Search Results (985)

Soils in industrially influenced areas are often exposed to elevated nickel (Ni) levels due to metallurgical and alumina production activities. In this context, this study evaluated bauxite residue (BR) as an amendment to mitigate Ni availability and mobility in five agricultural soils from the Attica region, Greece, selected according to their pH values. Apart from the pH, soil properties were greatly varied. A very small amount of 1% BR (w/w) was incorporated into soils and batch adsorption experiments with eight Ni concentrations ranging between 1 and 90 mg Ni L⁻¹ were performed, followed by the direct application of the Tessier sequential fractionation scheme. BR addition increased the Ni adsorption capacity of soils, particularly those of low and neutral pH. BR increased the pH of acid soils, thus increasing the negatively charged sites on soil colloids. The Langmuir b_L constant provided indications of advanced Ni surface precipitation in the presence of BR. However, the desorption results suggested that, in addition to pH, Fe-Mn free oxides, noticeably those of amorphous form, controlled Ni fractionation in the studied soils. The mobility factor (MF) showed that the availability of Ni was restricted in all soil–BR mixtures. Yet, the distribution of Ni among the chemically active phases was different depending mainly on Fe-Mn free oxide content. Due to its high content in iron oxides, BR assisted the retention of Ni in soils with low Fe-Mn oxide concentration and increased significantly the Ni proportion extracted from the reducible phase. However, in soils richer in Fe-Mn oxides, BR incorporation resulted in enhanced oxidizable and residual fractions, suggesting stronger Ni binding. The results demonstrate that even a low BR application effectively enhances Ni immobilization by increasing adsorption capacity, shifting Ni toward more stable geochemical fractions, and significantly reducing its mobility, highlighting its potential as a sustainable soil amendment for Ni-contaminated soils. Full article

Background: Anemia is common among patients with antiphospholipid syndrome (APS). It can persist alone (primary APS—pAPS) or with another associated disease (secondary APS—sAPS), predominantly systemic lupus erythematosus (SLE). There are no systematic reviews addressing the type of anemia (iron deficiency without anemia—IDWA, iron deficiency—IDA, and anemia of chronic disease—ACD) in these patients. Objectives: This study aimed to assess the type of anemia and to compare the usefulness of common diagnostic anemia parameters and their mutual relations. Methods: A cross-sectional study involving 163 patients was conducted at the University Clinical Center Bezanijska kosa from June 2022 to June 2024, including 79 patients with pAPS, 47 with sAPS and 37 patients diagnosed with SLE. We compared the usefulness of iron metabolism markers (serum iron—Fe; total iron-binding capacity—TIBC; ferritin; hepcidin) in the presence of inflammatory markers such as high-sensitivity (hsCRP) and IL6 in determining the type of anemia. Results: The most common types were IDA (61.9%) and IDWA (64.3%) in pAPS patients. In contrast, ACD was equally distributed across the three groups, with prevalences of 32%, 32%, and 36% (pAPS, sAPS, and SLE, respectively). A higher frequency of thrombosis was significantly associated with a high ferritin level ≥100 (p = 0.017) and high IL6 levels (p = 0.033) as well as fetal losses (p = 0.034 and p = 0.019, respectively). The logistic regression model identified ferritin as the only significant predictor of IDA (p = 0.023). For IDWA, both ferritin (p = 0.017) and hepcidin (p = 0.038) were significant predictors of this type of iron depletion. IL-6 levels were significantly correlated with ferritin and hsCRP levels (p = 0.004 and p = 0.007, respectively). In contrast, hepcidin did not show a statistically significant correlation with inflammatory markers. A total of 40% of patients with IDA had hepcidin levels below 10, and 48% of those with ACD had hepcidin levels above 10 (p = 0.036). Conclusions: It was found that iron deficiency anemia was the most common form in pAPS, while anemia of chronic disease was equally present across all patient groups. Ferritin emerged as an independent marker for identifying iron deficiency anemia in APS patients. Although hepcidin reflects a low-inflammatory state in APS, it proved to be a more valuable tool than ferritin in distinguishing the type of anemia, especially when ferritin levels were inconclusive. Clinical manifestations in APS patients correlated with inflammatory markers. Liver function or any drug used alone or in combination had no impact on anemia type. Full article

