ramipril , and GKT137831/GKT136901 (NOX4 inhibitors) significantly relieved DN by suppressing ER stress and its associated cell apoptosis [Under hyperglycemia, severe oxidative stress leads to sustained damage to mtDNA , which ipoptosis ,22,23,24Dioscorea villosa), fenugreek, smilax bockii warb, and dioscorea nipponoca makino. It has excellent pharmacological activity in the alleviation of hyperlipidemia, diabetes, diabetic kidney injury, and diabetic liver injury [Diosgenin (DIO), belonging to the category of steroidal sapogenin, is primarily obtained from wild yam . When they were cultured to 70–80% fusion, the cells were treated with different concentrations of HG  for 24 h, 30 mM HG for different durations , and DIO  for 24 h under HG (30 mM) condition. Mannitol (30 mM) was used as hypertonic control.Renal proximal tubular epithelial (HK-2) cells were provided by Purosa life technology . The HK-2 cells were cultured in DMEM media of 5.5 mM glucose  added with fetal bovine serum  and penicillin-streptomycin solution  at an incubator  fluorescent probe was used to measure O2O2 level in HK-2 cells. The HK-2 cells (approximately 2 × 106) were homogenized with lysate (200 µL) in an icy bath. Next, the obtained homogenate was centrifugated  to collect the supernatant fluid. Followed by, the supernatant was reacted with the test solution provided by the kit at 25 °C for 30 min. Finally, the absorbance at 560 nm of the samples was detected by the multifunctional microplate reader (Spark).The Hydrogen Peroxide Assay Kit (Beyotime) was used to detect the HThe cell culture and treatment refer to The expression of NOX4 in HK-2 cells was measured by immunofluorescence, as described by our previous study . In brieThe MtMP of the cells was measured by the JC-1 probe (Beyotime). Briefly, the HK-2 cells were cultured and treated as The special pathogen-free Sprague-Dawley (SD) male rats (200 ± 20 g) were provided by Chengdu Dossy Experimental Animals Co., Ltd . All rats were housed under a controlled condition of 20 ± 2 °C of temperature, 60–70% of humidity, and 12 h light/12 h dark cycle. After 1 week of adaptive feeding, the rats were randomly divided into the control group (fed with standard chow) and the DN group. The model construction refers to our previous description . Rats wiThe periodic acid and Schiff’s (PAS), hematoxylin and eosin (H&E), and Masson trichromatic dye analyses were processed according to our previous study . Then thp-CHOP (bs-5177R), PERK (bs-2469R), Caspase 12 (bs-1105R), and p-PERK (bs-3330R) were purchased from Bioss. The ATP6 primary antibody (AF6261) and the HRP-labeled secondary antibody (A0208) were purchased from Beyotime.The protein expressions of the kidneys and cells were detected by Western blot as described previously ,32. The p-PERK (bs-3330R) primary antibody was purchased from Bioss.The immunohistochemistry was performed as described by our previous study . The ATPp value less than 0.05 was considered as statistical significance.All data were presented as the mean ± standard deviation (SD). Significant differences between groups were analyzed by one-way ANOVA, followed by Duncan’s test for multiple comparisons using SPSS 20.0. A 2− and H2O2 are the two main ROS. The fluorescence intensity of DHE is positively correlated with the content of O2−. The results of DHE staining showed that HG treatment resulted in a significant increase of O2− content, while it was reversed in the DIO-treated groups  treatment for 24 h and 30 mM HG treatment for 6, 12, 24, and 48 h A,B. DIO d groups D. Additi-2 cells E. These Since the MRC is the primary site generating ROS, the protein levels of MRC complexes are important indicators to assess ROS production. In this study, the expressions of mitochondrial complex I-V  were significantly inhibited in HG-treated HK-2 cells, while these alterations were ameliorated in the DIO-treated groups . Therefo2O2 production. In this study, the NOX4 expression in HK-2 cells were detected by both Western blot and immunofluorescence. The data suggested that NOX4 expression was significantly up-regulated in HK-2 cells treated with HG and it was effectively inhibited by the co-treatment of DIO  significantly relieved the above pathological changes A,B. In aOxidative stress can cause severe damage to mtDNA and then cause mitochondria-mediated cell apoptosis. DAPI is common staining to detect cell apoptosis. The DAPI staining showed that HG exposure caused HK-2 cell apoptosis (dense or fragmented dense hyperchromatic nucleus) and DIO relieved the cell apoptosis A. The deTo further determine whether DIO ameliorates mitochondria-mediated cell apoptosis in vivo, the expressions of mitochondrial apoptotic proteins in rat kidney tissues were measured by Western blot and immunohistochemistry. Our results indicated that the up-regulation of mitochondrial pro-apoptotic proteins  were inhibited by DIO. While, the down-expression of Bcl2, an anti-apoptotic protein, was restored by DIO application . Therefop-CHOP, and Caspase 12) were up-regulated significantly in HK-2 cells treated with HG. DIO inhibited the up-regulation of these proteins. Consistent with the results obtained in vitro, the up-expressions of IRE1, p-PERK, ATF4, p-CHOP, and caspase 12 were relieved by DIO in vivo  and their downstream proteins including CHOP, ATF4, and caspase 12. Thus, these protein expressions were detected by Western blot and immunohistochemistry in this study. As shown in  in vivo . AltogetOxidative stress refers to the imbalance between the oxidative and anti-oxidant systems in the organism, making it prone to oxidation . NumerouThe current research focus is to develop natural antioxidants to prevent DN, such as vitamin C, vitamin E, vitamin D, resveratrol, quercetin, coenzyme Q10, curcumin, green tea extract, guava extract, etc. . DIO is 2− and H2O2 was observed in HK-2 cells exposed to HG. As expected, DIO significantly decreased O2− and H2O2 levels. Studies showed that ROS generated by the disordered NADPH oxidase and MRC under HG condition contributes to the progression of DN. In the condition of hyperglycemia, damaged mitochondria produce excessive electron leakage in MRC complex I and in the interface between MRC complex III and coenzyme Q [db/db mice and podocytes treated with HG. And the enhancement of MRC complex I expression can inhibit the production of ROS [2O2 in the kidneys [In the present study, DIO relieved the decrease in HK-2 cell viability after HG treatment. Next, the anti-oxidant activity of DIO was investigated. The excessive production of Oenzyme Q . Studiesn of ROS ,40. In t kidneys . The inc kidneys ,12,42,43 kidneys . In thisThe kidneys require massive mitochondria to provide energy, ensuring normal structure and function. The over-produced ROS derived from the defective MRC could in turn impair mitochondrial function . Oxidatip-PERK, p-CHOP, ATF4, IRE1, and caspase 12.In addition to mitochondria, ER stress can also cause cell apoptosis . Under nCollectively, our study indicated that DIO protected against diabetic kidney damage by inhibiting ROS production and cell apoptosis in vitro and in vivo. DIO inhibited ROS production mainly through down-regulating NOX4 expression and up-regulating the expressions of MRC complex I–V. Subsequently, the cell apoptosis regulated by mitochondria and ER stress was suppressed by DIO.


The effects of diet on the GM composition changed over time: in the first 12 weeks, α and β diversity differed considerably according to diet and weight increase. In contrast, at 24 weeks, the composition, although still different between groups D1 and D2, showed changes compared with previous samples, suggesting the beneficial effects of omega-3 fatty acids in D2. With regard to metabolic analysis, the results did not reveal relevant changes in biomarkers in accordance with AT studies showing an anti-inflammatory environment and conserved structure and function, which is in contrast to reported findings for pathogenic obesity. In conclusion, the results suggest that the constant and sustained administration of omega-3 fatty acids induced specific changes in GM composition, mainly with increases in Lactobacillus and Ligilactobacillus species, which, in turn, modulated the immune metabolic response of AT in this mouse model of obesity.Obesity is a chronic, relapsing, and multifactorial disease characterized by excessive accumulation of adipose tissue (AT), and is associated with inflammation mainly in white adipose tissue (WAT) and an increase in pro-inflammatory M1 macrophages and other immune cells. This milieu favors the secretion of cytokines and adipokines, contributing to AT dysfunction (ATD) and metabolic dysregulation. Numerous articles link specific changes in the gut microbiota (GM) to the development of obesity and its associated disorders, highlighting the role of diet, particularly fatty acid composition, in modulating the taxonomic profile. The aim of this study was to analyze the effect of a medium-fat-content diet (11%) supplemented with omega-3 fatty acids (D2) on the development of obesity, and on the composition of the GM compared with a control diet with a low fat content (4%) (D1) over a 6-month period. The effect of omega-3 supplementation on metabolic parameters and the modulation of the immunological microenvironment in visceral adipose tissue (VAT) was also evaluated. Six-weeks-old mice were adapted for two weeks and then divided into two groups of eight mice each: a control group D1 and the experimental group D2. Their body weight was recorded at 0, 4, 12, and 24 weeks post-differential feeding and stool samples were simultaneously collected to determine the GM composition. Four mice per group were sacrificed on week 24 and their VAT was taken to determine the immune cells phenotypes (M1 or M2 macrophages) and inflammatory biomarkers. Blood samples were used to determine the glucose, total LDL and HDL cholesterol LDL, HDL and total cholesterol, triglycerides, liver enzymes, leptin, and adiponectin. Body weight measurement showed significant differences at 4 (D1 = 32.0 ± 2.0 g vs. D2 = 36.2 ± 4.5 g,  Obesity is a major public health problem contributing to increased morbidity and mortality worldwide . It is vIn recent decades, numerous articles have been published linking specific changes in the GM to the development of obesity and its associated disorders ,8. The GIt is well established that both the GM structure and function are dynamic and strongly affected by diet nutrients, such as the content and composition of lipids. As a consequence, dietary lipids influence host physiology through interactions with the GM , althougFirmicutes, decreasing Bacteroidota [Proteobacteria [In this context, most of the studies