Perfusion fixation is widely used in neuroscience to prepare mammalian brain tissue for histological and ultrastructural analysis. Perfusion protocols are commonly assessed using macroscopic indicators such as gross appearance and neuroimaging, which assess the extent to which perfusate has been distributed throughout the brain. There is a critical need to determine to what extent these metrics can accurately predict high-quality ultrastructural preservation, particularly as new perfusion protocols are developed for connectomics. In this technical report, we describe evidence that these two measures can be decoupled by the addition of dehydrating agents to the perfusate solution. In three human brain donors and one canine brain donor perfused with a fixative solution containing 10% mannitol and 10% polyethylene glycol 35 kDa, macroscopic and radiological indicators of perfusion quality appeared adequate or favorable. However, electron microscopy revealed expanded extracellular space, shrunken cellular processes, and distorted cell membranes, consistent with an osmotic shock artifact resulting from severe hyperosmotic dehydration. Similar ultrastructural artifacts were observed in a canine brain donor perfused with 20% mannitol in 20% neutral buffered formalin without PEG. We compare these ultrastructural findings with findings from previously reported cases perfused with standard neutral buffered formalin without osmotic additives. These findings illustrate a risk of optimizing brain perfusion protocols designed to preserve neural circuitry based on macroscopic or radiological perfusion quality metrics alone, since these metrics can be satisfied while the ultrastructure is severely compromised.

α-Glucosidase is a key therapeutic target in the management of type 2 diabetes, as its inhibition delays glucose absorption into the bloodstream and thus attenuates postprandial hyperglycemia. Although several clinically approved antidiabetic agents function as α-glucosidase inhibitors, their clinical use is often constrained by gastrointestinal adverse effects. These side effects have driven efforts to identify novel α-glucosidase inhibitors with improved therapeutic index. Nonetheless, this increasing interest in the search for new compounds has been hampered by methodological difficulties associated with α-glucosidase inhibitor screening assays. Despite the considerable attention given to natural sources, especially polyphenolic compounds, the substantial differences in experimental setups across studies can compromise the reproducibility and comparability of results reported for the same molecules. This review aims to identify and summarize the methodologies and experimental conditions used to evaluate the inhibitory activity of polyphenols against α-glucosidase. A comprehensive analysis of 102 studies was conducted, focusing on key experimental parameters, including enzyme source and substrate type with their respective concentrations, temperature, pH, buffer, and incubation time employed in the assays. By pinpointing methodological inconsistencies and drawing attention to recurring best practices, this work underscores the importance of increased standardization to ensure reliable cross-study comparisons and to advance the rational development of polyphenol-based α-glucosidase inhibitors.

Ex vivo lung perfusion (EVLP) is now an established normothermic machine-perfusion strategy in lung transplantation, primarily enabling structured donor lung assessment, short-term preservation, and selective rehabilitation outside the recipient. Because the supply of transplantable lungs remains limited, clinically implemented EVLP protocols have increased donor-lung utilization by allowing transplant teams to evaluate marginal grafts under near-physiological ventilation and perfusion rather than relying exclusively on static cold storage. The perfusate is central to this process because it stabilizes colloid osmotic pressure, electrolytes, buffering capacity, metabolic substrates, and oxygen transport according to protocol requirements. This review critically summarizes the development of EVLP perfusates and separates clinically validated roles from experimental repair strategies. We first distinguish EVLP perfusates from cold preservation solutions and compare acellular, red blood cell-containing, and whole-blood approaches with attention to their physiological rationale, logistical constraints, and level of evidence. We then discuss protocol-specific additives, including anti-inflammatory agents, antioxidants, antimicrobials, anticoagulants, hormones, nutritional substrates, and cellular or gene-based therapies. Emerging interventions such as polymerized hemoglobin, cytokine adsorption, mesenchymal stromal cell-derived products, CRISPR-Cas-mediated interleukin-10 modulation, and prolonged or intermittent EVLP illustrate the expanding therapeutic potential of the platform. However, most of these strategies remain preclinical or early translational. Future perfusate development should therefore prioritize standardized outcome measures, evidence-based component selection, and careful separation of organ preservation, organ assessment, and organ rehabilitation.

