Polyethylenimine (PEI)-mediated transient transfection represents a pivotal methodology for recombinant protein production in HEK293 and derived cell lines. Despite extensive application, substantial potential for enhancement in transfection efficiency and exogenous protein expression persists, with the role of metal ions remaining inadequately characterized. We hypothesized that iron ions exert a concentration-dependent, biphasic regulation on PEI-mediated transfection, affecting both extracellular complex formation and intracellular gene expression. To test this hypothesis, we systematically investigated the impact of ferric ammonium citrate (FAC) on transient transfection efficiency and anti-PD-1 antibody production in Expi293F cells. Supplementation of iron-depleted basal medium with 10 μM FAC was identified as optimal, achieving 63.7% transfection efficiency for enhanced green fluorescent protein (eGFP) and an anti-PD-1 antibody yield of 55.0 mg/L. Transmission electron microscopy analysis revealed that FAC modulates transfection efficiency by altering PEI/DNA complex morphology. Quantitative real-time PCR demonstrated significant enhancement of intracellular DNA transcription by both FAC and calcium ions. The histone deacetylase inhibitor valproic acid further augmented transcription efficiency by 2.8-fold. Ultimately, anti-PD-1 monoclonal antibody production reached 171.0 mg/L in a 3-L bioreactor, representing a 10.6-fold improvement over initial conditions. This study provides preliminary evidence for establishing an efficient transient expression platform while offering initial mechanistic insights into how iron ions influence PEI-mediated transfection via complex morphology and transcriptional regulation.

Dialysate calcium (DCa) and magnesium (DMg) and the organic acid used in the acid concentrate have a profound combined influence on intradialytic mineral balance and may influence vascular calcification progression and treatment-related symptoms. Whereas Kidney Disease: Improving Global Outcomes recommends a DCa between 1.25 (DCa 1.25) and 1.50 mmol/l (DCa 1.50), the optimal DCa for an individual patient remains uncertain. No consistent mortality difference between DCa 1.50 and 1.25 has been observed. However, DCa 1.50 may induce a positive calcium balance, raising concerns about vascular calcification progression. Conversely, DCa below 1.25 mmol/l has been associated with a higher risk of sudden cardiac death. Compared with acetic acid (acetate dialysate, Acet-D), citric acid (citrate dialysate, Cit-D) reduces calcification propensity of serum, although the effect on vascular calcification progression is inconclusive. Whenever feasible, DCa should be individualized, ideally using estimates of calcium mass balance (CaMB). Because Cit-D generally results in a more negative CaMB than Acet-D solutions at the same nominal DCa, this difference should be considered when prescribing DCa. Increasing DMg from 0.50 to 0.75 mmol/l may improve surrogate cardiovascular outcomes although its effect on clinical outcomes remains to be defined. Overall, Acet-D with DCa 1.25 is a reasonable standard especially when using calcium-containing phosphate binders and in patients at risk for adynamic bone disease, whereas in patients with a high risk of sudden cardiac death, frequent intradialytic hypotension or with severe secondary hyperparathyroidism, DCa 1.50 may be a reasonable alternative. Regarding the choice of acid, the use of Cit-D represents a promising alternative pending further clinical evidence.

Fat-soluble micronutrients are essential to human health. However, their bioaccessibility may be impaired by pulse "antinutrients" such as phytates, saponins, and tannins. We investigated whether natural variations in chickpea antinutrient profiles could influence fat-soluble micronutrient bioaccessibility in a test meal. Nine chickpea batches with contrasted antinutrient contents were selected, cooked, and incorporated into test meals subjected to in vitro digestion. Bioaccessibility was determined by quantifying fat-soluble micronutrients in the micellar fraction and the digestate. Saponin profile was conserved in chickpeas after processing (R = 0.6120), whereas tannins showed an opposite trend (R = -0.7828), and no consistent link existed for phytates. Bioaccessibility of lycopene, cholecalciferol, α-tocopherol, γ-tocopherol, and zeaxanthin significantly differed between meals containing antinutrient-rich vs. antinutrient-poor chickpeas, while intermediate profiles yielded intermediate results. Overall, principal component analysis and Pearson correlations pointed to negative associations between saponins, phytates, and most fat-soluble micronutrient bioaccessibility.