in murine models used diets with a fat content of 60% or higher; these levels accelerated the development of obesity, generating exacerbated metabolic and physiological responses. However, Speakman et al. stated that HFDs with a 60% fat content represent a large distortion of a normal rodent chow, and the results obtained in these studies cannot be potentially extrapolated to humans . Additioeroidota ,27, and bacteria . On the bacteria . In addibacteria . HoweverMale C57BL/6J (B6) mice (6-weeks-old) were purchased from the Facultad de Ciencias Veterinarias of the Universidad Nacional de La Plata, Buenos Aires, Argentina. All animals were housed in isolation rooms at the Animal Facilities of the Facultad de Ciencias Químicas of the Universidad Católica de Córdoba. This research has the authorization of the Institutional Committee for the Care and Use of Laboratory Animals—CICUAL-FCQ- Universidad Nacional de Córdoba, Córdoba, Argentina. Resolution N° 939, EXP-UNC: 0023836/2018. The environmental conditions in the facility were set according to CICUAL Guidelines. All animal procedures were performed in accordance with the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.The mice were randomly divided into two groups of eight mice each and adapted for two weeks to the experimental conditions. Then, the mice were fed a low-fat diet, “Control Diet” , or an “obesity-induced diet” with medium fat content  for a period of 24 weeks. Both groups were maintained under a standard light cycle (12 h light/dark), with free access to water and food, and temperature conditions (21 ± 2 °C). The food was replaced every two days to keep it fresh.The body weight of the mice was evaluated at 0, 4, 12, and 24 weeks. DIO was defined by the cut-off point of the mean body weights of the mice at week 0 plus 3 standard deviations in both groups (D1 and D2).Control Diet composition (D1): 4% fat, 26% protein, 52% carbohydrate, 8% crude fiber, and 10% total minerals . Obesity-Inducing Diet composition (D2): 11% fat, 27% protein, 48% carbohydrate, 6% crude fiber, and 8% total minerals. Ingredients: Corn, wheat, rice, soybean meal, chicken by-product meal, beef fat and/or chicken oil preserved with mixed tocopherols, beef and egg meal, corn and/or wheat gluten meal, fish flour , and animal digest based on chicken and/or pork by-products and salt (Purina Nestlé). The lipid composition was as follows: D1, SFAs: 24.61%, MUFAs: 39.37%, PUFAs: 36.02%, omega-3 fatty acids: 20.29%, omega-6 fatty acids: 15.73%, and omega-3/omega-6 ratio: 1.29; and D2, SFAs: 25.81%, MUFAs: 35.38%, PUFAs: 38.81%, omega-3 fatty acids: 30.14%, omega-6 fatty acids: 8.67%, and omega-3/omega-6 ratio: 3.48.n = 10/each diet) with one mouse each in the animal laboratory facility under the conditions cited above under Animals and Experimental Design. The initial amount of feed was weighed (5 g/cage to record daily feed intake). Consumption was measured by the differential weighing of the food at 0 and 24 h after feeding using an Ohaus digital scale model CS200, precision ± 0.1 g. Two independent experiments were performed to obtain an acceptable trend prior to the statistical analysis of the data  [To assess the acceptance of both the D1 diet and D2 diet, we carried out a consumption assay to measure food intake by mice. Briefly, we used individual mouse cages (ificant) .The GM composition was studied in stool samples, collected individually in metabolic cages over a period of 6 h at weeks 0, 4, 12, and 24. The feces were frozen immediately after collection and stored at −40 °C until analysis.Stool samples were handled under a laminar flow hood using a sterile technique. Microbial DNA was isolated from 220 mg of stool using the QIAmp DNA Stool Mini Kit  following the manufacturer’s standard protocol. The DNA concentrations were measured using fluorometric quantitation with a Qubit 2 and the Qubit dsDNA high-sensitivity kit . The DNA was stored at −40 °C.Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Streptococcus group B; the latter was isolated and typified in the LACE Laboratory. A maximum of 12 barcoded 16S samples were sequenced on an Ion 316v2 chip using the Ion PGM Hi-Q view Sequencing Kit  according to the manufacturer’s instructions.Sequencing was performed using an Ion 16S Metagenomics Kit  on the Ion Torrent Personal Genome Machine (PGM) platform . The libraries were generated from 20 ng of fecal DNA with the Ion 16S Metagenomics Kit using a combination of two pools of primers targeting the V2, V4, and V8 hypervariable regions of the 16S rRNA gene in pool 1, and the V3, V6-7, and V9 regions in pool 2. The primers were partially digested and bar-codes were ligated to the amplicons, purified using the Agencourt AMPure XP beads  according to the manufacturer’s protocol, and stored at −20 °C. The concentration of each 16S library was determined by qPCR using the Ion Universal Library Quantitation Kit . The library was diluted to ~10 pM before template preparation. Template preparation of the barcoded libraries was performed using the Ion PGM Hi-Q View OT2 kit  and the Ion OneTouch 2 System . A mock community dataset was generated from mixed bacterial genomic DNA from ATCC strains, including http://huttenhower.sph.harvard.edu/galaxy/) accessed on 28 November 2022 [https://ncbi.nlm.nih.gov/bioproject/?term=PRJNA929200) accessed on 29 January 2023.The sequence quality control, annotation, and taxonomical assignment were performed using the DADA2 v1.22.0 , phyloseber 2022 , was appAfter 24 weeks of differential feeding, 4 mice per group were randomly selected and anesthetized using inhaled FORANE (isoflurane). Blood samples were obtained after 12 h of fasting by cardiac puncture in heparinized tubes, centrifuged at 3000 rpm, and the separated plasma was stored at −20 °C. The plasma triglyceride , total cholesterol , HDL-Cholesterol , glucose , aspartate aminotransferase activity , and alanine aminotransferase activity  levels were assessed using enzymatic kits (Roche Diagnostic) in a ROCHE Cobas 8000 auto-analyzer; and LDL-Cholesterol (LDL-c) was estimated using the Friedewald formula as:  in Hanks’ Balanced Salt solution (pH = 7.4). After the addition of 3 vol. PBS containing 5% FBS and filtration of the digested tissue through nylon mesh (70 μm), the filtrate was centrifuged at 200× ernatant .g for 5 min, and the remaining cells were suspended in PBS and exposed to FcBLOCK (BD Biosciences) for 20 min. Five hundred thousand SVF cells were washed in ice-cold FACS buffer (PBS-2%FBS) and incubated with fluorochrome-labeled antibodies for 30 min at 4 °C. Different combinations of the following antibodies were used: PeCy5-labeled: anti-CD11b, PE-labeled: anti-F4/80, APCCy7-labeled: anti-CD11c, PeCy7-labeled: anti-CD206, and APC/Alexa 647-labeled: anti-CD36. Cells were permeabilized with BD Cytofix/Cytoperm and Perm/Wash (BD Biosciences) to detect intracellular ROS according to the manufacturer’s instructions. Then, the cells were incubated with FITC/Alexa 488-labeled antibody for ROS. The cells were acquired on FACS Canto II (BD Bioscience). The results were expressed as the percentage of cells per gram of AT.The SVF of mouse epididymal AT was prepared as described above. Red blood cells were separated by centrifugation at 500× p-value of < 0.05 was considered significant. Comparative and differential analysis between variables was performed using the two-tailed Student’s t test for variables with a normal distribution (result expressed as the mean ± standard deviation (SD)), and the Wilcoxon or Friedman tests for variables without a normal distribution , as appropriate. Pairwise comparisons using the paired Wilcoxon signed-rank test were performed if the Friedman test yielded a significant result. The p-values were adjusted using the Bonferroni multiple-testing correction method. Correlations between the bacterial taxa’s abundances and metabolic parameters and cell populations in SVF were calculated using the corrplot package v 0.92 [p-value of <0.05 were considered significant. All data were represented using ggplot2 v3.4.0 [Statistical analysis was carried out and visualized using R v4.1.2 software . The alpe v 0.92 . For Lef2 v3.4.0  and ggpu2 v3.4.0 .p-value = 0.0331 and 0.0059), not showing a significant change after 24 weeks of feeding , respectively .As can be seen in ectively E–G. Is iThe comparative analysis of alpha diversity, measured as observed ASVs, Shannon and Simpson Indexes, between Groups D1 and D2 at weeks 0, 4, 12, and 24 is shown in The analysis within Group D1 only showed significant differences for Shannon and Simpson indexes when comparing week 4 and week 24 E. In conThe comparative analysis between diets at different times revealed no differences at week 0, in contrast to weeks 4, 12, and 24, when the groups clustered separately, indicating dissimilarities in the composition and taxonomic abundance of their microbiota A–D. SimiCampylobacterota, Cyanobacteria, Deferribacterota, Desulfobacterota, and Firmicutes and lower abundance of Bacteroidota and Proteobacteria in Group D2 compared with D1 at week 4; different changes were detected at week 12, when D2 presented a higher prevalence of Deferribacterota, Desulfobacterota, and Firmicutes and lower prevalence of Actinobacterota, Bacteroidota, and Proteobacteria compared with Group D1; meanwhile, at week 24, Group D2 showed a higher abundance of Desulfobacterota and Pastescibacteria and lower abundance of the Cyanobacteriota Phylum in comparison with the control group. To determine which taxa were enriched in the different groups, linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) was applied. First, the effect of diet in each sampling week was compared and, later, each group was analyzed over time to determine specific changes not associated with diet. Significant differences at the levels of phylum, class, order, family, and genus were found among the different groups. Only enriched genera will be mentioned below to simplify interpretation.Escherichia/Shigella, Orsenella, and Parasutterella in contrast to group D2, presenting higher abundance of the Eubacterium eligens group, Mycoplasma, and Lachnospiraceae UC5-1-2E2 genera ; glyoxylate and dicarboxylate metabolism; glycerolipid metabolism; carbon fixation pathways in prokaryotes; and oxidative phosphorylation for amino acid, carbohydrate, lipid, and energy metabolism was shown in Group D1. In addition, a high prevalence of the biosynthesis of antibiotics, biosynthesis of secondary metabolites, carbon metabolism, and pantothenate and CoA biosynthesis was observed. In contrast, a high prevalence of pathways related to carbohydrate metabolism, such as amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, galactose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, and