Siderophores are classically understood as microbial iron-acquisition metabolites: low-molecular-weight ligands secreted by bacteria to solubilize and transport Fe(III) under iron-limited conditions. In this review, we expand that paradigm by highlighting an emerging and underappreciated chemical axis—boron coordination by siderophores—that links terrestrial (soil/rhizosphere) and marine microbiomes. Across diverse bacterial taxa, siderophore production is widespread and central to competitive fitness because Fe(III) is poorly soluble and frequently sequestered in environmental or host matrices. Yet in boron-rich settings (seawater and borate-enriched soils), the same oxygen-donor architectures that support Fe(III) chelation can also engage boron chemistry. We synthesize evidence that carboxylate/α-hydroxyacid (dicitrate-type) and catecholate siderophores can form tetrahedral borate/boronate complexes, whereas hydroxamate siderophores generally lack the vicinal dianionic O,O motif required for stable boron binding. Structurally characterized examples—including vibrioferrin, rhizoferrin, and petrobactin—demonstrate that boron complexation is experimentally observable by ESI-MS and multinuclear NMR and can be modulated by pH and microenvironment. Integrating these findings with datasets on boron-tolerant bacteria, we propose that when iron is scarce and boron is available, boron–siderophore complexation becomes chemically feasible and may influence microbial physiology by altering ligand conformation, metal selectivity, and potentially extracellular signaling behavior—especially in marine systems where borate is abundant at oceanic pH. Overall, this review frames boron-binding siderophores as a cross-ecosystem phenomenon and a promising conceptual bridge between environmental boron geochemistry, microbial metal economy, and metalloid-mediated signaling. Full article

Novel porous geopolymer (IGP&SS), possessing mesoporous structure and a compressive strength of 9.40 MPa, was synthesized through alkali activation of double solid wastes such as iron tailings and steel slag. To overcome the high activation energy barrier of oxidants for refractory pollutant treatment, the IGP&SS was designed to efficiently activate peroxymonosulfate (PMS) under visible-light irradiation, generating reactive radicals for the rapid degradation of methylene blue (MB). The system achieved nearly complete removal within 30 min. To enhance MB removal, the effects of key factors including IGP&SS dosage, PMS dosage, initial MB concentration, temperature, and pH on the degradation process were systematically investigated. Quenching experiments revealed that several reactive oxygen species contributed to MB degradation, with the order of contribution being •OH > ¹O₂ > SO₄•⁻ > •O₂⁻. Mechanistic studies indicated that the efficient MB degradation was primarily attributed to the flexible Fe(II)/Fe(III) redox cycling in IGP&SS, which accelerated PMS activation and radical generation. X-ray photoelectron spectroscopy (XPS) analysis of the post-reaction catalyst confirmed its structural robustness, revealing a characteristic binding energy shift in the O 1s peak to 530.8 eV and a quantitative redistribution of iron species (Fe(III) content increasing from 40.4% to 57.0%). Given its outstanding performance, demonstrated stability, and eco-friendly preparation, IGP&SS holds great promise for PMS-based advanced oxidation processes in dye wastewater treatment, offering a sustainable approach for high-value utilization of iron tailings and steel slag while alleviating resource scarcity. Full article

Ferroptosis is an iron-dependent programmed cell death (PCD) implicated in cancer therapy response, yet its transcriptional control remains unevenly characterized and often centered on a limited subset of transcription factors (TFs) rather than systematically addressing TF families. The Activating enhancer-binding Protein-2 (AP-2) family of TFs is a plausible but understudied regulatory node linking oncogenic programs to ferroptosis, with prior research limited to AP-2α and AP-2γ, suggesting anti-ferroptotic and pro-tumorigenic roles. Thus, the present study aimed to provide a family-wide analysis of the relationships between AP-2 and ferroptosis across tumors in which this PCD type is considered biologically and clinically relevant. The research integrates ferroptosis gene modules with AP-2 targetomes, tumor–normal expression comparisons, survival stratification, ferroptosis scoring, cross-cohort functional analyses, and signaling pathway projection extending canonical ferroptosis circuits with AP-2–associated non-canonical elements. Consistent associations between AP-2 expression, prognosis, and ferroptosis score were observed in five tumor cohorts: cervical squamous cell carcinoma, glioblastoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, and thyroid carcinoma. In addition, cross-cohort clustering highlighted genes enriched in redox- and lipid-metabolism programs linked to apoptosis and autophagy-dependent death. Among the candidates emerging from these analyses, ferroptotic markers (LOX, PTGS2, and NQO1) and AP-2–linked nodes such as CD36, DUOX1, EPHA2, MUC1, PTPRC, SNAI2, and TP63 warrant targeted functional and binding validation to infer whether these associations reflect direct AP-2 regulatory mechanisms. Most importantly, AP-2–centered research appears to be a valuable area for guiding studies of tumor-specific ferroptosis vulnerability or resistance. Full article

