Red blood cell (RBC) transfusion aims to restore tissue oxygen delivery, yet a stored unit’s capacity to unload oxygen depends on 2,3-diphosphoglycerate (2,3-DPG), the principal allosteric modulator of hemoglobin–oxygen affinity. Randomized trials comparing fresher with older units have been consistently null, and all have used storage duration as the exposure variable. We asked whether storage age reflects the metabolic state of individual units. Twenty-four leukodepleted packed RBC units were sampled serially from day 3 to day 42 of standard cold storage. 2,3-DPG was quantified by liquid chromatography–mass spectrometry (LC–MS) with isotopically labelled internal standards, pH was measured in parallel, and trajectories were modelled by linear mixed effects with unit-level random intercepts. At day 3, 2,3-DPG spanned an approximately 18-fold range across units of identical age (0.8–14.7 µmol/g Hb; median 4.0): some were already near-depleted at the earliest sampled timepoint, while others retained near-physiological concentrations. This heterogeneity collapsed rapidly—median values fell to 20% of day-3 levels by day 7 and by more than 95% by day 14 (β = −0.298 µmol/g Hb per day, p < 0.001)—and were below the limit of detection in most units after day 17. pH declined monotonically to 6.40 ± 0.04 by day 42 (β = −0.0163 units per day, p < 0.001). Units of identical storage age therefore differ markedly in the levels of the metabolite that governs oxygen unloading, and are depleted long before the 42-day expiry limit, suggesting that storage duration alone may incompletely capture the metabolic state of stored red blood cell units.
Autism spectrum disorder (ASD) research has largely treated early regression, late decline after a period of stability, gradual increases in symptom severity, late diagnosis, and the sex difference in diagnostic timing as separate topics. These phenomena differ in definition, evidence, and plausible mechanism, and diagnostic and recognition processes may explain much of the late-diagnosis and sex-timing findings. This narrative, hypothesis-generating review asks whether a cumulative oxidative and inflammatory burden, arising from environmental chemical exposure, infection and autoimmune activity, and chronic psychosocial stress, and modified by genetic susceptibility, one-carbon metabolism, the gut microbiome, and individual buffering capacity, could contribute to some of these trajectories. We grade the evidence for each link in four tiers (human ASD data, other human data, experimental models, and our hypotheses). Most links rest on associations with ASD risk or on experimental data; no study has linked these exposures to regression or late diagnosis, and oxidative stress may be a consequence or correlate rather than a cause. We therefore define operational terms, derive predictions that could refute the model, and outline the prospective, sex-stratified evidence needed to test it.
In humans, six common non-synonymous exonic single-nucleotide polymorphisms in ALDH1L1 lead to ten enzyme haplotypes. Since ALDH1L1 is a metabolic regulator and a candidate tumor suppressor, we studied how haplotype variants affect metabotypes, proliferation and motility. Using lentiviral delivery to A549 and PC3 cells, we have generated two cell panels, with each cell line in the panel carrying one of ten ALDH1L1 haplotype variants. Untargeted metabolomics revealed strong differences in metabotypes between cells carrying ALDH1L1 variants. Certain ALDH1L1 variants had decreased ability to regulate one-carbon metabolism and downstream pathways, including amino acid and glutathione metabolism, nucleotide biosynthesis, and the TCA cycle. The direct comparison of A549 and PC3 cells using an untargeted metabolomic dataset also showed that the two cell lines have vastly different metabotypes. In agreement with this finding, the analysis of A549 and PC3 ALDH1L1 haplotype panels suggested that the ALDH1L1-linked metabolic regulation is strongly influenced by specific cellular metabotypes and genetic background. ALDH1L1 variants also exhibited reduced inhibition of cellular proliferation and motility compared to the most common haplotype, which supports further investigation of their potential relevance to individual risk of metabolic disorders.
Head and neck squamous cell carcinoma (HNSCC) develops resistance to cisplatin. We investigated whether JIB-04, an inhibitor of Jumonji histone demethylases, enhances cisplatin activity in established HNSCC cell lines. Four UT-SCC cell lines originating from primary tumors and matched lymph-node metastases were treated with JIB-04 and cisplatin, alone or combined. Viability was assessed by MTT assay, and drug interactions were evaluated using the Highest Single Agent model. In UT-SCC-24A cells, we assessed BrdU incorporation, LDH release, cell-cycle distribution, and apoptosis. RNA sequencing was performed after 24 h at the same drug concentrations used in functional assays. JIB-04 reduced viability in all HNSCC lines at submicromolar concentrations, with moderate effects in BJ fibroblasts. HSA analysis identified a localized positive interaction in UT-SCC-24A, where the overall HSA score was 4.25, whereas local scores reached approximately 16–18 within a narrow low-dose range. Co-treatment further reduced DNA synthesis and increased S/G2-M accumulation and apoptosis, without significantly increasing LDH release. RNA sequencing revealed JIB-04-associated induction of integrated stress response/ATF4- and DNA damage response-related genes, alongside suppression of proliferation- and cell-cycle-associated genes. The combination retained these transcriptional features. These findings support the further investigation of JIB-04–cisplatin combinations in selected HNSCC cell-line models.
Reis–Bücklers corneal dystrophy (RBCD) is an early-onset TGFBI-related corneal dystrophy associated with the TGFBI R124L variant. It is characterized by recurrent corneal epithelial erosion, hyaline-like deposits involving Bowman’s layer and the anterior stroma, and progressive subepithelial corneal opacity. However, how mutant TGFBI protein (TGFBIp) accumulates in the cornea remains unclear. In this study, we established induced pluripotent stem cells (iPSCs) from two affected members of an RBCD family and one unaffected family member. The patient-derived lines retained the R124L variant, whereas the line established from the unaffected mother did not. All three lines showed pluripotency and trilineage differentiation, had normal karyotypes, and were confirmed by STR analysis to match their donors. After 4 weeks of SEAM induction, all three iPSC lines developed early SEAM-like cultures containing PAX6- and p63-positive cells, demonstrating their capacity for early differentiation toward the corneal epithelial lineage. TGFBIp was also detected in extracellular and pericellular regions by immunofluorescence. In silico analysis detected no major structural difference between WT and R124L TGFBIp in the high-confidence CROPT and FAS1 domain regions, whereas surface analysis showed local changes in electrostatic potential and molecular lipophilicity around residue 124. These lines can be used to study TGFBIp deposition and epithelial–stromal interactions in RBCD.