Targeting redox homeostasis represents a promising strategy to overcome adaptive mechanisms that sustain cancer cell survival under therapeutic stress. Here, we evaluated a pharmacological sensitization strategy based on quercetin (Q) preconditioning followed by treatment with 3',5'-dimaleamylbenzoic acid (DMAB), aimed at exploiting glutathione-dependent vulnerabilities in cervical cancer cells. Q and DMAB exhibited synergistic interactions across a 6 × 6 concentration matrix, with a marked leftward shift in the dose-response profile within the IC50-proximal range. Combined treatment was associated with significant depletion of intracellular glutathione (GSH) and a reduction in the GSH/GSSG ratio, consistent with disruption of redox buffering capacity. Functionally, Q + DMAB reduced HeLa cell viability to 23% at 48 h and markedly increased apoptotic cell death (75%), accompanied by S-phase cell cycle arrest and inhibition of cell migration. Notably, cytotoxic effects were attenuated in nonmalignant epithelial cells. In vivo, combined administration significantly reduced tumor burden (69%) and improved survival in a murine L5178-Y lymphoma model, without overt signs of systemic toxicity. Collectively, these results demonstrate that Q enhances the antitumor activity of DMAB through modulation of glutathione-dependent redox homeostasis and support the potential of redox-directed combination strategies in cancer therapy.

Potassium- and phosphorus-containing food additives represent large and functionally diverse classes of ingredients used to control flavor, texture, water dynamics, emulsification, pH, leavening, and shelf stability. Potassium-based additives, numbering over 70 approved compounds in the United States and European Union, are used as functional salts, buffering agents, antimicrobial preservatives, nonnutritive sweeteners, and leavening components, with expanding application in reformulated and reduced-sodium food products. Phosphorus-containing additives, encompassing more than 50 approved inorganic and organic phosphate compounds, constitute another major category of multifunctional ingredients used for emulsification, leavening, moisture retention, pH control, metal ion sequestration, and nutrient supplementation. Among these ingredients, many contain both potassium and phosphorus. These myriad additives differ substantially in chemical form, reactivity, solubility, and technological function, leading to distinct formulation roles and regulatory classifications. Despite their prevalence, dietary intake of potassium and phosphorus can be under-recognized, as ingredient labels typically declare additive names without quantifying the associated potassium or phosphorus contents. This review provides a structured reference for industry professionals and researchers by systematically categorizing potassium- and phosphorus-containing food additives according to technological function, food usage, chemistry, and regulatory status across major food safety authorities.

Residual proteins from fining agents such as egg albumen and animal gelatin may persist in wine, raising food safety and labelling concerns. This study develops a metrologically traceable HPLC-HRMS workflow for the absolute quantification of proteotypic peptides from ovalbumin, ovotransferrin, lysozyme, and collagen α1/α2, using isotope-dilution mass spectrometry in parallel reaction monitoring mode. Method development, calibration hierarchy, and uncertainty evaluation were fully established in ammonium bicarbonate buffer, achieving good calibration linearity (chi-square values below 1) and LOQs of 0.63 to 1.16 mg/L. Wine was evaluated only as a post-extraction matrix effect, through matrix-matched calibration in a reference red wine. Expanded uncertainties were comparable between buffer (28.6-36.6%) and wine extract (27.8-36.2%), indicating that matrix effects had limited impact despite peptide-dependent signal suppression. This framework offers a solid, SI-traceable analytical foundation, established in buffer, for future validation in fortified wine samples, supporting quantitative allergen assessment in winemaking.