Background: Oral squamous cell carcinoma (OSCC) remains clinically challenging, particularly in advanced disease, where treatment resistance limits therapeutic outcomes. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a potential anticancer vulnerability. Galectin-1 (Gal-1/LGALS1), a β-galactoside–binding lectin frequently overexpressed in OSCC, is associated with tumor progression and unfavorable prognosis; however, its involvement in ferroptosis regulation remains incompletely understood. Methods: To investigate whether Triptolide (TPL) influences ferroptosis-associated responses through Gal-1 modulation, OSCC cell lines (SAS and HSC-3) were treated with TPL and analyzed for cell viability, lipid reactive oxygen species (ROS) accumulation, and glutathione peroxidase 4 (GPX4) expression. Publicly available The Cancer Genome Atlas (TCGA) datasets were examined to evaluate Gal-1 expression patterns and survival associations. An OSCC xenograft mouse model was further used to assess the antitumor effects of TPL and changes in ferroptosis-related markers in vivo. Results: TPL treatment reduced cell viability and increased lipid ROS accumulation in OSCC cells, accompanied by downregulation of GPX4 expression. Gal-1 expression was also decreased following TPL exposure in vitro and in xenograft tumors. Analysis of TCGA data revealed that elevated Gal-1 expression was significantly associated with poorer overall survival in OSCC patients. Conclusions: These findings indicate that TPL induces ferroptosis-associated responses in OSCC and suggest that this effect is partly mediated through modulation of Gal-1 expression. Gal-1 may represent a clinically relevant factor influencing ferroptosis susceptibility, and targeting this pathway warrants further investigation as a potential therapeutic strategy for OSCC. Full article

Excessive oxidative stress can cause irreversible cytotoxic damage to both healthy and cancer cells through the induction of reactive oxygen species (ROS) mediated lipid peroxidation. Ferroptosis has recently been shown to promote lipid peroxidation due to the over-accumulation of iron. Although cancer cells possess elevated antioxidant capacity to neutralize chemotherapy-induced oxidative stress, the co-delivery of polyphenol compounds such as curcumin (CUR) can overwhelm these defenses by elevating intracellular ROS levels to a toxic threshold, thereby increasing anticancer efficacy. In this study, we evaluated the potential of CUR to chemosensitize doxorubicin (DOX) towards the DOX-resistant lung cell line (H69AR). Our results demonstrated that the combination of DOX and CUR resulted in a concentration-dependent behavior, where low-dose concentrations exhibited antagonistic effects, while high-dose IC₅₀-equivalent concentrations shifted towards synergism. The combination induced significantly greater mitochondrial dysfunction, ATP depletion, cytochrome C release, and caspase-3 activation. This also resulted in excessive ROS generation, intracellular iron overload, and lipid peroxidation, accompanied by a reduction in antioxidant enzymatic activities. Pretreatment with N-acetyl-L-cysteine (ROS inhibitor) and ferrostatin-1 (ferroptosis inhibitor) further supported the involvement of oxidative stress and ferroptosis in modulating apoptosis and DNA fragmentation. Molecular docking analyses supported the binding of CUR and DOX to key ferroptosis regulators. This study shows the potential of CUR to sensitize DOX-resistant cancer cells through ferroptosis-linked-oxidative stress targeting. Full article

