Polysulfides play an important role in regulating many physiological processes, making their study relevant in health and disease. This study aims to improve their evaluation method by determining alkaline-labile sulfane sulfur (ALSS) and to test it on various biological samples, as well as on models of hyperhomocysteinemia and oxidative/reductive stress. The ALSS assay is based on the photometric determination of H₂S, which is generated from polysulfides under alkaline conditions. The limit of quantitative detection was 3.3 nmol, interday reproducibility was 5.7% (blood plasma), and the yields of H₂S precipitation and product extraction were 97.8 and 89.7%, respectively. The specificity of the approach, determined with two different methods of sulfide destruction, was 80% on a model mixture of bovine albumin or blood plasma. A small but noticeable matrix effect was revealed. ALSS of control rats (n = 15) and human plasma (n = 10) was 133 (128; 135) and 177.6 (171.8; 183.6) μmol/g protein, respectively. Decreased ALSS levels were observed in rats with mild hyperhomocysteinemia (n = 14) and in patients with acute stroke (n = 20). Under 24h oxidative stress, ALSS decreased from 14.8 ± 1.3 to 10.4 ± 1.0 μmol/g protein (p = 0.0055) in the THP-1 culture, while 96h reductive stress caused it to increase from 10.9 ± 0.9 to 15.9 ± 2.0 μmol/g protein, p = 0.00034. The method presented in this paper allows for the assessment of ALSS in various biological matrices and has demonstrated sensitivity to oxidative stress in vitro and hyperhomocysteinemia in vivo. This makes it a promising indicator of sulfur metabolism disorders in pathological conditions.

The Unfolded Protein Response (UPR) determines cell fate under endoplasmic reticulum (ER) stress, yet its temporal dynamics across different cell types and senescence states re-main poorly understood. In this study, we investigated UPR kinetics in endothelial and T-lymphoblastoid cell lines with distinct ER volumes, as well as in senescent FRSN cells undergoing ER expansion. Variations in ER volume were confirmed via confocal microscopy and flow cytometry. Using the ‘gene response time’ metric, we demonstrated experimentally and through mathematical modeling that the BiP response kinetics closely correlate with variations in ER volume. Applying this metric to presenescent FRSN cells with a significantly expanded ER, we found that their ER stress response machinery maintained a response time similar to that of exponentially growing FRSN cells, but exhibited a lower amplitude and a prolonged lag phase, leading to chronic ER stress signaling such as a protracted XBP1(S) expression profile. Scanning ion-conductance microscopy (SICM) revealed a significant decrease in the stiffness of the near-surface layer of presenescent cells, reflecting a rarefaction of the actin cortex and possibly cytoplasmic dilution. We propose that this structural remodeling could be associated with active intracellular transport impairment that weakens the temporal ‘tethering’ of the proteome to the genome upon cellular senescence. Consequently, the stress response machinery fails to scale proportionally with cell enlargement during senescence, creating a state of ‘functional dilution’. This prevents enlarged senescent cells from mounting a scaled response, resulting in prolonged stress signaling and increased vulnerability to proteotoxic stress.

Crohn's disease (CD) is a chronic inflammatory bowel disorder driven by dysregulated immune responses, epithelial barrier dysfunction, and progressive tissue damage. Despite advances in biologic therapies, treatment resistance and loss of response remain common. The activating receptor NKG2D, expressed on natural killer cells and T cell subsets, recognizes stress-induced ligands (MICA, MICB, ULBP) upregulated on intestinal epithelial cells under inflammatory conditions. In CD, sustained ligand expression promotes NKG2D-dependent cytotoxicity, pro-inflammatory cytokine release (IFN-γ, IL-17), and lymphocyte trafficking into the mucosa, perpetuating a cycle of epithelial injury and chronic inflammation. Preclinical colitis models and clinical trials with anti-NKG2D monoclonal antibodies (e.g., NNC0142-0002) demonstrate reduced disease activity, improved histological outcomes, and induction of remission in moderate-to-severe CD patients. However, challenges remain, including potential impairment of immune surveillance against infections and tumors, patient heterogeneity, and pathway redundancy. Future directions include biomarker-guided patient selection, combination therapies with anti-TNF or anti-integrin agents, and next-generation biologics such as bispecific antibodies. Understanding the cellular and molecular basis of NKG2D signaling in mucosal inflammation positions this receptor as a promising, mechanistically anchored therapeutic target in Crohn's disease.