Integrative network analysis identifies candidate genes shared between ferroptosis and cuproptosis pathways in rheumatoid arthritis pathogenesis

by Abbas Ahmad, Shehzad Khalil, Douglas Law, Patricio R. De los Ríos-Escalante, Mostafa A. Abdel-Maksoud, Saeedah Almutairi, Aljawharah Fahad Alabbad, Waheed Ahmad, Ayaz Ahmad

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial inflammation, progressive joint destruction, and systemic immune dysregulation. Recent findings suggest that disturbed metal homeostasis and regulated cell death pathways, including ferroptosis (iron-dependent lipid peroxidation) and cuproptosis (copper-dependent mitochondrial proteotoxic stress), contribute to RA pathogenesis. In this study, we used an integrative bioinformatic approach combining weighted gene co-expression network analysis (WGCNA), differential expression analysis, functional enrichment, protein-protein interaction (PPI) network construction, and immune cell infiltration deconvolution to identify key metal-dependent cell death regulators in RA. Using bulk RNA-seq data from peripheral CD14⁺ monocytes (GSE294225) from 15 healthy controls and 9 patients with active RA (DAS28 > 2.7), we identified 1,410 significantly differentially expressed genes (DEGs) (adjusted P < 0.05, log2 fold change > 1). WGCNA revealed an RA-associated module enriched in oxidative stress, mitochondrial dysfunction, and cell death pathways. Overlap analysis of ferroptosis- and cuproptosis-related gene sets distinguished three upregulated hub genes, including FTH1 (ferritin heavy chain 1), SOD2 (superoxide dismutase 2), and CDKN2A (cyclin-dependent kinase inhibitor 2A) as key candidate regulators. These genes showed high module membership, significant differential expression in RA monocytes, and notable associations with immune infiltration patterns, including increased pro-inflammatory monocytes/macrophages and reduced regulatory T cells. Functional enrichment also highlighted oxidative stress response, iron and copper homeostasis, mitochondrial respiration, and cellular senescence. The single-cell analysis further showed that these hub genes are predominantly expressed in the RA synovial macrophages and fibroblasts, two major mediators of joint pathology. Together, these findings indicate that there may be an association between ferroptosis-related pathways and cuproptosis-related pathways in RA and suggest that FTH1, SOD2, and CDKN2A are candidate biomarkers and candidate therapeutic targets.

Endophytic <i>Aspergillus fumigatus</i> modulates growth, oxidative stress, and cadmium accumulation in okra under salinity and heavy-metal stress

by Sobia Khan, Salman Khan, Afshan Afshan, Muhammad Arif, Humaira Gul, Muhammad Hamayun, Mamoona Rauf, Douglas Law, Bandar M. Almunqedhi, Mohamed A. El-Tayeb, Ahmed Othman Alsabih, Waheed Ahmad, Patricio R. De los Ríos-Escalante, Ayaz Ahmad

Abiotic stress factors, such as salinity and heavy metals (HM), significantly reduce okra (Abelmoschus esculentus L.) yield, an important vegetable crop cultivated globally. The symbiotic relationship between endophytic fungi and plants enhances plant growth and helps plants overcome such stresses. This study was designed to isolate and examine endophytic fungi from Ziziphus lotus roots for their growth-promoting potential under salt (250 mM NaCl) and cadmium (100 ppm Cd) stress. The growth of okra was significantly enhanced by inoculation with Aspergillus fumigatus (SOA), resulting in a 28.9% increase in plant height (33.24 cm vs. 25.79 cm in the control) and improved biomass, chlorophyll content, and phytohormone regulation. Chlorophyll A content rose by 108.4% in SOA-treated plants (4.894 mg/g FW) relative to control (2.348 mg/g FW), while shoot dry weight increased more than threefold (4.365 g vs. 1.42 g in control). Abscisic acid (ABA) content decreased in SOA-treated plants, indicating a reduced stress response, whereas enhanced gibberellic acid (GA) and indole-3-acetic acid (IAA) content promoted growth. Biochemical analysis revealed higher accumulation of lipids, sugars, phenols, and flavonoids, resulting in improved stress adaptation. Oxidative damage was reduced through high antioxidant enzyme activities (CAT and POD). SOA modulated cadmium uptake, thereby reducing heavy metal toxicity. These findings showed that endophytic fungi have the potential to enhance plants’ resilience and provide a promising controlled-environment approach to enhance crop productivity in metal- and salt-contaminated soils.