starch and sucrose metabolism; a high capacity for streptomycin biosynthesis, sulfur metabolism, peptidoglycan biosynthesis, glycerolipid metabolism, and polyketide sugar unit biosynthesis; and a high ability for xenobiotics biodegradation, including chloroalkane and chloroalkene degradation, naphthalene degradation, and polycyclic aromatic hydrocarbon degradation, were described for Group D2. D.To evaluate the relationship among the changes detected in the GM composition, immune cells phenotype in the VAT, and metabolic markers, we analyzed four mice per group that were randomly sacrificed after 24 weeks post-differential feeding.p-value = 0.0039 and 0.0007). Regarding the metabolic state, it can be seen in The characterization of immune cells in the VAT showed that mice fed with diet D2 had a significantly lower proportion of cells of myeloid lineage (CD11b+), total macrophages (CD11b+ F4/80+), and pro-inflammatory M1 macrophages (CD11b+ F4/80+ CD206-CD11c+) compared with those fed with diet D1. A marked decrease in ROS production was detected in all of these cells and a similar pattern was found in CD36+ expression, indicating an anti-inflammatory microenvironment in the VAT of group D2. No significant difference in the prevalence of anti-inflammatory M2 macrophages (CD11b+ F4/80+ CD206+ CD11c−) was observed. . SupplemIn order to determine whether there was a relationship between metabolic parameters, immune cells present in VAT, and the bacterial genera of the GM, we performed a correlation analysis among these attributes. Lactobacillus and Ligilactobacillus were negatively correlated with pro-inflammatory immune parameters, such as ROS production by total leukocytes and macrophages, M1 macrophages, and CD11b+ cells. Similar results were found for Anaerostipes, Anaerovorax, Christensenellaceae R-7 group, Eubacterium nodatum group, Lachnoanaerobaculum, Lactobacillaceae HT002, Lachnospiraceae ASF356, Lachnospiraceae UCG-004, Negativibacillus, Oscillospiraceae NK4A214 group, and Peptococcus. In contrast, Bilophila, Anaeroplasma, Alistipes, Lachnospiraceae GCA-900066575, and Lachnospiraceae FCS020 group were positively correlated with ROS production by immune cells in VAT. Additionally, Negativibacillus, Anaerotruncus, Lactobacillus, Eubacterium nodatum group, Lachnospiraceae ASF356, Anaerovorax, Peptococcus, Anaerostipes, and Lachnospiraceae UCG-004 were negatively correlated with the prevalence of pro-inflammatory M1 macrophages in VAT, while Rikenellaceae RC9 gut group, Alistipes, and Lachnospiraceae GCA-900066575 were positively correlated.It can be seen that the abundances of Alistipes and Lachnospiraceae GCA-900066575 were correlated inversely with the mice body and VAT weights. Moreover, these genera plus Rikenellaceae RC9 Gut Group were negatively correlated with the leptin levels. In contrast, the abundances of Butyricicoccaceae UCG-009, Candidatus Saccharimonas, Harryflintia, Lachnospiraceae AC2044 group, Lachnospiraceae ASF356, Lachnospiraceae UCG-004, Lactobacillaceae HT002, Lactobacillus, Monoglobus, Negativibacillus, Peptococcus, and Ruminococcaceae Incertae Sedis were positively correlated with the body weight and leptin levels.The abundances of Anaerostipes, Christensenellaceae R-7 group, Eubacterium nodatum group, Harryflintia, Lachnoanaerobaculum, Lachnospiraceae ASF356, Lachnospiraceae UCG-004, Lactobacillaceae HT002, Lactobacillus, Ligilactobacillus, Negativibacillus, and Oscillospiraceae NK4A214 group were positively correlated with some of the lipid levels studied . Only Ruminococcaceae Incertae Sedis was correlated with LDL-c.Odoribacter, Anaeroplasma, Prevotellaceae NK3B31 group, Eubacterium eligens group, Angelakisella, Bilophila, and Lachnospiraceae FCS020 group showed a significant negative correlation with the glucose levels, unlike the genus Muribaculum, which showed a positive correlation. There is increasing evidence that the GM plays a fundamental role in the regulation of homeostatic mechanisms and metabolism to exert either beneficial or detrimental effects on the host’s health. Different studies have shown that the qualitative and quantitative composition of the diet is a key factor modulating the host microbiota structure and function, or leading to gut dysbiosis, which may impact the development or prevention of certain NCDs . Indeed,Firmicutes and a decrease in Bacteroidota, which is in agreement with findings reported previously [Campylobacterota [Cyanobacteria [Deferribacterota, and Desulfobacterota [Eubacterium eligens group,Eubacterium nodatum group,Lachnospiraceae AC2044 group,Prevotellaceae group NK3B31,Ruminococcaceae UBA1819, among others. Furthermore, we found that mice with a higher dietary fat intake had higher abundances of Oscillibacter,Harryflintia,Mucispirillum,Flavonifractor, and Anaerotruncus. These bacteria have been related to inflammatory processes and the development of different chronic diseases in numerous reports. Lam et al. reported a significant increase in the abundance of Oscillibacter in HFD-fed mice compared with LFD-fed mice. At the same time, these changes were associated with weight gain, as well as being negatively correlated with gut permeability [Harryflintia under a HFD [Mucispirillum, a potentially pathogenic genus, has been reported in different conditions associated with gut inflammation [Flavonifractor and a decrease in the Christensenellaceae family have been associated with the development of affective disorders in humans; these findings are also associated with greater oxidative stress and low-grade systemic inflammation [Anaerotruncus, a butyrate producer, exhibited higher abundance in mice fed with diet D2; to note, this genus has also been associated with the development of obesity [We observed that, after a feeding period of 12 and 24 weeks, mice fed with diet D2 showed a significant increase in body weight and obesity. Remarkably, these results reveal that it is feasible to induce obesity with a considerably lower fat content than that traditionally used in HFD models, generating an experimental model and changes that better reflect the biological conditions leading to human obesity . Besideseviously ,14,45,46acterota , Cyanobabacteria , Deferriacterota ,50, wereacterota ,51,52. Weability . Similarer a HFD . On the ammation  and relaammation . Additioammation . Anaerot obesity . Overall obesity , leadingBilophila and Desulfovibrio genera was also observed, contrary to previous reports, where levels of Bilophila wadsworthia, a sulfite-reducing pathobiont associated with increased intestinal inflammation, were found when mice were fed a diet enriched with milk fat [Desulfobacter spp., Desulfovibrio spp., and Bilophila wadsworthia) in colonic mucosa compared with mice fed with LFD  after 20 weeks of feeding [Muribaculum, associated with intestinal barrier function markers, was also detected [With regard to mice fed with diet D1, they showed a dominance of mainly SCFA-producing taxa and lactic acid producers, both considered beneficial bacteria. Surprisingly, a higher prevalence of milk fat . Similardetected .Firmicutes and Bacteroidota abundances, although the differences between groups D1 and D2 were not statistically significant. Su et al. showed that the contribution of high doses of PUFAs, especially EPAs and DHAs, reversed the changes produced by HFD in the abundance of Firmicutes and Bacteroidota within a period of 18 weeks [Lachnospiraceae AC2044 group, Lachnospiraceae ASF356, Lachnospiraceae UCG-003, and Lachnospiraceae UCG-004, which are related to the production of SCFAs, and a decrease in the abundance of Faecalibacterium. In our cohort, we also observed an enrichment of Anaerostipes, one of the most abundant butyrogenic bacteria of the healthy microbiota and a high consumer of lactate in the colon [Akkermansia and Lactobacillus compared with the GM from mice fed with D1; the former has the ability to improve the intestinal microenvironment, increasing the thickness and maintaining the barrier function of the intestinal mucosa [Clostridiaceae, Sutterellaceae, and Akkermansiaceae families, among other changes that were reversible after a washout period [Lactobacillus, which is known to decrease intestinal inflammation and provide competitive resistance against pathogens [Interestingly, at week 24, the changes observed in the composition of the GM in Group D2 were different to the alterations described earlier. In line with different reports, these findings may be attributable to the sustained dietary intake of omega-3; we detected a correction of the 18 weeks . Additiohe colon . Remarkal mucosa . Additiol mucosa . This ist period . Similarathogens ,66.Harryflintia, Lachnospiraceae AC2044 group, Lachnospiraceae ASF356, Mucispirillum, Ruminococcaceae UBA1819, and Tyzzerella could be considered a microbial signature for GM in mice fed with a fatty-acid-rich diet, as they presented the same distribution profile in the differential analysis between diets over time.The different impact of diet over time observed in our study is probably due to the fact that the proportion of the saturated and/or omega-6 fatty acid component of D2 would favor the growth of bacteria associated with the development of metabolic endotoxemia throughout the first 12 weeks of differential feeding, favoring the development of obesity and potential associated metabolic disturbances. Later, throughout weeks 12 and 24, the effect of omega-3 fatty acids became predominant, favoring the growth of bacteria with probiotic potential, which would contribute to controlling the intestinal inflammation, modulating both metabolism and the immune system, as observed by the presence of an anti-inflammatory environment in VAT ,61. FurtOur study also explored the metabolic activities of GM from Groups D1 and D2; the differential feeding generated distinct functional profiles in each group at different times, indicating that the content of dietary fatty acids influenced the functional profile of the microbiota in a time-dependent manner. However, knowledge of the connections between omega-3 and the GM metabolic activities is still limited . Even soLactobacillus and Ligilactobacillus with the production of ROS by total leukocytes, CD11b+ cells, and M1 macrophages. It is important to note that these genera presented differential abundances at week 24 after feeding D2, showing that the effect of long-term intake of omega-3 on the modulation of the intestinal microbiota favored immunomodulation in VAT. In this context, Huang et al. demonstrated that Lactobacillus-induced anti-inflammatory macrophages associated with REG3γ in the intestinal lamina propria may play a role in adipose tissue homeostasis