The prevalence of urinary stones range between 1 and 14% in different countries around the world. Men account for approximately 67% of cases, but the gender gap is narrowing over time. A study conducted in the Russian Federation showed that 22.2% of subjects interviewed with a questionnaire reported a history of urolithiasis. Stone formation begins with the urinary crystallization of less soluble solutes contained in urine (calcium oxalate and/or calcium phosphate, uric acid, struvite, and cystine), which often depends on urinary pH values. The most common calcium oxalate stones can grow on calcium phosphate plaques (Randall's plaque) that emerge on the surface of the papilla or on calcium oxalate plugs that form in the collecting ducts due to excessive concentrations of calcium or oxalate. Randall's plaques form due to a proinflammatory process initiated by local macrophages with activation of fibroblasts, matrix modeling, and interstitial calcium phosphate deposition. The inhibitory effect of crystallization and crystal aggregation was studied in vitro demonstrating a reduction in the nucleation inhibition index and in aggregation inhibition index in renal stone formers compared to healthy controls (2.15% vs 6.15% and 34.15% vs 62.15%, respectively). Urolithiasis is a cluster of metabolic phenotypes interacting through urinary supersaturation. The metabolic work-up consists in the identification of high-risk patients requiring full metabolic profiling by basic metabolic panel, two 24-hour urine collections, stone analysis (when available), and software assessment of Relative Supersaturation (RSS) for calcium oxalate, calcium phosphate and uric acid. The endpoint of therapy is to reduce RSS not just to achieve "normal" lab values. The most active agents used for the prevention of calcium oxalate stones are phytate, potassium citrate, and magnesium. Phytate stands as the most potent inhibitor of both calcium oxalate and calcium phosphate crystallization exerting its therapeutic effect without modifying urinary pH. It is the ideal inhibitor of calcium oxalate crystallization, particularly in cases of severe hypercalciuria, because it does not increase the risk of secondary calcium phosphate crystallization. Potassium citrate has a weaker direct inhibitory potential at the concentrations achieved in standard clinical practice and tends to increase urinary pH, posing a risk of inducing calcium phosphate precipitation. Magnesium does not affect urinary pH, but its inhibitory effect of calcium oxalate crystallization is negligible at conventional clinical doses [...].

Calcium supplementation to prevent or treat hypocalcemic symptoms during leukapheresis is not standardized. Institutions vary in practice. Within an institution, there is also nursing variability. Recognizing the need to harmonize our practices, the apheresis nurses at our institution launched a process improvement initiative to develop a standardized Citrate Reaction Grading and Treatment Chart for leukapheresis procedures. Based on nurse surveys, interdisciplinary collaboration, and clinical data, the chart enhances recognition and management of hypocalcemia-related symptoms, aiming to improve patient safety and streamline care across apheresis practices.

Viral infections are a widely recognized cause of mortality. Recently it has been demonstrated that vitamin D plays an important immuno-modulatory role in both innate and adaptive immune responses against infections, by reducing excessive inflammation and enhancing defense mechanisms. Specifically, exacerbation of infections may cause sepsis, characterized by a dysregulated immune response with hyperinflammation and immune exhaustion. Vitamin D can modulate these processes through various mechanisms, such as enhancing antiviral protection, promoting anti-inflammatory responses, protecting endothelial barriers, and modulating T-cells. This has been demonstrated for various viral infections, including influenza viruses, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus. Although vitamin D deficiency has been associated with increased susceptibility to viral infections and immune cells of infected patients are highly responsive to vitamin D, the clinical benefits of its supplementation to vitamin D-deficient infected individuals are uncertain and, most importantly, direct evidence linking vitamin D to viral sepsis specifically remains particularly limited, with most mechanistic inference extrapolated from non-septic viral infection and bacterial sepsis literature. Based on these assumptions, this review will outline the present understanding about vitamin D regulatory effects on immune system and the possible connection between its serum levels and viral infections, while also addressing controversial issues.

