Endogenous molecules are proposed as biomarkers to assess drug transporter function, with changes in their concentrations reflecting altered transporter activity. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed, comprising a hydrophilic interaction liquid chromatography (HILIC) method and a reversed-phase liquid chromatography (RPLC) method both with MS/MS, using a single supernatant to quantify plasma biomarkers of clinically relevant drug transporters: coproporphyrin I (CPI), 4-pyridoxic acid (PDA), N1-methylnicotinamide (NMN), and 1-methyladenosine (m1A). Optimization resulted in a 5-min HILIC method for PDA, NMN, and m1A using a pH 9 mobile phase containing 10 mM ammonium acetate and 0.04% ammonium hydroxide to achieve acceptable peak shapes and retention, with 5% methanol added to the organic phase to maintain salt solubility. For CPI and CPIII, a 3.5-min RPLC method at pH 2.7 enabled baseline separation of the two isomers with a low gradient steepness of 0.032 at 60 °C. Protein precipitation provided lower limits of quantification of 0.18-4.00 ng/mL across the different biomarkers, while parallelism experiments confirmed 3 × charcoal-stripped K2EDTA human plasma as a suitable surrogate matrix. Fit-for-purpose validation demonstrated intra- and inter-assay accuracy and precision and confirmed assay selectivity. The final method was applied to plasma samples from healthy volunteers, resulting in a geomean of the baseline levels of 0.52 ng/mL for CPI, 5.5 ng/mL for PDA, 14.7 ng/mL for NMN, and 28.9 ng/mL for m1A, consistent with previously published data. The variation in the baseline levels was mainly described by sex for CPI and by the sex-age interaction for PDA.

Single-crystal materials are essential for fundamental catalysis studies and advanced technologies; however, synthesizing single crystals that simultaneously integrate mesoporosity, tunable size, and well-defined surfaces remains challenging. Here, we report a wet-chemical, template-free strategy to synthesize mesoporous quasi-single-crystal CeO2 superparticles (SPs). Operando wide- and small-angle X-ray scattering combined with theoretical modeling reveal a nonclassical growth mechanism driven by the oriented attachment of nanocluster building blocks. This process yields high-surface-area quasi-single crystals that preferentially expose energetically favorable (111) facets on both the external surfaces and internal pore walls, thereby enhancing the stabilization of active sites when used as heterogeneous catalyst supports. Using this approach, we demonstrate broad applicability by synthesizing highly dispersed precious metal-loaded CeO2 (M/CeO2, M = Pd, Pt, Rh, or Ir) and metal-doped CeO2 (Cu-CeO2) SPs. Moreover, Pd/CeO2 SPs exhibit effective stabilization of Pd clusters in CO2 hydrogenation to methanol, delivering a high methanol selectivity of 85% and markedly improved durability compared with conventional supports.

Sexually deceptive orchids mimic female mating signals of their pollinators to achieve pollination. In Australia, multiple orchid genera exploit male thynnine wasps as pollinators, raising the possibility of convergent evolution of floral traits. This study investigates the chemical basis of attraction in Spiculaea ciliata and its pollinator Thynnoturneria abspiculus, uncovering the use of shared chemical attractants. Flowers of S. ciliata were collected in the field and extracted in dichloromethane and methanol. Gas chromatography-mass spectrometry (GC-MS) and field bioassays were used to identify and test candidate floral semiochemicals. Compound identity was confirmed through co-injection and peak enhancement with a synthetic standard, and behavioural responses of pollinators to candidate compounds were assessed with the aid of dummies loaded with candidate compounds. Spiculea ciliata attracts male T. abspiculus using a single semiochemical emitted from its labellum, identified as 4-hydroxy-3-(methylthio)benzaldehyde. This compound alone elicited sexual behaviours in male wasps comparable to those triggered by whole floral extracts. 4-Hydroxy-3-(methylthio)benzaldehyde is also known from distantly related Caladenia orchids and their pollinators, indicating repeated evolution of this unusual aromatic sulfurous semiochemical. These findings demonstrate convergent evolution of chemical signalling systems in sexually deceptive orchids and their thynnine wasp pollinators.