and be involved in high-fat-diet-mediated resistance to obesity [Christensenellaceae family, which also increased in Group D2, it has been shown that certain species have powerful immunomodulatory properties [Alistipes and Lachnospiraceae GCA-900066575 showed an inverse correlation with the mice body and VAT weight. This has been also reported in humans by Tavella et al., who stated that a high abundance of Alistipes was associated with a lower proportion of VAT and healthier metabolic profile in older adults [Alistipes, such as Alistipes shahii and Alistipes sp., was significantly lower in obese subjects in comparison to normal-weight subjects [Rikenellaceae RC9 Gut Group and Oscillospiraceae UCG-003 were correlated inversely with the triglyceride level, which is in agreement with previous findings [Lactobacillus was correlated positively with the TC, TG, and HDL-c levels, which is in opposition to reports where mice treated with probiotics, Lactobacillus curvatus alone or together with Lactobacillus plantarum, fed with a HFD showed reduced cholesterol in the plasma and liver [Lachnospiraceae UCG-004 also showed a positive correlation with HDL-c, similar to results reported by Lan et al. in the Chinese population [Although obesity is characterized by the inflammation of AT, with an important infiltrate of mainly pro-inflammatory M1 macrophages ,6, our f obesity . Regardioperties , correlaoperties ,80, althsubjects . In contsubjects ,54. Addisubjects ,83,84. Wfindings . Moreovend liver . In addipulation .Lactobacillus and Ligilactobacillus, promoted by the sustained intake of omega-3 PUFAs, which, in turn, contributed to the generation of an anti-inflammatory microenvironment in the VAT with a marked decrease in pro-inflammatory cells and ROS production. Despite these findings, our study has some limitations: the sample size to determine the microbiota composition, metabolic markers, and immune cell profile was small (n = 8 and n = 4), respectively. On the other hand, we only analyzed the bacterial taxonomy up to the genus level; thus, it was possible that information on the existence of some species/strains that could be considered microbial biomarkers associated with the contribution of omega-3 is missing. The results obtained do not allow us to determine the mechanism underlying the relationships among nutrition, GM, and immune-metabolism. These observations need further validation by performing studies of metabolic parameters and immunological profiles in VAT at different time intervals and considering a diet of medium fat composition without omega-3 supplements.Taken together, these results suggest that the administration of a diet with medium amounts of saturated fat supplemented with omega-3 fatty acids contributed to the development of obesity, associated with specific changes in the composition and diversity of the intestinal microbiota, metabolic pathways profiles, and VAT immuno-metabolism in a time-dependent manner. The stabilization of weight gain with preserved metabolic state noted by week 24 after differential feeding was probably due to beneficial changes in the intestinal microbial communities, such as the increased abundance of probiotic bacteria, such as Lactobacillus and Ligilactobacillus species, which, in turn, modulated the immune metabolic response of AT in this mouse model of obesity.Remarkably, these results reveal that it is feasible to induce obesity with a considerably lower fat content than that traditionally used in HFD models, generating an experimental model and changes that better reflect biological conditions contributing to human obesity. The constant and sustained administration of omega-3 fatty acids induced specific changes in the GM composition, mainly with increases in The identification of different taxonomic signatures associated with the healthy functionality of VAT and metabolic state in obesity could be a new, promising tool to design treatment or prevention strategies for obesity and associated comorbidities, focusing on the modulation of the intestinal microbiota through the consumption of omega-3 fatty acids.


Glioblastoma (GB) and brain metastases (BM) are the most common brain tumors in adults and are invariably associated with a dismal outcome. These highly malignant tumors share common features including increased invasion and migration of the primary or metastatic brain cancer cells, whose triggering mechanisms are largely unknown. Emerging evidence has suggested that the ubiquitin-conjugating enzyme E2C (UBE2C), essential for controlling cell cycle progression, is overexpressed in diverse malignancies, including brain cancer. This review highlights the crucial role of UBE2C in brain tumorigenesis and its association with higher proliferative phenotype and histopathological grade, with autophagy and apoptosis suppression, epithelial-to-mesenchymal transition (EMT), invasion, migration, and dissemination. High expression of UBE2C has been associated with patients’ poor prognosis and drug resistance. UBE2C has also been proven as a promising therapeutic target, despite the lack of specific inhibitors. Thus, there is a need to further explore the role of UBE2C in malignant brain cancer and to develop effective targeted therapies for patients with this deadly disease. The most common brain tumors in the adult population include glioblastoma (GB), a primary brain tumor arising from cells within the brain, and brain metastases (BM), secondary lesions that arise from systemic cancers that disseminate to the brain during disease progression. Even though brain tumors are rare, accounting for 3% of all cancer types, patients have increased mortality and severe disabilities ,2. StandGlioblastoma is the most frequently diagnosed malignant primary brain tumor in adults. It is a grade 4 tumor according to the classification of the World Health Organization (WHO) and the most aggressive type of glioma . CurrentInvasion is a main feature of GB through the infiltration of cancer cells in the brain parenchyma, leading to therapeutic resistance and tumor recurrence ,10,11. GAnother common brain tumor in the adult population is BM. BM arise from the dissemination of cancer cells from a systemic primary tumor to the brain and are estimated to be 10 times more frequent than primary brain tumors. Additionally, the incidence of BM is thought to be increasing, possibly as a result of the improvement in diagnostic methods and better treatment of systemic cancer ,16. ApprMYC, YAP1, and MMP13 genes were amplified in BM in comparison with their corresponding primary lung tumors, suggesting that these genes may be driving BM [p53, KRAS, DSC2, WNT–TCF, HB–EGF, COX2, BMP-2, src, serpins, and cathepsin-S [Dissemination is a complex multi-step process that comprises genetic, epigenetic, and biological alterations of cancer cells. This metastatic cascade starts with the migration and invasion of cancer cells into adjacent tissues, followed by intravasation into the bloodstream and colonization of a new secondary site . Recentliving BM . Other ghepsin-S ,30,31,32Despite previous attempts to study the mechanisms that underlie the invasion and dissemination of cancer cells to the brain and within the brain, these remain poorly understood. Uncovering these mechanisms is an unmet medical need critical for the development of novel therapeutic approaches that can improve patients’ survival and quality of life.The ubiquitin system has been gaining significant relevance in cancer research due to its role in several cellular processes. In the last years, growing data have been exhibiting the human ubiquitin-conjugating enzyme E2C (UBE2C) as a relevant player in tumorigenesis and a valuable biomarker in several types of cancers. In this review, we aim to discuss the role of UBE2C in brain cancer invasion and dissemination and its potential therapeutic targeting in brain tumor patients.Damaged proteins, either caused by intrinsic or extrinsic factors, affect different cell functions, and consequently cell viability. The ubiquitin proteasome system (UPS) is responsible for eradicating these damaged proteins and maintaining protein homeostasis. Additionally, the UPS controls the concentrations of cell cycle regulators according to the cell phase, regulating cell cycle progression ,34.Protein degradation starts with the ubiquitination of damaged proteins, an ATP-dependent process that requires three types of enzymes, namely ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and ubiquitin ligases (E3), which operate consecutively in a cascade. The E1 enzyme activates the ubiquitin molecule through ATP hydrolyzation followed by adenylation of the glycine residue present in the C-terminal of ubiquitin and its linkage to the cysteine active site of E1. Subsequently, ubiquitin is transferred to the E2 enzyme, allowing the conjugation between E2 and E3, which in turn recognizes and binds the target protein while catalyzing the ubiquitin transfer to the latter. These enzymes operate in a methodical manner to polyubiquitinate the substrates so that these can be recognized and degraded by the proteasome ,34.Dysregulation of the UPS leads to an accumulation of damaged proteins that have key roles in DNA damage repair, cellular differentiation, cell cycle control, apoptosis, and other vital functions. This dysregulation has been associated with several diseases, namely neurodegenerative disorders and different types of cancer, including brain tumors . InteresUBE2C gene  is located at chromosome 20q13.12 and can generate 7 transcript variants, being the longest transcript translated into a protein composed of 179 amino acids with a total molecular weight of 19.65 kDa [The 9.65 kDa ,45. The 9.65 kDa ,45,46.UBE2C is located in the nucleus and cytoplasm of cells, being scarcely expressed in normal tissues, at both RNA and protein levels . UBE2C iAdditionally, UBE2C promotes the inactivation of the mitotic cell cycle spindle assembly checkpoint (SAC), necessary to guarantee the appropriate separation of the sister chromatids into two daughter cells, averting aneuploidy . This isUBE2C overexpression triggers whole chromosome instability and consequent missegregation and aneuploidy [UBE2C overexpression may play an important role in tumorigenesis and tumor progression [euploidy . Its accgression .UBE2C has been described in many human cancers, namely breast, lung, thyroid, endometrial, renal, prostate, pancreatic, esophageal, hepatocellular, gastrointestinal, and brain tumors [The overexpression of n tumors ,52,53,54n tumors ,47,52,54UBE2C knockdown leads to inhibition of proliferation and invasion in breast cancer cells, as well as a decrease of p-AKT, p-mTOR, and hypoxia-inducible factor 1-α (HIF-1α) and