Hepatitis C virus (HCV) chronically infects over 50 million people worldwide and poses a significant risk to global health. The HCV NS3/4A complex, a bifunctional enzyme comprising a protease and a helicase domain, is indispensable for viral replication and immune evasion, making it a pivotal target for direct-acting antiviral agents (DAAs). Here, we summarize its structural features, functional mechanisms, and implications in drug design and protein engineering (e.g., nanopore sequencing applications). The NS3 protease domain is activated by the NS4A cofactor, which mediates viral polyprotein processing and relies on a zinc-binding site for structural stability. The C-terminal helicase domain catalyzes ATP-dependent 3′→5′ unwinding, and allosteric crosstalk between the protease and helicase domains dynamically modulates the enzymatic activity, balancing unwinding velocity and processivity. Beyond supporting viral replication, NS3/4A cleaves MAVS to abolish RIG-I/MDA5 signaling but spares TRIF, leaving TLR3-mediated immunity intact; it also modulates host lipid and iron metabolism, contributing to HCV pathogenesis. Notably, structural and functional studies of NS3/4A lay a solid theoretical foundation for developing novel therapeutic strategies. Currently, DAAs targeting NS3/4A have achieved high sustained virologic response rates; however, resistance-associated substitutions remain a major clinical challenge, particularly in genotype 3 infections. Emerging therapeutic strategies targeting NS3/4A include allosteric inhibition and proteolysis-targeting chimeras (PROTACs)-mediated degradation. Full article

Background: Endocrine complications remain a major cause of long-term morbidity in patients with transfusion-dependent β-thalassemia (TDT), with hypogonadism being the most frequently reported abnormality. Although iron overload is central to disease pathophysiology, its relationship with reproductive endocrine function in well-chelated adult women remains unclear. Methods: This retrospective longitudinal study evaluated endocrine function in 15 adult women with transfusion-dependent β-thalassemia major over a two-year follow-up period at a tertiary care center. Age, hormonal profiles, ovarian reserve markers, and clinical reproductive characteristics were assessed at baseline and follow-up. An age-matched control group of 22 healthy women was included. Endocrine and biochemical evaluation comprised gonadotropins (follicle-stimulating hormone and luteinizing hormone), estradiol, thyroid-stimulating hormone, prolactin, anti-Müllerian hormone, hemoglobin, serum iron, total iron-binding capacity, vitamin B12, folate, 25-hydroxyvitamin D, and cardiac and hepatic MRI T2* assessment of iron burden. Results: Hypogonadism was clinically prevalent, while other endocrine axes largely remained within reference ranges during follow-up. No newly emerging overt endocrine disorders were identified. Reproductive hormone levels showed no significant temporal changes and were comparable to those of healthy controls. AMH levels demonstrated marked interindividual variability and did not consistently correlate with systemic or imaging-based iron indices. Conclusions: In well-chelated adult women with transfusion-dependent β-thalassemia, reproductive endocrine parameters appear biochemically stable over short-term follow-up, yet clinically relevant hypogonadism persists. AMH variability may reflect subtle ovarian reserve impairment not captured by conventional gonadotropin measurements, supporting the need for longitudinal, phenotype-oriented endocrine surveillance. Full article

Spi-C is a member of the ETS (E26 transformation-specific) family of transcription factors, a group of proteins that regulate gene expression in animals by binding to specific DNA sequences. Spi-C has emerged as a central regulator of macrophage adaptation to iron exposure, inflammatory stress, and tissue injury. Studies show that Spi-C programs iron-recycling macrophages by promoting expression of key iron-handling genes, thereby supporting iron efflux, safe intracellular iron storage, and the development of red pulp macrophages critical for systemic iron recycling. Its expression is strongly induced by heme and iron, enabling macrophages to respond adaptively to increased heme turnover, whereas Spi-C deficiency leads to impaired iron recycling and pathological iron accumulation. Beyond iron homeostasis, Spi-C is increasingly recognized as a regulator of inflammatory disease, functioning as an anti-inflammatory and tissue-protective factor across multiple models, including lipopolysaccharide (LPS)–induced systemic inflammation and colitis, where Spi-C deficiency leads to enhanced cytokine production, increased tissue injury, and impaired repair. By integrating NF-κB-driven inflammatory cues with metabolic adaptation, Spi-C maintains macrophage homeostasis across tissues. This short review summarizes these known functions and provides a forward-looking perspective that Spi-C may also regulate macrophage susceptibility to ferroptosis, an iron-dependent form of cell death implicated in diverse inflammatory and degenerative conditions. Full article

