Abstract Tracheal smooth muscle tone is regulated by diverse receptor systems that play key roles in respiratory diseases. Evaluating competitive receptor antagonism in isolated tissue preparations is therefore essential for characterizing novel therapeutic agents. However, methodological variability and the lack of a standardized functional framework often compromise reproducible assessment and mechanistic differentiation of candidate compounds. This article presents a standardized organ-bath protocol for evaluating receptor-mediated contraction and relaxation in isolated tracheal smooth muscle preparations. The method is intended to determine whether natural products, plant extracts, bioactive constituents, or synthetic compounds modify airway responses through receptor blockade, receptor stimulation, or non-receptor-mediated mechanisms. The protocol incorporates three complementary approaches: (1) addition of a test agent after establishment of an agonist-induced contractile or relaxant response; (2) comparison of test-agent responses in tissues pre-incubated with a receptor-selective antagonist versus non-incubated tissues after KCl-induced precontraction; and (3) cumulative concentration–response curve analysis in the presence of saline, a reference competitive antagonist, or graded concentrations of a test agent. Tracheal rings are mounted in Krebs–Henseleit solution under controlled organ-bath conditions, and responses are recorded isometrically. Quantitative outcomes include the half-maximal effective concentration (EC50), maximum response (Emax), Hill slope, and concentration ratio minus one (CR − 1), which quantifies concentration–response curve displacement. A parallel rightward shift with preserved Emax supports competitive antagonism, whereas reduced Emax, altered slope, or non-parallel displacement suggests non-competitive, mixed, or downstream functional mechanisms. This protocol provides a reproducible framework for mechanistic airway pharmacology and preclinical screening of candidate bronchodilatory and bronchoprotective agents.
Abstract Sialylation at the terminal ends of cell-surface glycoproteins and glycolipids plays a pivotal role in dictating cellular interactions, human health, and pathological processes. This crucial modification is mediated by a family of twenty distinct human sialyltransferases whose exquisite substrate specificities and stereochemistry make them high-priority therapeutic targets. Consequently, robust techniques to detect and quantify sialyltransferase activity are critical for translational research and inhibitor screening. To address this need, we present a detailed protocol for the chemo-enzymatic synthesis of a C5 alkyne-functionalized CMP-Sia donor substrate tailored for bioorthogonal click chemistry. This synthesis is coupled with the development of a simple, highly sensitive, and reproducible microplate-based assay designed to perform in-depth enzymatic characterization of individual sialyltransferase activities. The protocol eliminates common throughput limitations by streamlining substrate preparation and optimizing microplate detection metrics. Our results demonstrate that this assay provides exceptional sensitivity and consistency across diverse sialyltransferase isoforms. Ultimately, this unified method establishes one of the most robust, easily replicable, and high-throughput platforms currently available for sialyltransferase profiling and small-molecule inhibitor screening.
Abstract Fish scales are a calcium-phosphate-rich by-product of food processing that can be converted to hydroxyapatite (HAp), offering a circular-bioeconomy pathway for the development of oral biomaterials. Although an established extraction and characterization basis exists for HAp derived from Rohu (Labeo rohita) scales, direct investigations of fish-scale nano-HAp on enamel remain limited. This protocol provides optimized calcination and fluoride-functionalization conditions for nano-HAp derived from L. rohita scales, comprehensively characterizes the optimized material, formulates pH-standardized gels (pH 7.0 ± 0.1 at 25°C) with an analytically determined final-gel total fluoride target of 1450 ± 75 ppm for the fluoride-containing groups, and compares its capacity to repair artificial early enamel lesions against fish-scale nHAp, commercial synthetic nHAp, fluoride-matched free-fluoride controls, and a vehicle control. Gel fluoride will be determined after validated complete digestion and confirmed against an independent ion-chromatography method; the fluoride controls will be matched to the measured fluoride content of the functionalized-gel batches. The study is structured across three linked phases. Phase I will prepare fish-scale HAp at calcination temperatures of 800, 900, and 1000°C, followed by wet planetary milling and controlled fluoride ion exchange at four nominal fluoride levels (0, 0.75, 1.45, and 2.15 wt%), with three independent synthesis batches per experimental condition. Phase II will select an optimized fluoride-functionalized nHAp using prespecified multi-response desirability criteria, followed by comprehensive characterization of phase composition, crystallinity, morphology, primary and hydrodynamic particle size, surface charge, Ca/P ratio, fluoride incorporation, trace element content, thermal behavior, surface area, and fluoride release profile. Phase III will employ 120 enamel specimens obtained from 60 sound permanent premolars, randomized to six treatment groups (n = 20 per group) within a 14-day pH-cycling model. The primary outcome measure is percentage surface microhardness recovery (%SMHR). Secondary outcomes include lesion mineral loss and depth, cross-sectional hardness, Ca/P ratio, surface roughness, mineral deposition, and resistance to a secondary acid challenge. This study evaluates the complete translational pathway from fish-processing waste to a chemically verified, fluoride-functionalized biomimetic material. By including a fluoride-matched physical-mixture control, the design is specifically configured to distinguish a genuine functionalization effect from the independent contributions of HAp and soluble fluoride.