an increase of phosphorylated phosphatase and p-PTEN levels. UBE2C is thought to promote breast cancer proliferation by activating the AKT/mTOR signaling pathway, a known key player in metastasis [Breast cancer is the most frequently diagnosed cancer, with an 11.7% global incidence, and is the leading cause of cancer death in women ,80. Up ttastasis ,83. UBE2tastasis ,60. Moretastasis .UBE2C has a positive correlation with tumor grade, being overexpressed in poorly differentiated tumors [MALAT1, leading to the inactivation of WNT, PI3K/AKT, and MAPK/ERK, thus inhibiting proliferation, migration, and invasion of lung cancer cells [MDR1) gene [ABCG2, and ERCC1 [p53 mutational status and inversely correlate with EGFR mutational status [Lung cancer is the second most common cancer, with a global incidence of 11.4%, and is the leading cause of cancer-related deaths. The five-year survival for patients with lung cancer is 10–20% . One of d tumors ,87. The er cells . AdditioR1) gene , ABCG2, nd ERCC1 . In NSCLl status . Moreovel status . Recentll status .UBE2C upregulation is associated with tumor invasion, dedifferentiation, and poor prognosis [UBE2C downregulation, suppression of p53 proteasomal degradation, and inhibition of cell proliferation and invasion [UBE2C in the HuH6 hepatoblastoma cell line and in the HB-243 cell line from a patient-derived xenograft decreased cell viability by up to 44% and cell migration by 65% [UBE2C knockdown impairs invasion and migration [UBE2C has been described as a promoter of migration and invasion of tumor cells in several other types of cancer ,72,89,90rognosis . On the invasion . In hepan by 65% . In osteigration . This efigration ,95,96. Rigration .An association between UBE2C and p53, a tumor suppressor protein involved in the G2/M checkpoint and apoptosis of cells with damaged DNA , is descUBE2C knockdown leads to increased apoptosis induced by cisplatin and reduced phosphorylation of ERK, AKT/PKB, and p38 [UBE2C [The PI3K/AKT/mTOR/signaling pathway has been correlated with UBE2C levels in several cancers, including thyroid, gastric, and pancreatic cancer. In gastric and cervical cancer,  and p38 ,100. In  and p38 . Finally and p38 . Recentl8 [UBE2C . Other s8 [UBE2C ,73,102. 8 [UBE2C . On the UBE2C overexpression is only found in cancer cells, and not in normal tissues [The role of UBE2C has been studied in brain tumors, including gliomas, meningiomas, and BM. UBE2C expression was analyzed in astrocytic tumors, and authors concluded that GB exhibits high levels of this protein compared with low-grade astrocytomas, indicating a correlation between UBE2C expression and glioma tumor grade. It was also shown that  tissues ,103,104. tissues . UBE2C transcription by binding to its promoter regions and is associated with poor prognosis in gliomas [It has been shown that the Forkhead box transcription factor M1 (FoxM1) triggers  gliomas . FoxM1 h gliomas . Interes gliomas ,106. Upr gliomas ,108. The gliomas .UBE2C overexpression correlates with higher histological grade, increased proliferation, and poor prognosis [UBE2C function in meningioma cells with different grades, it was reported that silencing this gene inhibits proliferation, migration, and invasion. Moreover, UBE2C knockdown induces apoptosis by increasing Bax and caspase-3 and by decreasing Bcl-2 levels [RIZ1, a tumor suppressor gene, is markedly decreased and regulates UBE2C in a c-Myc-dependent manner in malignant meningioma [In meningiomas, rognosis . When ex2 levels . It was ningioma ,111. UBE2C overexpression increases migration and invasion of breast and lung cancer cells. In addition, mice orthotopically injected with UBE2C-overexpressing cancer cells develop increased leptomeningeal dissemination, an aggressive phenotype of brain metastatic disease [UBE2C as a signature gene in the proliferative molecular subgroup [Recently, Paisana et al. reported the association of UBE2C and BM . The aut disease . Anothersubgroup .The effects of UBE2C dysregulation in cancer are highlighted in The best approach to target the UPS in order to achieve better selectivity with less toxicity is through inhibition of the E2 enzymes or E3 ligases, which have been reported as dysregulated in several cancers. E2 enzymes are abnormally translated in a variety of cancers, being responsible for tumor progression and unfavorable biological and clinical characteristics, which emphasizes the relevance of UBE2C as a potential therapeutic target. Until now, UBE2C targeting has been limited to experimental studies, making the development of UBE2C inhibitors essential for patient care ,114.UBE2C knockdown using interference RNA inhibits proliferation and promotes activation of the tumor suppressor p53 and apoptosis in U251 glioma cells [UBE2C knockdown suppresses glioma cell growth by 40% in comparison to control cells and induces autophagy through inactivation of the PI3K/Akt/mTOR pathway [ma cells . It was  pathway . UBE2C silencing inhibits ERK and AKT phosphorylation and increases the levels of phosphorylated PTEN, which acts as a negative regulator of the PI3K/AKT pathway [UBE2C depletion in triple-negative breast cancer cells decreases proliferation, which is substantially enhanced in the presence of paclitaxel, suggesting a possible new combination therapy [UBE2C expression in estrogen receptor-positive breast cancer cells [In breast cancer cells,  pathway . UBE2C i pathway . UBE2C d therapy . Intriguer cells . UBE2C silencing decreases cell proliferation in vitro and in vivo and results in G2/M arrest, increased cell apoptosis, and reversed cisplatin resistance. In addition, cyclin B1, CDK1, and Bcl-2 levels are downregulated, while Bax expression is upregulated [UBE2C depletion impairs the ERK1/2 signaling pathway in vitro, while decreasing tumor volume in vivo. Additionally, the ERK1/2 inhibitor U0126 reduces ERK1/2 phosphorylation and reverses the effect of UBE2C on cell proliferation, migration, and invasion [UBE2C knockdown suppresses the growth of xenografted tumors [UBE2C downregulation increases the sensitivity to chemotherapeutic agents including doxorubicin, 5-fluorouracil (cellular thymidylate synthase inhibitor), and the multikinase inhibitor sorafenib [UBE2C silencing also sensitizes colorectal cancer cell lines to bortezomib (proteasome blocker) and oxaliplatin , inducing apoptosis [UBE2C knockdown decreases cell proliferation, migration, and invasion, promotes apoptosis, and reduces cisplatin resistance [In ovarian cancer, egulated . In gastinvasion . Similard tumors . In hepaorafenib . UBE2C spoptosis . In thyrsistance . UBE2C dsistance .Interestingly, the manipulation of miRNAs, namely miR661-3p and miR-381-3p, has shown the ability to target UBE2C in NSCLC and prostate cancer cells, respectively, leading to reduced proliferation, invasion, and metastasis ,121.UBE2C-depleted prostate cancer cells reduces the degradation rate of cyclin B1, suggesting less ability to recover from the mitotic arrest induced by docetaxel. Therefore, UBE2C seems to be a key player in the regulation of mitotic slippage and a mediator in docetaxel resistance in prostate cancer [UBE2C-driven orthotopic mouse xenografts of breast and lung cancer BM. Interestingly, early treatment with oral dactolisib prevents leptomeningeal dissemination in vivo [CCI-779, an mTOR inhibitor, significantly decreases UBE2C in prostate cancer cell lines. CCI-779 inhibits the recruitment of androgen receptor-coactivator to the UBE2C enhancers and, consequently, reduces the loading of RNA polymerase II and transcription. In addition, CCI-779 inhibits cell proliferation and invasion in vitro and decreases tumor growth in mouse xenografts . Moreovee cancer . Treatmee cancer . Recentl in vivo .A study on cervical carcinoma analyzed the structure, binding energy, chemical properties and drug-like properties of UBE2C and identified 2,4-diimino-1-methyl-1,3,5-triazepan-6-one, a sulfuric acid compound with 5,6-diamino-2,4-pyrimidinediol (1:1) and 7-alpha-d-ribofuranosyl-2-aminopurine-5’-phosphate as possible inhibitors . NonetheA novel in vitro biochemical assay that exploits the ability of UBE2C to ubiquitinate itself in particular conditions was developed to study the UBE2C ubiquitin-conjugating activity. This platform provides a rapid and sensitive time-resolved fluorescence resonance energy transfer-based assay that might be used to identify UBE2C inhibitors and can be scalable into an automated high-throughput screening layout . FurtherDespite being a rare condition, malignant brain tumors cause a dismal prognosis and reduced life expectancy. Invasion and dissemination of cancer cells into the brain are important features of aggressiveness in brain cancer that urgently need a better therapeutic intervention aiming to improve patient prognosis .UBE2C is a key enzyme during the cell cycle and has been considered a proto-oncogene and a tumor biomarker. High expression of UBE2C is observed in several different types of cancer and correlates with histological grade, poor prognosis, resistance to therapy, and relapse. Moreover, there is evidence of the role of UBE2C in proliferation, EMT, invasion, and migration in several malignancies, including brain tumors. Due to its characteristics, UBE2C has the potential to be used as a diagnostic and prognostic biomarker, including in liquid biopsies from cancer patients. However, most studies reporting the role of UBE2C in brain tumors are relatively recent, meaning this is an understudied subject. Particularly, an in-depth focus on the relevance of UBE2C in the tumor microenvironment is lacking to understand the best clinical strategy in UBE2C-driven carcinogenesis.UBE2C can also be considered a promising therapeutic target. Several reports have highlighted the interaction between UBE2C and the PI3K/AKT/mTOR signaling pathway in driving tumor progression and metastases through a yet unknown mechanism. The evidence of efficacy from diverse PI3K/AKT/mTOR inhibitors in UBE2C downregulation and EMT reversion sheds light on possible novel therapeutic options. However, although genetic silencing and indirect inhibition have been successfully tested, there is still an unmet need to identify UBE2C-specific inhibitors with therapeutic efficacy. One of the most prominent challenges consists of the three-dimensional structure of E2 enzymes. The catalytic site of these enzymes is located in a region that unlikely allows specific interactions with small molecules with high affinity . MoreoveIn conclusion, despite the established relevance of UBE2C in cancer, its role in brain tumors should be further investigated to assess its potential value as a diagnostic and prognostic marker, as well as a potential therapeutic target.