Background/Objectives: Females are generally more resistant to ischemia-related ferroptosis than males, due to differences in iron metabolism, antioxidant pathways, and sex hormone-mediated regulation of ferroptosis suppressors. This has not been systematically studied in a human donor liver model. To investigate the effect of sex on ferroptosis and oxidative stress pathways in non-utilized donor livers (NDLs), we assessed patterns of gene expression in NDLs under ex vivo normothermic machine perfusion (NMP). Methods: We utilized the PROTECT dual-circuit ex vivo NMP system to assess three male and two female NDLs undergoing 6 h NMP. Perfusate and tissue samples were collected at baseline and 6 h of NMP. Malondialdehyde (MDA) levels were quantified as biochemical markers of iron overload and lipid peroxidation, respectively. Ferroptosis-related gene expression was assessed using molecular assays. Comparisons between male and female NDLs were used to determine the influence of sex on ferroptosis and oxidative injury during NMP. Results: NMP was successfully performed on NDLs (n = 5) from three male (56.3 ± 5.7 years) and two female donors (46.5 ± 0.7 years, p = 0.15). The fold-change in the oxidative stress marker MDA was comparable between female (1.2 ± 0.6) and male (1.0 ± 0.4) NDLs after 6 h NMP (p = 0.76). All livers showed upregulation of ferroptosis-related genes (Hypoxia-inducible factor 1 alpha, Iron Responsive Binding Elements 2, Ribosomal Protein L8, Ferritin Heavy Chain 1, Acyl-CoA synthetase family member 2, ATP synthase membrane subunit c locus 3, Heme-oxygenase 1, NAD(P)H Quinone Dehydrogenase 1, Tetratricopeptide Repeat Domain 35, Nuclear Factor Erythroid 2 Related Factor 2). ACSF2 expression was significantly higher in female NDLs compared with males undergoing 6 h NMP (3.6 ± 3.0 vs. 1.0 ± 0.7-fold change, p = 0.04). There were no sex-based significant differences observed in the expression of other ferroptosis-related genes (HIF-1α, IREB2, RPL8, FTH-1, ATP5G3, HO-1, NQO1, TTC35, and NRF2) between male and female NDLs. No gene reached statistical significance after false-discovery-rate (FDR) correction. Conclusions: Normothermic machine perfusion of NDLs was feasible, and no sex-related differences were observed in MDA levels or most ferroptosis-related gene expression after 6 h. Although ACSF2 showed higher expression in female livers, this was not significant after multiple testing correction, highlighting the need for larger studies to explore sex-dependent ferroptosis signaling during liver preservation. Full article

Anthropogenic CO₂ emissions have accelerated climate change, prompting the need for effective capture technologies. Adsorption using clay-based sorbents offers an eco-friendly alternative, although performance often requires enhancement. This study explored mechanochemical modification of two halloysite-rich kaolin clay samples—iron-poor (Hal) and iron-rich (HalFe)—using locust bean gum and quillaja saponin and compared their CO₂ uptake with the calcined counterparts (CHal, CHalFe). All samples were characterized using standard techniques, and their CO₂ uptake was measured volumetrically across 0.1–20 bar and 15–35 °C. Modified sorbents showed enhanced mesoporosity and binding sites, increasing CO₂ uptake by up to 26% at 20 bar (11.85 mg/g) and 125% at 1 bar (2.25 mg/g). Calcination, however, reduced surface area and sorption capacity. Isosteric heat values remained within the physisorption range, as supported by FTIR, XRF, and XPS, which showed no bulk carbonate formation. These sorbents show lower CO₂ uptakes than conventional ones. Yet their low costs, abundance, biocompatibility, and solvent-free synthesis indicate strong potential for large-scale applications, especially for low-pressure implementations such as landfills. Further detailed studies on kinetics, thermodynamics, and sorbent regeneration are needed. Spent sorbents can potentially be repurposed for subsequent use in other applications, e.g., water treatment, construction materials, thereby minimizing waste production and supporting circular economy principles. Full article