Vitamin D plays a fundamental role in calcium and phosphate homeostasis, skeletal development, immune regulation, and numerous metabolic processes in both humans and animals. Serum 25-hydroxyvitamin D [25(OH)D] is the accepted biomarker of vitamin D status, but its physiological concentrations vary considerably among animal species due to differences in metabolism, diet, ultraviolet B (UVB) exposure, and environmental conditions. This review summarizes current knowledge on vitamin D status across vertebrate species and identifies the major determinants responsible for interspecies variation. A structured literature search was conducted using Google Scholar, PubMed, Scopus, and Web of Science. Studies reporting serum or plasma 25(OH)D concentrations in companion animals, livestock, wildlife, reptiles and fish were evaluated, together with publications addressing vitamin D metabolism, analytical methods, and supplementation. Available evidence demonstrates marked differences in serum 25(OH)D concentrations among vertebrate species. Vitamin D status is primarily influenced by UVB exposure, dietary intake, husbandry conditions, season, and species-specific metabolic adaptations. In companion animals such as dogs and cats, dietary intake is the predominant source of vitamin D, whereas in livestock both UVB exposure and supplementation substantially influence circulating 25(OH)D concentrations. Wild animals often show greater seasonal variation in reported 25(OH)D concentrations, although comparisons with domestic counterparts are complicated by differences in diet, supplementation, environmental exposure, and analytical methods. Across multiple species, supplementation with 25-hydroxyvitamin D3 appears more effective than cholecalciferol in increasing circulating 25(OH)D concentrations. Reported vitamin D status differs among vertebrate species and cannot be interpreted using universal reference values, particularly when analytical methods and study conditions differ. Species-specific physiology, environmental factors, and nutritional management should all be considered when evaluating serum 25(OH)D concentrations. Future studies should prioritize standardized analytical methodologies, establish species-specific reference intervals, and expand research on wildlife to improve understanding of vitamin D physiology across vertebrates.

Female reproductive disorders are closely associated with ovarian oxidative stress and lipid dysregulation, but the upstream triggers remain poorly defined. In this study, we found that Cyp27b1, a key mitochondrial enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25(OH)D3) to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is an important regulator of ovarian lipid homeostasis and redox balance. In the cyp27b1[-/-] zebrafish model, cyp27b1 deletion impaired ovarian development and caused mitochondrial dysfunction and oxidative stress, all of which were alleviated by exogenous 1,25(OH)2D3 treatment. Lipidomic analysis revealed an abnormal accumulation of triglycerides (TG) and diglycerides (DG) in the knockout group, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE), key membrane lipid components, were significantly reduced, disrupting membrane integrity and energy homeostasis. Dual-luciferase reporter assays showed that 1,25(OH)2D3 increased pnpla2 promoter activity through a predicted vitamin D response element, thereby promoting TG hydrolysis in cyp27b1[-/-] ovaries. In contrast, insufficient 1,25(OH)2D3 production increased Pparγ expression and promoted abnormal lipid deposition. Exogenous 1,25(OH)2D3 treatment reduced lipid deposition and restored Pparγ and Pnpla2 expression toward normal levels. In conclusion, our findings suggested that Cyp27b1 deficiency impaired ovarian development through lipid overload associated oxidative damage. These results linked impaired Cyp27b1 mediated 1,25(OH)2D3 production to ovarian lipid metabolic disturbance and redox imbalance. Lipid metabolic changes involving pnpla2 and pparγ may represent potential regulatory targets for 1,25(OH)2D3 deficiency associated ovarian pathologies.