Plants uptake and assimilate nitrogen from the soil in the form of nitrate, ammonium ions, and available amino acids from organic sources. Plant nitrate and ammonium transporters are responsible for nitrate and ammonium translocation from the soil into the roots. The unique structure of these transporters determines the specificity of each transporter, and structural analyses reveal the mechanisms by which these transporters function. Following absorption, the nitrogen metabolism pathway incorporates the nitrogen into organic compounds via glutamine synthetase and glutamate synthase that convert ammonium ions into glutamine and glutamate. Different isoforms of glutamine synthetase and glutamate synthase exist, enabling plants to fine-tune nitrogen metabolism based on environmental cues. Under stressful conditions, nitric oxide has been found to enhance plant survival under drought stress. Furthermore, the interaction between salinity stress and nitrogen availability in plants has been studied, with nitric oxide identified as a potential mediator of responses to salt stress. Conversely, excessive use of nitrate fertilizers can lead to health and environmental issues. Therefore, alternative strategies, such as establishing nitrogen fixation in plants through diazotrophic microbiota, have been explored to reduce reliance on synthetic fertilizers. Ultimately, genomics can identify new genes related to nitrogen fixation, which could be harnessed to improve plant productivity. Soil erosion, compaction, acidification, and contamination are significant factors causing soil degradation, affecting 50% of agricultural land and food production ,2,3. Con3−) translocation and intake are positively connected as well [4+) application promotes iron (Fe) uptake [3− triggers the chlorosis symptoms of Fe deficiency [3− uptake, which promotes the synergetic transfer of cations, including K+, sodium (Na+), cadmium (Cd2+), and zinc (Zn2+) while inhibiting the absorption of anions like chloride (Cl−) and sulfate (SO4−) [3− and NH4+ concentrations have unique impacts on plant performance. While the usage of NH4+ reduces the effects of drought on plant development, NO3− application has a reverse influence [N is a vital macronutrient for plants and a crucial component of amino acids that serve as the building blocks of enzymes and proteins in plants. Additionally, N is a part of the chlorophyll molecule, an essential factor in photosynthesis for absorbing sunlight energy, promoting plant growth and grain yield. Even the roots contain this essential element because proteins and enzymes control water and nutrient intake . Recentl as well . The pla as well . Regardi) uptake ; in contficiency . Sulfur ficiency , is corrficiency . Alternaficiency . In addie (SO4−) . Under dnfluence .3− or NH4+ form, followed by a structural investigation of the transporters of these two forms. Upon reaching their destination, NO3− and NH4+ are exposed to various metabolic pathways . Next, N assimilation occurs under the intersection with glutamine synthetase (GS) and glutamate synthase or glutamine 2-oxoglutarate aminotransferase (GOGAT). Then, a comprehensive survey of the role of N in plant adaptation/tolerance to abiotic stresses, either drought or stress, is discussed. Finally, the detrimental effects of nitrogen mineral fertilization and the possibility of microbial application and genomic approaches are summarized.In this review, we aim to provide a detailed story of the N journey starting from its uptake from soil either in NO3− and NH4+), which are transported across the root plasma membrane by different families of transporters. Under normal soil conditions, N is mainly available in the form of NO3−, and four families of transporters mediate NO3− uptake, namely NRT1, NRT2, chloride channel (CLC-1), and slow anion channel-associated 1 homolog 3 (SLAC1/SLAH). These transporters have distinct characteristics in affinity, capacity, regulation, and localization [4+ uptake is facilitated by ammonium transporters (AMTs), high-affinity transporters expressed mainly in the root hairs and epidermis [4+ is the dominant form of N in flooded or acidic soils, and AMTs-mediated acquisition is crucial for the N demand of plants growing in such conditions [N is an essential macronutrient for plants, and it can be acquired from the soil in inorganic forms . There are 53 NRT1 genes in Arabidopsis and 93 in rice [3− root uptake. The NRT1 family can transport NO3− and other substrates such as hormones, nitrite, amino acids, peptides, chloride, glucosinolates, and jasmonate-isoleucine [3− conditions. In Arabidopsis, four NRT2 transporters  function in nitrate influx and account for 95% of nitrate uptake under low NO3− concentrations [3− from different sources to different tissues. For example, NRT2.4 and NRT2.5 absorb NO3− from the soil root hairs, while NRT2.1 and NRT2.2 transport it from the apoplast to the cortex and endodermis cells. Moreover, NRT2.5 is induced by long-term starvation and enhances NO3− uptake from both shoots and roots of mature plants [3− transport in plants. The NRT3 family consists of two members: NRT3.1 and NRT3.2. These transporters form a complex with NRT2 transporters and regulate their activity and stability [Plants acquire NOfamilies . The NRT in rice . These goleucine . The NRTtrations . Howevere plants ,34. Furttability .3− transceptor (transporter/receptor) NRT1.1 regulates the expression levels of many NO3− assimilation pathway genes by sensing the external NO3− concentration and modulating the root growth accordingly [3− transporters are regulated through phosphorylation, mediated by calcium-dependent calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) [3− concentrations, NRT1.1 activates the expression of Arabidopsis nitrate regulated 1 (ANR1), a transcription factor that promotes lateral root growth and proliferation [3− concentrations, NRT1.1 inhibits lateral root development by controlling auxin levels and meristem activation [3− signals from NRT1.1 to the nucleus, calcium acts as a secondary messenger that modulates the expression of NO3−-responsive genes. Three calcium-dependent protein kinases  and their partner CBLs are involved in this process [NOordingly ,37. NO3−e (CIPK) ,39. The e (CIPK) . At highferation . At low tivation . To tran process .3− from the xylem and phloem, which affects the distribution and availability of NO3– within the plant. For example, NRT1.5, NRT1.8, and NRT1.9 are expressed in the xylem and phloem, and they function in the influx/efflux, removal, and loading of NO3− from/to the root vascular tissues, respectively [3– concentration, and interaction with other genes. For instance, only NRT1 transporters have been successfully overexpressed in both leaves and roots to enhance NUE, while the role of NRT2 transporters in this process is still unclear [3– uptake and NUE under low nitrate conditions [Several NRTs are involved in the loading and unloading of NOectively ,44. Othe unclear . Recentlnditions . Anothernditions . Severalnditions ,48,49,503−-responsive genes. According to reports, these TF families act as essential regulators of the plant N response, which encompasses MADS-box [Moreover, some genes that regulate or interact with NRTs have been identified as potential targets for improving NUE in plants. For example, the transcription factor OsNAC42 activates the expression of several NRTs and increases nitrate assimilation and NUE in rice. The nitrate reductase gene OsNR2 converts nitrate to ammonium, affecting the expression of NRTs and other nitrogen-related genes in rice. The nitrate transporter OsNPF4.5 is regulated by a microRNA (miR827) and modulates nitrate distribution and remobilization in rice. These studies demonstrate the complexity and diversity of nitrate transport and signaling in plants as well as provide valuable insights for improving NUE and crop production . NotablyMADS-box , NLP [53MADS-box , LBD [57MADS-box , MYB [59MADS-box , TCP [61MADS-box , and zinMADS-box .3– permeases, while others are in histidine transport [NRT1, NRT2, and NRT3 have different structural conformations . As a reransport . The NRTransport .Archaea to Homo sapiens. MFS proteins target a diverse range of substrates in both directions across the membrane, including ions, carbohydrates, lipids, amino acids and peptides, nucleosides, and other small molecules, in many cases catalyzing active transport by converting the energy stored in a proton electrochemical gradient into a concentration gradient of the substrate [Arabidopsis, the functional unit of the high-affinity nitrate influx complex is most likely a tetramer composed of two NRT2 and NRT3 subunits each [NRT2 members belong to the PF07690 (MSF) family, representing the largest family of secondary transporters, with members ranging from ubstrate . NRT3 caubstrate . NRT3 isubstrate . In Arabits each .3− and NH4+ are the principal inorganic nitrogen sources absorbed by plant roots [4+ requires less energy than NO3− assimilation, NH4+ is the preferred form of N uptake when plants grow under N deficiency [4+ uptake is more efficient, high absorbed amounts of NH4+ by the plant can be hazardous [4+ uptake system in plants receives special attention [4+ from symbiotic fungi to plants [4+ from roots to shoots [4+ in leaves and reproductive organs [4+ transport [4+ [4+ intake on plant growth and development, AMTs activities are fine-tuned not only at the transcriptional level through the involvement of at least four transcription factors [As mentioned earlier, NOnt roots ,74. Becaficiency ,76. Althazardous ,78,79; tttention ,81,82. Ro plants ,84, delio shoots , transmie organs ,87,88, aransport ,90. Asidport [4+ ,92,93. T factors ,95,96,97 factors ,99, pH [ factors , and end factors .4+ transporter domain (PF00909) comprises two structural copies of five helices plus one extra C-terminal helix. It has been defined as an 11-times-spanning membrane channel. Ideal NH4+ transporter domain is composed of 11–12 transmembrane spots, with feature sequences “D (F Y W S) A G (G S C) X2 (L I V) (E H) X2 (G A S) (G A) X2 (G A S) (L F)” at transmembrane spot 5 and “D D X (L I V M F C) (E D G A) (L I V AC) X3 H (G A L I V) X2 (G S) X (L I V A W) G” at transmembrane region 10 [The NHegion 10 . AMTs caegion 10 ,85,102. 