Prostate cancer is the second most frequently diagnosed malignancy in men globally, with over 1.4 million new cases and approximately 397,430 deaths in 2022. Despite available treatments, incidence and mortality remain high, underscoring the need for novel, cost-effective therapeutic options. Myrianthus arboreus P. Beauv. (Cecropiaceae) is traditionally employed in Cameroonian herbal medicine for cancer-related conditions. This study assessed the anti-prostate cancer activity of aqueous and methanolic bark extracts of M. arboreus on LNCaP, DU145, and PC3 human prostate cancer cell lines and in a benzo(a)pyrene (B[a]P)-induced prostate cancer rat model. In vivo, 48 male Wistar rats were randomized into six groups (n = 8) receiving distilled water, casodex (13.5 mg/kg), or M. arboreus methanolic extracts (50, 100, or 200 mg/kg) orally for 196 days following B[a]P-induced carcinogenesis. In vitro, both extracts inhibited cancer cell growth in time- and concentration-dependent manner, with M. arboreus methanolic extracts showing greater potency. M. arboreus methanolic extracts promoted apoptosis in LNCaP and DU145 cells, suppressed clonogenicity, migration, and invasion in DU145 cells, and enhanced adhesion to collagen and fibronectin matrices. M. arboreus methanolic extracts significantly reduced tumor incidence, mass, volume, and PSA levels, and reversed B[a]P-induced oxidative stress, inflammation, dyslipidemia, and hepatorenal dysfunction. These findings indicate that M. arboreus methanolic extracts exerts potent chemopreventive and anti-proliferative effects in prostate cancer models, supporting its development as a plant-based therapeutic candidate against prostate cancer.

Despite the legal prohibition of alcohol in Iran, consumption of non-standard and often toxic alcoholic beverages continues to present a significant public health challenge. Methanol poisoning and associated mortality underscore the urgent need to address alcohol-related harms through ethical and population-centered strategies. This analysis employs the three core goals of contemporary public health-autonomy, well-being, and equality-integrating both duty-based and outcome-oriented ethical reasoning to evaluate current responses and guide interventions. A public health approach is necessary to address ethical issues such as disproportionate impact on young adults and socioeconomically vulnerable groups. A public health centered approach that emphasizes prevention, protection, and health promotion provides opportunities to reduce harm, improve health outcomes, and empower individuals even within restrictive legal and cultural environments. Lessons from national experiences in harm reduction and public health interventions, such as programs for substance addiction and sexually transmitted disease control, particularly HIV control, demonstrate that effective and ethically grounded strategies are achievable even when such certain behaviors such as sex work or alcohol consumption are legally sanctioned or even criminalized. These experiences highlight the moral responsibility of public health authorities to protect vulnerable populations while promoting collective well-being through inclusive, evidence-based, and context-sensitive measures.

Metamorphosis in class Insecta is defined as the drastic transition of an organism from a larval stage to a morphologically very different adult form. Insects such as Drosophila melanogaster undergo metamorphosis, which requires precise temporal control, exerted by ecdysteroids, the primary regulators of these developmental transitions. The active form of ecdysone is 20-hydroxyecdysone (20E), which acts in pulses that trigger the onset of different developmental stages. A massive pulse of 20E occurs between the prepupal and pupal stages, triggering pupariation. Precise quantification of 20E levels is critical for understanding how this hormonal signaling governs the developmental timing of different stages. This protocol is sensitive for detecting low levels of 20E and describes a methanol-based extraction method coupled with a competitive ELISA to quantify whole-body 20E levels in third-instar Drosophila larvae. Further, this protocol enables detection of differences in ecdysone titers among genotypes affecting ecdysone biosynthesis, particularly at the late third-instar larval stage. This protocol can also be utilized for disease-causing insects (Aedes aegypti), as well as economically relevant beneficial insects (Apis mellifera), to study the effects of ecdysteroid analogs during development. Key features • 20E-dependent Ecdysone signaling induces morphological changes in insects known as metamorphosis. • This protocol provides details for 20E quantification, based on the competitive ELISA between unconjugated and HRP-conjugated 20E. • Spectrophotometry-based color intensity is directly proportional to the conjugated 20E and inversely proportional to the amount of 20E present in the sample.