Dalbavancin hydrochloride (DB) is a semi‑synthetic lipoglycopeptide antibiotic whose pharmacopeial impurity analysis relies on mobile phases containing non‑volatile phosphate buffers. Although effective for separation, these buffers are incompatible with mass spectrometry (MS), impeding structural characterization of trace impurities. In this study, a heart‑cutting two‑dimensional liquid chromatography/high‑resolution MS (2D‑LC/HRMS) method was developed to overcome this limitation. The first dimension employed a dual‑column serial system with a phosphate‑based mobile phase to achieve high‑resolution separation of dalbavancin and its related substances. Target fractions were online transferred to a second dimension with MS‑compatible conditions for desalting and accurate mass analysis. Using this approach, 22 components-including five major components (DB‑A0, DB‑A1, DB‑B0, DB‑B1, and DB‑B2) and 17 impurities (e.g., DB‑R1, DB‑R2, DB‑D0, DB‑D1, DB‑C0, DB‑C1, RRT0.94, and RRT0.96)-were identified based on accurate mass and tandem MS fragmentation patterns, with confirmation by reference standards where available. To support routine quality control, three complementary high-performance LC methods were developed and fully validated according to ICH guidelines for the quantification of related substances, specific impurities DBa/RRT1.12/RRT1.15, and the highly polar impurity DB‑R6. Forced‑degradation studies under thermal, photolytic, acidic, alkaline, and oxidative conditions revealed distinct degradation behaviors: DB‑R1 increased markedly under thermal, acidic, and photolytic stress; DB‑R2 was the primary alkaline degradation product; DBa and RRT0.96 increased markedly under oxidative conditions; and DB‑R6 increased markedly under acidic conditions. This integrated analytical workflow provides a robust platform for both impurity characterization and routine quality control of DB, supporting process optimization and stability assessment.

Ubiquitin-like modifier activating enzyme 1 (UBA1) occupies the apex of the canonical ubiquitination cascade and supplies activated ubiquitin to a broad network of E2 conjugating enzymes and E3 ligases. Although traditionally regarded as an essential housekeeping enzyme, human genetics has revealed a more instructive function for UBA1: it determines whether proteostatic stress is buffered, converted into inflammatory adaptation, or progresses to cell death. Somatic UBA1 mutations causing VEXAS syndrome provide a natural experiment in this threshold biology. Partial and compartment-selective loss of UBA1 activity permits hematopoietic cells to survive while reducing ubiquitination capacity, activating stress-response pathways, altering cell-death thresholds, and promoting innate immune dysregulation. Single-cell and functional studies further demonstrate early inflammatory activation in UBA1-mutant hematopoietic progenitors, myeloid-biased differentiation, monocyte dysfunction, and inflammasome-associated signaling. Conversely, profound pharmacological UBA1 inhibition with agents such as TAK-243 drives proteotoxic collapse and is being explored as an anticancer strategy. Here, we propose that UBA1 functions as a proteostasis-innate immunity checkpoint whose biological output is determined by residual enzymatic activity, subcellular distribution, lineage context, and clone size. This framework reconciles inflammatory disease caused by partial UBA1 dysfunction with the antitumor effects of UBA1 inhibition and suggests a mechanism-based approach to therapeutic intervention in VEXAS syndrome and malignancy.

Pharmaceuticals are increasingly found as emerging environmental contaminants in water. Many of these compounds may interact with humic acid and other forms of natural dissolved organic matter, which affects properties such as their bioavailability and persistence. Ultrafast affinity extraction (UAE) has been previously applied to study the binding of drugs to proteins for biomedical applications. In this study, UAE was adapted and used to examine humic acid binding to ciprofloxacin and norfloxacin, two antibiotics that have been found in the environment. Microscale columns containing entrapped humic acid within porous silica were prepared and used to capture the non-bound and free fractions of these drugs in solution-phase mixtures containing soluble Aldrich or Leonardite humic acid. The results were used to acquire both the apparent dissociation rate constants and binding constants for the drugs with these types of humic acid. The distribution equilibrium constants measured for this binding at pH 7.0 in 0.10 M potassium phosphate buffer and at 25 °C were 3.6-5.6 × 10[4] L/kg for Aldrich humic acid with ciprofloxacin or norfloxacin and 7.4-8.3 × 10[4] L/kg for Leonardite humic acid. These binding constants fit well with those determined using other techniques. The apparent dissociation rate constants measured by UAE in the same pH 7.0 buffer and at 25 °C were 0.10-0.17 s[-1] for Aldrich humic acid with ciprofloxacin or norfloxacin and 0.052-0.16 s[-1] for Leonardite humic acid. Advantages of this method included its speed, need for only small amounts of soluble drugs and binding agents, and ability to provide information on both binding strength and dissociation rates for these systems. This approach is a general one that could also be applied for binding and rate studies with other emerging contaminants and soluble binding agents for environmental analysis and research.