Abstract Semaglutide and tirzepatide are typically evaluated through dose escalation, yet many patients in routine care do not reach a recommended maintenance dose due to tolerability, access, cost, supply, or individualized goals. Here, we used repeated 0.25 mg only semaglutide and 2.5 mg only tirzepatide prescription records as real-world proxies for sustained treatment at the lowest approved dose, a prescribing pattern that may overlap with, but does not establish, intentional microdosing. We analyzed de-identified electronic health records (EHRs) from a federated system of 29 million patients. Among 490 072 semaglutide-treated patients, 814 met the stringent sustained low-dose criterion of at least three distinct 0.25-mg prescriptions spanning at least 6 months from the first to the last qualifying prescription and among 322 442 tirzepatide-treated patients, 1016 met the analogous criterion of at least three distinct 2.5-mg prescriptions spanning at least 6 months. In a matched head-to-head comparison aided by AI-enabled curation of clinical notes, sustained low-dose tirzepatide showed higher 24-month adverse event rates (P < .05) than sustained low-dose semaglutide for constipation (32.6% vs. 22.4%), acute kidney injury (2.7% vs. 0.4%), muscle cramps (8.3% vs. 4%), lumbar disc disease (3.3% vs. 0.3%), dyspnea on exertion (9.6% vs. 6.8%), lentigo (4.7% vs. 0.8%), and actinic keratosis (3.3% vs. 0.6%), whereas sustained low-dose semaglutide (vs. tirzepatide) had higher rates of including otitis (5.6% vs. 2.1%), diaphoresis (5.5% vs. 3.2%), and ankle swelling (6.9% vs. 3.7%). Sustained low-dose tirzepatide was associated with greater weight loss than semaglutide (mean 12-month percent weight change −5.5% vs. −2.2%; P < .05 at all timepoints). Analyses of prescription patterns supported the findings from clinical notes. For example, renal-supportive and hyperkalemia therapies increased after low-dose tirzepatide (5.6% to 9.5% at 12 months, P = .016) but not after low-dose semaglutide (8.2% to 6.6%, P = .382). Taken together, these observational associations motivate prospective studies to better characterize the outcomes and pharmacology of sustained initiation-dose semaglutide or tirzepatide, and related microdosing studies.
Abstract Caenorhabditis elegans is widely used in genetics, neurobiology, aging, toxicology, host–microbe research, and whole-animal screening, yet culture conditions are often treated as technical details rather than biologically active variables. Solid agar-based and liquid suspension systems expose animals to different spatial, nutritional, mechanical, and physicochemical environments that can influence locomotion, feeding, metabolism, stress responses, development, gene expression, and experimental reproducibility. This critical narrative review compares the principles, advantages, limitations, and applications of these two major culture regimes and evaluates how culture-dependent variables affect biological interpretation. Solid systems provide spatial organization, localized chemical and microbial cues, and high individual-level resolution, making them particularly suitable for behavioral, developmental, neurobiological, and longitudinal studies. Liquid systems facilitate more readily standardized population density, nutrient and compound exposure, synchronized biomass production, molecular analyses, automation, and high-throughput screening. However, they introduce specific sources of variability associated with swimming, oxygenation, agitation, vessel geometry, and population density. Comparative and recent studies indicate that culture and exposure conditions can alter the magnitude and, in some cases, the detectability of biological and toxicological phenotypes. Emerging approaches incorporating microfluidics, defined media, automated imaging, and controlled microbial environments increasingly combine features of both formats while improving experimental precision. We therefore propose that solid and liquid systems should be considered complementary regimes that should not be assumed to be biologically equivalent. Deliberate culture selection and comprehensive reporting of culture conditions are important for reproducibility, cross-platform comparability, and mechanistic interpretation in C. elegans research.