Building on recently developed ion mobility spectrometry (IMS) instrumentation, this work presents the first chiral and achiral analyses of multicomponent mixtures and real-world samples using supercritical fluid chromatography (SFC) coupled to soft X-ray IMS. The implementation of ultra-fast polarity switching IMS technology, previously developed for hyphenated gas chromatography (GC), enables a new application of dual-polarity detection and extends the range of detectable compounds. Additionally, the quasi-simultaneous detection of charged analyte species formed by soft x-ray ionization in both positive and negative ion modes within a single chromatographic measurement enables comprehensive analysis with improved confidence in compound identification based on polarity-dependent ionization efficiency. To evaluate the capabilities and limitations of SFC-X-ray-IMS, the influence of varying proportions of methanol as a modifier on the amount of the reactant ions and, subsequently, on the amount of analyte ions was investigated. Reliable detection of analyte ion species of opposite polarities was demonstrated in the simultaneous separation of the enantiomers of Tröger's Base and hydrobenzoin. Finally, the system was employed for the determination of spiked nitrosamines and the semi-quantitative assessment of nicotine in tobacco-free nicotine pouches.

One of the most commonly used culinary spices, black pepper (Piper nigrum L.) is also a rich source of bioactive phytochemicals. The present study aims to investigate the oleoresin extracted from black and white pepper using a Soxhlet extraction system and describe its physicochemical characterization, GC-MS analysis, and antioxidant and antibacterial activity evaluation. The antioxidant capacity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, and the antibacterial activity was determined by the agar well diffusion method. The extract had the highest antibacterial activity against Gram-positive Bacillus subtilis, with zones of 20 mm and 16 mm for black and white pepper extracts, respectively. The antioxidant activity of both extracts was found to be high, with DPPH radical scavenging activities of 86.80% for black pepper extract and 87.62% for white pepper extract. The oleoresin yield was 4.2% for black pepper and 3.2% for white pepper. GC-MS analysis showed a variety of phytochemical constituents such as alkaloids, terpenoids, fatty acids, and sterols. Major alkaloids of black pepper were found as piperine (8.54%) and yohimbine (12.27%), while the sterols were the most abundant class of compounds present in white pepper (28.67%). The total alkaloids present in the oleoresins of black pepper and white pepper were approximately 35%. This indicated that the oleoresins may serve as a natural source of biologically active compounds.

Steryl glucosides are minor lipid contaminants in biodiesel that can cause fuel filter plugging and adversely affect fuel quality, even at trace concentrations. Reliable determination of steryl glucoside is therefore essential for biodiesel quality control. Solid-phase extraction (SPE) is widely used for sample pretreatment prior to chromatographic analysis; however, conventional silica-based SPE protocols typically require hazardous organic solvents for cartridge preconditioning. In this study, a controlled silica hydration-based SPE method was developed for the pretreatment of steryl glucoside in crude biodiesel prior to high-performance size-exclusion chromatography coupled with evaporative light-scattering detection using a 100-Å Phenogel column. The effect of water volume (0.05-0.50 mL) used for silica preconditioning was systematically investigated. Following SPE, retained steryl glucoside was recovered by heptane washing and elution with acetone/methanol (9:1, v/v). Preconditioning with 0.075 mL of water provided the highest steryl glucoside concentration (106.29 µg/mL), approximately four times the concentration obtained with non-preconditioned silica (27.06 µg/mL) and substantially higher than those obtained with conventional heptane- (53.70 µg/mL) and acetone/methanol-preconditioned (76.73 µg/mL) cartridges. The method exhibited limits of detection and quantification of 8.07 and 24.47 µg/mL, respectively. The enhanced extraction efficiency is attributed to controlled hydration of surface silanol groups, which establishes a balanced physicochemical environment favorable for steryl glucoside adsorption while substantially reducing the consumption of hazardous organic solvents. The proposed controlled silica hydration-based SPE method provides a simple, environmentally friendly, and mechanistically guided pretreatment strategy for the determination of steryl glucoside in biodiesel and may also be applicable to the extraction of other polar lipid analytes.