Advantageous functions have been attributed to amyloid β, which helps explain its expression despite a propensity to aggregate. Besides supporting cognitive processes, it has antimicrobial activity, e.g., amyloid β can entrap pathogens or disrupt their membranes. Since iron is an essential element for invading organisms, limiting its availability is an antimicrobial strategy. This can be achieved by various means, such as reducing circulating iron, as is the case for anemia of inflammation or anemia of chronic disease, which may occur in Alzheimer’s disease. The protein lactoferrin both sequesters iron and generates proteolytic fragments with antimicrobial properties, and amyloid β may have similar traits. Amyloid β, which is derived from proteolytic cleavage of amyloid precursor protein, directly inhibits microorganisms. In addition, it binds redox-active metals, such as iron and copper. After being generated, amyloid β can enter the interstitial fluid and undergo clearance by a variety of mechanisms (e.g., glymphatic system, transport across the blood–brain barrier, and uptake by microglia or astrocytes). This clearance, together with its small size and iron-binding properties, positions amyloid β to perform a surveillance function to access, capture, and export labile iron. By removing extraneous iron, amyloid β also helps to limit metal-catalyzed reactions that cause tissue damage. In summary, besides preventing the aggregation and neurotoxicity of amyloid β, the clearance of amyloid β from the CNS may serve a surveillance function to remove loosely bound iron to avert injury by redox reactions and enable amyloid β to function as a mammalian siderophore making iron unavailable to invading microorganisms. Full article

The removal of toxic trace metals such as cadmium (Cd²⁺) and lead (Pb²⁺) from wastewater is critical due to their persistence, bioaccumulation, and adverse health effects. In this study, a novel composite adsorbent was synthesized by integrating activated carbon with nickel–iron-layered double hydroxides (NiFe-LDH) and immobilizing the resulting nanocomposite within Polyether sulfone (PES) beads to improve stability, handling, and recyclability. The material was evaluated under varying pH, initial metal concentration, and contact time conditions. The adsorption behavior was investigated using four isotherm models and two kinetic models. The composite beads exhibited maximum adsorption capacities of 1.784 mg g⁻¹ for Cd²⁺ and 5.882 mg g⁻¹ for Pb²⁺. The Cd²⁺ adsorption followed the Langmuir isotherm model (R² = 0.995), indicating a homogeneous monolayer adsorption, whereas Pb²⁺ adsorption was best described by the Freundlich model (R² = 0.955), suggesting heterogeneous surface interactions and multiple binding sites. The kinetic analysis showed that the adsorption of both metals followed a pseudo-second-order model, supporting chemisorption as the dominant rate-controlling mechanism. The AC@NiFe-LDH/PES beads demonstrated high efficiency, structural integrity, and ease of recovery over multiple cycles, highlighting their potential as a sustainable and environmentally friendly adsorbent for trace metal removal from contaminated water. Full article

Soil salinization and alkalinization critically constrain alfalfa (Medicago sativa L.) productivity, yet the regulatory mechanisms underlying its responses to salt–alkali stress are not fully understood. In this study, the alfalfa variety “Zhongmu No. 1” was used as experimental material. The seeds were subjected to salt stress (75 mM NaCl), alkali stress (15 mM NaHCO₃), and combined salt–alkali stress (50 mM NaCl + 5 mM NaHCO₃) in dishes, with ddH₂O serving as the control (CK). After 7 days of germination, the seedlings were transferred to a hydroponic system containing Hoagland nutrient solution supplemented with the corresponding treatments. Following 32 days of stress exposure, leaf and root tissue samples were collected for morphological and physiological measurements, as well as mRNA and miRNA sequencing analyses. Physiological assays revealed significant growth inhibition and increased electrolyte leakage under stress conditions. Transcriptome profiling identified over 5000 common differentially expressed genes (DEGs) in both leaves and roots under stress conditions, mainly enriched in pathways related to “iron ion binding”, “flavonoid biosynthesis”, “MAPK signaling”, and “alpha-Linolenic acid metabolism”. MiRNA sequencing detected 453 miRNAs, including 188 novel candidates, with several differentially expressed miRNAs (DEMs) exhibiting tissue- and stress-specific patterns. Integrated analysis revealed 147, 81, and 140 negatively correlated miRNA–mRNA pairs across three treatment groups, highlighting key regulatory modules in hormone signaling and metabolic pathways. Notably, in the ethylene and abscisic acid signaling pathways, ERF (XLOC_006645) and PP2C (MsG0180000476.01) were found to be regulated by miR5255 and miR172c, respectively, suggesting a post-transcriptional layer of hormonal control. DEM target genes enrichment pathway analyses also identified stress-specific regulation of “Fatty acid degradation”, “Galactose metabolism”, and “Fructose and mannose metabolism”. qRT-PCR validation confirmed the expression trends of selected DEGs and DEMs. Collectively, these findings reveal the complexity of miRNA–mRNA regulatory networks in alfalfa’s response to salt–alkali stress and provide candidate regulators for breeding stress-resilient cultivars. Full article