3− in the root that needs to be converted to NO2− to make organic compounds such as amino acids. The cytosolic NO2− is toxic to the cytosol and needs to be transported to the chloroplasts, where it is reduced to NH3 by NIR [2− needs to be actively transported across the membrane of the chloroplasts by a specific transporter to avoid accumulation and toxicity. Several types of plastids and plasma membrane-localized NIR have been described in Escherichia coli, Cyanobacteria, green algae, red algae, Aspergillus nidulans, diatoms, and a haptophyte, but there was no evidence of a nitrite-specific plastid and plasma membrane transporter in higher plants [3− and NO2− enter the plasma membrane via similar transporters due to previously observed competitive reduction of NO2− absorption by NO3− and vice versa [Cytosol is the first cellular compartment to encounter NO3 by NIR . Cytosolr plants ,104. Conce versa .2− in vitro [2− transporter in the chloroplasts of plants and belongs to the proton-dependent oligopeptide transporter (POT) family [2− depending on the energy availability. In 2013, Kotur et al. used 13NO2− to characterize nitrite influx and nitrite-specific transporter into roots of Arabidopsis thaliana [2− transport activity from Arabidopsis, and these two genes were detected based on homologs genes from cyanobacterial genomes [In 1988, Brunswick and Cresswell found that illuminated chloroplasts take up NOin vitro . In 2007) family . CsNitr1thaliana . Fortunathaliana . Further genomes .2− than the cyanobacterial proteins [2− uptake activity than the chloroplasts isolated from the wild-type Col-0 plants. AtNITR2;2, expressed in roots and not detectable in shoots, had a putative transit peptide similar to that of AtNITR2;1 and demonstrated low but significant activity of NO2− transport in the cyanobacterial cell. Finally, it was discovered that NO2− increased the expression of AtNITR2;1 and AtNITR2;2 under the direction of NIN-like proteins, indicating that these two proteins are nitrate-inducible parts of the nitrite transport system of plastids.AtNITR2;1 and AtNITR2;2 are HPP-domain-containing proteins . These tproteins . AtNITR23−, NO2−, and NH4+), with the remaining 6% in the atmosphere  and a trace (0.006%) in the hydrosphere and biosphere . N is the fourth most prevalent element in the biosphere after oxygen, carbon, and hydrogen and is an essential component of total biomass [3−, NO2, nitrous oxide (N2O), NO, dinitrogen (N2), and NH4+) [N is among the most widely distributed elements in the lithosphere, atmosphere, hydrosphere, and biosphere . The lit biomass ,111. The biomass ,114,115,nd NH4+) ,117,118.3− is the most oxidized form of N, while NH4+ is the most reduced form. Many microorganisms, mainly microbes, drive the global N cycle by metabolizing N through a variety of redox processes for energy transduction, detoxification, or assimilation via various sub-pathways such as N fixation , assimilatory NO3− reduction , dissimilatory NO3− reduction , denitrification , nitrification , complete nitrification, complete ammonia oxidation (comammox) , and anammox .NOMoreover, each sub-pathway from these previous sub-pathways consists of a list of genes and their encoded enzymes that are involved in the various biogenic processes in the nitrogen cycle. The nitrogen fixation path consists of nitrogenase molybdenum-iron protein alpha chain (NIFD), vanadium-dependent nitrogenase alpha chain (VNFD), and nitrogenase delta subunit (ANFG) genes ,120,121.2 to NH3 by oxidoreductase reaction. NH3 is widely used in different vital pathways for living organisms, such as arginine biosynthesis , purine metabolism , alanine, aspartate, and glutamate metabolism . Also, NH3 is involved in glycine, serine, and threonine metabolism  as well as cyanoamino acid metabolism , glyoxylate and dicarboxylate metabolism , and lipoic acid metabolism . NH3 is a component in biosynthesis of secondary metabolites , microbial metabolism in diverse environments , carbon metabolism , biosynthesis of amino acids , proximal tubule bicarbonate reclamation , collecting duct acid secretion , protein digestion and absorption , Vibrio cholerae infection , and epithelial cell signaling in Helicobacter pylori infection  [In this sub-pathway, these previous genes were used to convert Nap05120) ,120,121.2 to N2 or NH3 using nitrite reductase enzyme [3− to N2 through NO2, NO, and N2O. Furthermore, assimilatory and dissimilatory NO3− reduction paths have different genes, such as nitrate reductase/nitrite oxidoreductase, alpha subunit , NAPA, nitrite reductase (NADH) large subunit (NIRB), nitrite reductase (cytochrome C-552) (NRFA), ferredoxin-nitrate reductase (NARB), nitrate reductase (NAD-(P)-H) (NR), assimilatory nitrate reductase catalytic subunit (NASC and NASA), nitrite reductase (NAD-(P)-H) (NIT-6), ferredoxin-nitrite reductase (NIRA), and nitrite reductase (NAD(P)H) large subunit (NASD and NASB) that can convert NO2 to NH3 [In addition, the anammox path consists of hydrazine dehydrogenase (HDH), hydrazine synthase subunit (K20932), and nitrite reductase (NO forming) (NIRK) genes, and these previous genes can convert NOe enzyme ,123. In e enzyme ,125,126.2 to NH3 ,129,130.3− that is reduced by NR to NO2−, which is then imported into the chloroplast and reduced further by NIR into NH4+. NO2− reduction to NH4+ occurs in the stroma of chloroplasts and is catalyzed by the enzyme NIR. NIR reduces NO3− to NO2− using electrons from NAD(P)H, and the reduced ferredoxin (Fdred) generated due to the photosynthetic electron transport is the electron donor for NO2− reduction. NH4+ is combined with glutamate to be assimilated into glutamine [3−, N, light, growth conditions, hormones, N metabolites, and phosphorylation [N uptake for plant growth has been extensively studied and discussed previously . N uptaklutamine . NR is rrylation ,177,178.4+ assimilation, where the enzyme GS facilitates this conversion [4+-responsive GS/GOGAT isoenzymes reduces NH4+ assimilation, particularly in the roots, resulting in impaired growth [4+-responsive forms of GS/GOGAT have a central role in the primary assimilation of NH4+ within the roots [4+ availability, it is plausible to infer that plants possess a regulatory transcriptional network that modulates their gene expression in response to NH4+ availability [In various biological systems, the interconversion of glutamate and glutamine plays a central role in N assimilation, transport, and recycling. GS and GOGAT are vital enzymes orchestrating these interconversions, ensuring optimal N utilization and maintaining N homeostasis . The roonversion ,180. Folnversion ,182,183.d growth ,186,187.he roots . As the lability .Plant GS (EC6.3.1.2), a highly conserved enzyme, catalyzes glutamine synthesis from glutamate and ammonium ions, conserving the N atom in an organic molecule. This conversion is fueled by the energy released through the hydrolysis of ATP to ADP, making GS a crucial component of N metabolism across different life forms . In addiConversely, the NADH-dependent variant, also present within plastids, is primarily concentrated in non-photosynthesizing cells. The reductant is sourced from the pentose phosphate pathway . Notably4+ in leaves, whereas GS1 is associated with recycling N within the plant. However, the relative activity levels exhibited by GS1 and GS2 are subject to variation based on species, specific plant organs, N sources, developmental stages, and prevailing environmental conditions, including abiotic stress factors. This variability underscores the intricate involvement of these isozymes, reflecting a multifaceted role [Arabidopsis [4+ supply. These isoenzymes’ expression at transcript and protein levels highlights their specificity in NH4+ assimilation.According to subcellular distribution patterns, investigations into plant GS enzymes have identified two main categories. The initial group, designated as GS1, is primarily situated within the confines of the cytoplasm. Conversely, the second group, GS2, is predominantly found within plastid structures ,193. Varted role . In wheated role . Employited role  successfted role . In addited role  as well bidopsis ,199 are 2 or assimilated organic C and (b) the energy obtained by the TCA cycle and mitochondrial electron transport chain. To create the amino acids glutamate, glutamine, and aspartate, the primary absorption of inorganic N (NH4+) needs carbon skeletons in the form of keto acids  as well as energy in the form of ATP and NADPH [3− assimilation mechanism in eukaryotic microalgae and plant leaves: NAD (P) H-nitrate reductase catalyzes NO3− reduction into NO2− in the cytosol of cells, which is required for (a) NO3− transport into the green cell, and (b) NO3− reduction into NO2− in the cytosol of cells. In addition, in Cyanobacteria, ferredoxin-nitrate reductase catalyzes the reduction of NO3− to NO2−, (c) NO2− transport into the chloroplast of cells via NAR1 family and its subsequent reduction into NH4+ in a six-electron reaction catalyzed by ferredoxin-nitrite reductase, and (d) the process mediated by the ATP-dependent GS- GOGAT cycle, which results in the formation of L-glutamate by the incorporation of NH4+ into the carbon skeleton of 2-OG [4+ as well as via the amination of 2-OG catalyzed by NAD(P)H glutamate dehydrogenase [NAD(P)H-GDH [Microalgae are unicellular aquatic microorganisms with sizes between 1 µm and 2 mm. They can generate biomass using light and carbon dioxide through the photosynthetic process. Due to their remarkable tolerance for environmental stress, they can be grown in various environmental conditions, including freshwater, saltwater, or wastewater . After Cnd NADPH . There a of 2-OG . Microal(P)H-GDH ,203.Plants must endure abiotic stressors, including drought, salt, and severe temperatures, because they are immobile. The spread of plants is significantly constrained by these stresses, which also change plant growth and development and lower agricultural output . Since N3− and NH4+ have impacts on plants’ underwater limitations. The application of NH4+ alleviates the effect of drought on plant development, while NO3− has the opposite impact [4NO3) improved water-use efficiency (WUE) by increasing plant dry mass and decreasing water loss [Drought stress significantly damages crops worldwide, particularly in arid and semi-arid regions . Hence, e impact . In thate impact . It has ter loss .4+ is essential in promoting plant drought tolerance by increasing root numbers (surface area) and subsequently improving and facilitating water uptake. Also, decreased aerenchyma development may contribute to NH4+-promoted drought tolerance [3− on drought responses is linked to NO3− transport/assimilation in plants. Numerous NO3− transport/assimilation-related genes, such as NRT2.5, GS, and GOGAT, are repressed in response to drought stress [−1) can increase the tolerance of drought stress in wheat plants combined with the highest yield [Additionally, N may lessen drought’s inhibitory effects on photosynthesis and prevent C starvation . On the olerance . The inft stress . Interest stress . Plant nt stress . The uset stress ,212. Thet stress ,214. Appst yield .Under drought stress, GS1 and GS2 exhibited differential expression among different plant organs and species, with a prominent increase of GS1 in roots and leaves, while the total GS activity showed a decrease in potato root ,216. A c2 or reduction from NO2−, have been described in plants [Like all organisms, plants must respond to various extreme environmental cues by responding to various internal signals. NO is a small lipophilic molecule that diffuses through plant cell membranes as active signals to thrive and survive under those conditions. In addition, NO is a relevant signal molecule in many plant processes. Several pathways for NO production, either oxidative in the presence of On plants . NO reguMoreover, several studies showed the involvement of NO in reproductive processes, control of development, and the regulation of physiological responses such as stomatal closure. NO also regulates the expression of several genes involved in the synthesis of and response to pathogen attacks as well as reproductive mechanisms that operate during pollen recognition by the stigma ,226,227.2− can be reduced to NO by a family of five molybdenum (Mo)-containing enzymes, which include NR, xanthine oxidase reductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mARC [Drought induces the synthesis of abscisic acid (ABA), which regulates several critical plant processes like seed germination and stomatal conductance, with the help of signaling molecules like NO and ROS . Interesand mARC ,235.Medicago truncatula, and they study addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti [4+, the functional role of NR was eliminated [In a study by Berger et al. (2020) three NR genes  were discovered in the genome of meliloti . These rmeliloti . Howeveriminated . Moreoveiminated . It was iminated . Moreoveiminated . Figure +) and chlorine (Cl−) concentrations increase in saline soil and affect the natural physiological process in plants [− competes with NO3−, and NH4+ competes with Na+ and creates ion toxicities and inequalities, which can limit the processes of N uptake, transport, and assimilation [Salinity stress has a destructive impact on the metabolism and productivity of crops worldwide, resulting in the remarkable conversion of agricultural arable soil into unproductive land . The hign plants . N applin plants ,242. Mormilation ,244. Salmilation .Interestingly, it was reported that the interaction between salt stress and N had an impact on plant growth, and the effect of N on salt-exposed plants was dependent on the N rate . In the 3− resulted in an increase of Na+ uptake and loading of the sodium into the xylem, increasing root inhibition caused by the salinity. Na+ and NO3− co-transportation was suggested based on NO3−-dependent Na+ uptake data at various Na+ concentrations [3−-dependent transport mechanisms in salt-stress environments encourage Na+ ion uptake and loading into the xylem, which may represent a main pathway for Na+ accumulation in Arabidopsis shoots [On the contrary, it was reported that the application of NOtrations . Similars shoots .It is worth noting here that GS isoforms imply a multifaceted role for GS in navigating through various dimensions of abiotic stress tolerance ,248. ForPhragmites communis) plants, and poplar trees [+/Na+ ratio, the Na+/H+ antiport in the tonoplast, and proton pump activity [Triticum aestivum [It has been demonstrated that NO can act as a signal to induce the resistance of salt stress by raising the K:Na ratio in plant cells, which is based on an increase in the H1-ATPase activity of plasma membrane in calluses from maize seedlings, reed  at a critical value of 10 mg L−1 N [−1 N [3−-rich water has been associated with methemoglobinemia (blue child syndrome) and different forms of cancer [3−-contaminated drinking water.Nutrient deficiency and/or availability are crucial to plant production ,256. Oneollution . The impundwater ,260. Sinmg L−1 N . In Arge N [−1 N . Consumpf cancer ,263,264.3− levels in ten rivers within a Kenyan tea plantation for three years. Water samples were obtained before and after fertilizer application in 2004, 2005, and 2006. For three years, there was no established trend between surface water nitrate levels and the time of fertilizer applications. However, the highest nitrate–nitrogen levels were in thr river Temochewa during the first fertilizer applications (4.9 mg L−1 to 8.2 mg L−1). Furthermore, fertilizer application increased NO3− levels, and the study indicated that initial nitrate–nitrogen levels in most rivers were high, causing surface water contamination [A notable study by Maghanga et al. (2013) was conducted to monitor changes in surface water NOmination .Rhizobia is a powerful adaptation strategy that enables these plants to reduce their dependence on synthetic fertilizers and thrive in N-deficient soils. Improving the microbial activities that boost plant growth is necessary to increase food production [Rapid human population growth and declining agricultural soils intensify resource competition, affecting human food hunger and sustainable agriculture systems worldwide . Hence, oduction . In thatoduction .Bacterial endophytes associated with crops, i.e., corn and wheat, can produce various sugar compositions, such as arabinose , to stimImproving the sustainability of biological nitrogen fixation (BNF) to cereal crops is a future reason to enhance crop productivity, representing a significant breakthrough in N-fixation research. To achieve this goal, engineering key regulators of BNF by activating the symbiotic signaling pathway between diazotrophic bacteria and cereal crops is a promising strategy for sustainable development.Rhizobium–plant symbiosis is now becoming challenging with diverse genetic modifiers of the symbiosis relationship not only in the genomic variability of partners but also in the soil microbiota. In that sense, there is a future direction to improve the specificity of the partnership during symbiotic colonization using different methods  by eliminating other microbes. Therefore, identifying and developing bioinoculant strains and genotypes by adapting and evolving elite strains in both lab-scale analyses and trials to open-field applications to increase crop yield becomes crucial. Fortunately, establishing new omics techniques such as metabolomics, metagenomics, metatranscriptomics, and metaproteomics will help in-depth research and identify the rhizospheric microbes using next-generation sequencing approaches . AccordiFertile soil is a primary component in the backbone of sustainable agriculture; however, land degradation and rapid desertification cause an estimated global loss of 24 billion tons of fertile arable land . Noticea9 microbial cells g−1 dry soil) and microbial diversity  [Rhizobium, Azotobacter, and Bacillus subtilis) [Rhizobia from the soil into nodules BNF. BNF is an eco-friendly and manageable strategy by harnessing nitrogen-fixing endophytic and free-living rhizobacteria to increase N levels in agricultural land by converting N2 to the fixed form, NH3 [3 inside root nodules by legumes account for about 65% of N utilization in global agriculture via N-fixing Rhizobia [Rhizobia strains  was studied on the growth and nodulation of three cowpea (Vigna unguiculata) genotypes in semiarid regions of Kenya. Field trials were performed in a randomized complete block design with three replications, and the symbiotic efficiency (SE) of Rhizobium isolates was evaluated. In the field, Rhizobia inoculation significantly increased nodulation and shoot DW compared to the uninoculated controls. Interestingly, Rhizobia inoculation significantly increased yields, whereas inoculation with native isolates recorded a 22.7% increase compared to uninoculated control in the first season and a 28.6% increase in yield in the second season. This study concluded that the efficient native Rhizobia in smallholder farms has promising potential to improve cowpea yield under a changing climate. In particular, R. tropici clone H53, Mesorhizobium sp. WSM3874, and R. pusense strain Nak353 showed more superiority in all the tested parameters [Bacillus, Pedobacter, Chryseobacterium, and Flavobacterium [Soils are habitats for diverse microorganisms that are either harmful (pathogenic) or beneficial (symbiotic) to plants, comprising organic substances, water, and nutrients. Food production requires essential nutrients, metabolites, and water that the soil provides to plants ,278,279.ry soil) ,282. Rhiry soil) ,284,285.ubtilis) ,286. Accubtilis) ,289,290.orm, NH3 . The bioRhizobia . AccordiRhizobia . The inorameters . A recenrameters  and exhirameters ,297. N-lrameters ,299. A lacterium .Rhizobia and leguminous plants is a significant research area to reduce the dependency on chemical fertilizers. Bradyrhizobium, Azospirillum, and Rhizobium had high plant-growth-promoting capacity and N-fixing efficiency, indicating abundant functional microbial resources in extreme soil environments [Azospirillum, Burkholderia, and other N-fixing bacteria had little success in the field [Applying the symbiotic association between ronments ,302. On he field . Thus, d2 into NH4+ [Streptomyces thermoautotrophicus. The complete genomic sequence of S. thermoautotrophicus and the precise amino acid sequence of this fourth type of nitrogenase have not been determined [A. vinelandii is one of the extensively researched diazotroph with sequenced genomes that can fix N in an aerobic culture. It has served as a model for N-fixation for decades due to several factors, including (i) its genetic adaptability; (ii) its capacity to fix N in aerobic growth conditions; (iii) its nutritional flexibility, which is demonstrated by its capacity to fix N via three different pathways; and (iv) its capacity to adapt its metabolism to a variety of nutrients and media additives; and (v) the genome’s comprehensive sequencing and manual editing [A. vinelandii has a single circular chromosome with 5,365,318 base pairs in its genome, which is expected to code for 5051 proteins [Pseudomonas stutzeri is the closest companion of A. vinelandii with a sequenced genome [P. stutzeri grows aerobically and can only fix N in microaerobic conditions [2), A. vinelandii can catalyze N fixation in an oxygen-sensitive process [A. vinelandii may modify its respiration rate to maintain a low amount of cytoplasmic oxygen while N-fixing is taking place [The identification of new genes involved in N fixation, investigation of the genome organization of N-fixing species, and the discovery of novel diazotrophic species have all been made possible by plant and microbial genomics. The primary genes responsible for N fixation encode nitrogenase enzymes, which play a crucial role in converting Nnto NH4+ ,305. Thetermined ,307. The editing ,309. A. d genome . Moreovenditions ,312. Alt process ,314. Itsng place . Five teng place ,307. Theng place ,317,318.N is an essential macronutrient required for the growth and development of all living organisms. N uptake, transport, assimilation, and metabolism are finely tuned in plants. Additionally, N serves as a key mediator in plant–microbe interactions, fostering beneficial relationships through symbiotic partnerships that enhance nutrient availability. Moreover, N plays a pivotal role in a plant’s response to various environmental stresses, such as drought, salinity, and disease. Understanding N dynamics is critical for optimizing crop yields and sustainability, as it impacts the health and productivity of individual plants and the entire ecosystem and food security.Consequently, harnessing N in agriculture addresses the challenges of feeding a growing global population while minimizing the environmental footprint of farming practices. In this sense, improving NUE by plants is a promising strategy to reduce the negative impact of using synthetic fertilizers. BNF is an eco-friendly and manageable strategy to increase N levels in agricultural land. Thus, improving BNF by genome editing could replace synthetic fertilizers to reduce dependency on chemical fertilizers. In addition, discovering new plant microbiomes and understanding their structure, abundance, and diversity will open a new avenue to better understanding the agricultural scenario to sustain global food security under limited N conditions. Despite understanding the role of N in plant biology, the precise molecular mechanisms governing N sensing in plants are still unclear, which could lead to the development of more efficient N-use strategies.2 and methane (CH4) emissions. The gaseous C leak from thawing permafrost is being extensively researched in order to determine the extent of the permafrost–carbon feedback to the climate [4+ and NO3− and driving nitrification and denitrification, the two primary processes producing greenhouse gas in soils [2 gas [2O emissions, another potent greenhouse gas. The significance and amount of N2O released from arctic soils are unknown. There is a crucial need for a combination of in situ field investigations, laboratory experiments, and cutting-edge metagenomic techniques to better understand the role of microorganisms in climate change and seasonal changes in bacteria.Furthermore, investigating the effects of climate change on N availability and its environmental impact becomes increasingly crucial. For example, arctic land areas are expected to warm, resulting in significant permafrost thawing ,320 and  climate ,324. Per climate . Organicin soils , and thes [2 gas . In addi