Production of human papillomavirus (HPV) virus-like particles (VLPs) in Komagataella phaffii is often constrained by methanol-associated toxicity and suboptimal feeding strategies during induction. In this study, a microbioreactor-based approach was employed to systematically design and evaluate mixed-substrate fed-batch strategies for enhanced HPV16 L1 VLP production. Four methanol-based feeding compositions, including methanol-only and combinations with glycerol and sorbitol, were investigated under pulse and linear feeding modes. Among the tested conditions, a linear feeding strategy using 70% methanol and 30% glycerol (70 M + 30G) resulted in the highest biomass (DCW: 90.36 g/L) and VLP titer (42 mg/L), corresponding to a two-fold improvement over methanol-only feeding. Enhanced performance was attributed to reduced methanol-induced metabolic stress and improved substrate utilization, leading to more stable dissolved oxygen (DO) profiles and sustained cellular activity. In contrast, pulse feeding resulted in significant DO fluctuations, indicating transient metabolic imbalances and lower overall productivity. The optimized feeding strategy was successfully translated to a 1 L stirred-tank bioreactor, achieving a DCW of 120 g/L and a VLP titer of 49 mg/L, with a ~ 2.3-fold increase compared to the control. The consistency between microscale and bench-scale results demonstrates the predictive capability of microbioreactor systems for fed-batch process development. Overall, this study presents a rational and scalable feeding strategy that improves recombinant VLP production while reducing methanol usage, providing a practical framework for efficient bioprocess design in K. phaffii.

Conventional extraction of bioactive alkaloids relies on volatile organic solvents. This study evaluated 21 deep eutectic solvents (DESs) through experimental screening and multi scale computational profiling to extract piperine (PIP) from Piper nigrum and hydroxy sanshools (HAS and HBS) from Zanthoxylum bungeanum. For rigid PIP, a choline chloride/1,2-butanediol (1:4, 20% water) DES achieved 60.11 mg/g (a 1.53-fold increase over methanol). For flexible sanshools, a urea/acetic acid (1:2, 30% water) DES yielded 59.36 mg/g (HAS) and 14.61 mg/g (HBS). Molecular dynamics revealed that extraction efficiency depends on topological and electrostatic complementarity. Specifically, ChCl/1,2-BD extracts PIP via synergistic hydrogen bonding and van der Waals interactions, whereas UR/HAc solubilizes sanshools through network reorganization, anchoring polar terminals while accommodating flexible chains. This work establishes three generalizable principles for rational DES design: electrostatic complementarity matching, spatial topology adaptation for rigid frameworks, and van der Waals compensation for flexible chains.

The authors compared Cydonia oblonga fruits from two regions of Transylvania with regard to their polyphenol content, total acidity, and vitamin C levels. The polyphenol content was determined using the Folin-Ciocalteu method, which is based on a redox reaction involving electron transfer from phenolic compounds to the Folin-Ciocalteu reagent. The results ranged from 365.96 ± 1.7 to 422.93 ± 2.3 mg GAE/100 g dry weight in aqueous extracts and from 397.50 ± 1.9 to 998.78 ± 4.5 mg GAE/100 g dry weight in methanolic extracts. These values are in agreement with those previously reported in the literature. No significant differences in polyphenol content were observed either between the harvested samples or between the solvents used. However, the analytical method and storage duration of the fruits may influence the measured polyphenol content. The flavonoid content of the fruits was determined using the method described in the 10th edition of the Romanian Pharmacopoeia. The results ranged from 30.9 ± 1.02 to 34.0 ± 1.05 mg QE/100 g dry weight for aqueous extracts and from 19.2 ± 0.98 to 20.7 ± 0.87 mg QE/100 g dry weight for methanolic extracts. The total acidity of the fruit extracts was measured by acid-base titration with potentiometric endpoint detection. The samples showed slight differences depending on the harvesting location and the extraction solvent (methanol or water). The vitamin C content was determined by bromatometric titration using p-ethoxychrysoidine as the endpoint indicator. The ascorbic acid content was found to depend on both the geographical origin of the samples and the nature of the extraction solvent.