Fusobacterium nucleatum subsp. animalis (FNA) plays a prominent role in oral microbial ecology and is increasingly implicated in systemic diseases such as colorectal cancer. However, its genetic intractability has significantly hindered functional studies to understand its pathogenic mechanisms. To address this challenge, we developed a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains. This system features inducible control of plasmid replication via a theophylline-responsive riboswitch regulating repA, and counterselection via the MazF toxin under the control of an anhydrotetracycline-inducible promoter. In this chapter, we provide a detailed, step-by-step protocol for implementing this system using FNA strain 7_1 as a model. We illustrate the procedure by deleting the luxS gene, which encodes S-ribosylhomocysteine lyase-an enzyme involved in AI-2 quorum sensing and biofilm regulation. Our protocol provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.

Accurately predicting hepatic clearance remains a critical hurdle in preclinical pharmacokinetics, especially for metabolically stable compounds and emerging modalities such as proteolysis targeting chimeras (PROTACs) that fall in beyond-rule-of-five chemical space. In this study, we developed and evaluated a 3-dimensional hepatic spheroid-array system that improves clearance prediction for both small molecules and PROTACs. Using a panel of 15 small molecules and 9 PROTACs, we compared in vitro clearance predictions from the spheroid-array model with conventional suspension hepatocytes. The spheroid-array predicted human hepatic clearance within a 2-fold range for 9 of 15 and within a 3-fold range for 12 of 15 small molecules. The average absolute fold error was lower for the spheroid system than the suspension system (1.74 vs 3.52). Notably, unlike the suspension system, the spheroid-array quantified clearance for several low-clearance compounds such as warfarin and theophylline. Regression correction improved prediction accuracy for both, with a significant improvement for the suspension model. For several PROTACs, metabolic turnover could not be measured in the suspension system, highlighting its limited suitability for highly stable compounds. In contrast, the spheroid system demonstrated significant metabolic activity, enabling characterization of hepatic clearance for these otherwise stable PROTACs. Together, these findings establish the spheroid-array system as a sensitive and physiologically relevant tool for preclinical clearance assessment across traditional and emerging therapeutic modalities. SIGNIFICANCE STATEMENT: We developed a 3-dimensional hepatic spheroid-array that shows excellent viability and functional activity for 3 weeks. The system showed better hepatic clearance predictivity with a lower average absolute fold error than the suspension system. Our work also first time validates the spheroid-array system for beyond-rule-of-five molecules showing the possibility for evaluating clearance of proteolysis targeting chimeras. This work establishes spheroid as a reliable tool for preclinical hepatic clearance assessment.

The diaquadichlorotheophylline-copper(ii) coordination complex, [Cu(theophylline)(H2O)2Cl2], was synthesized via slow solvent evaporation and comprehensively characterized through experimental and computational approaches. X-ray powder diffraction and Rietveld refinement confirmed a triclinic crystal system (space group P1̄(C [1] i)) with pentacoordinate copper(ii) in a square-pyramidal geometry. Thermal analyses revealed thermal stability up to 358 K, with dehydration requiring 89.5 kJ mol[-1] per H2O molecule. Vibrational spectroscopy (Fourier transform infrared and Raman) combined with density functional theory calculations (PBE1PBE/6-311++G(d,p)) provided suitable mode assignments and demonstrated excellent agreement between experimental and calculated spectra, including solvent effects using the IEFPCM model. Frontier molecular orbital analysis yielded the highest occupied molecular orbital-lowest unoccupied molecular orbital gaps of approximately 3.8 eV and electrophilicity indices of approximately 6.8 eV, indicating good chemical stability and biological potential. Ultraviolet-visible-near infrared spectroscopy revealed characteristic d-d transitions for pentacoordinate copper(ii) and π → π*/n → π* transitions from the theophylline ligand. Molecular docking predicted a strong in silico binding affinity to deoxyribonucleic acid (K i = 5.85 µM, binding free energy = -7.14 kcal mol[-1]), consistent with an intercalative mode and moderate affinity to bovine serum albumin (K i = 40.78 µM, binding free energy = -5.99 kcal mol[-1]). Cytotoxicity assays against PC-3 (prostate), MDA-MB-231 (breast), and HCT-116 (colorectal) cancer cell lines revealed dose-dependent activity with half-maximal inhibitory concentration values ranging from 2.91 to 3.77 µM and selectivity indices greater than 1 compared to the non-tumorigenic GM07429A cell line, with preferential activity against breast cancer. These results indicate that diaquadichlorotheophylline-copper(ii) is a promising anticancer candidate with favorable selectivity for malignant cells over healthy tissue, warranting further preclinical evaluation.

Accurate assessment of haemostasis is essential in both research and clinical practice. Thrombin generation assays (TGA) provide a comprehensive evaluation of coagulation; however, results can be influenced by preanalytical conditions, including the choice of blood collection tubes. Citrate-theophylline-adenosine-dipyridamole (CTAD) tubes are recommended by the manufacturer of the Ceveron[®] alpha TGA system due to their ability to inhibit platelet activation. Nevertheless, CTAD tubes are costly, less widely available, and not manufactured in small volumes suitable for specific patient populations, such as paediatric cohorts. To evaluate whether standard citrate tubes can serve as a viable alternative, we prospectively compared TGA parameters from blood collected in 3.2% sodium citrate tubes versus CTAD tubes in 12 healthy volunteers. Assays were performed using low and high phospholipid micelle concentrations (RC low/RC high). Significant differences between citrate and CTAD were observed for all parameters with the RC low reagent. In contrast, only lag time showed significant differences with the RC high reagent. In conclusion, while citrate tubes may be used for TGA, the type of blood collection tube can influence measurements under specific assay conditions and should be considered a relevant factor when comparing and interpreting TGA results.

Acute mountain sickness (AMS) is a common altitude-related condition affecting individuals ascending to high elevations, presenting with headache, nausea, fatigue, and dizziness. Although several pharmacological agents have been proposed for AMS prophylaxis, their comparative effectiveness remains insufficiently established. A systematic review and network meta-analysis were conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Major electronic databases were searched for randomized controlled trials (RCTs) assessing pharmacological interventions for AMS prophylaxis. Data from 46 eligible studies were extracted using Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and analyzed with R software (R Foundation for Statistical Computing, Vienna, Austria). The primary outcome was AMS incidence. Network geometry was mapped using network plots; treatment effects were calculated as odds ratios (ORs) with 95% confidence intervals (CIs); and interventions were ranked using surface under the cumulative ranking (SUCRA) probabilities. Risk of bias was assessed, and certainty of evidence was determined by CINeMA (Confidence in Network Meta-Analysis). The analysis incorporated 46 studies evaluating multiple pharmacological agents. SUCRA rankings identified inhaled budesonide as the most efficacious agent for AMS prevention, followed by acetazolamide and theophylline. Most studies demonstrated low risk of bias. Acetazolamide exhibited narrower CIs, reflecting a more robust and reliable evidence base. CINeMA showed overall moderate certainty of evidence. Inhaled budesonide had the highest probability of being the most effective pharmacological agent for AMS prevention, with acetazolamide and theophylline also demonstrating substantial efficacy. Acetazolamide remains the best-evidenced option. Additional high-quality RCTs are required to consolidate treatment hierarchies and evaluate agents with limited current data.

C2-alkylated imidazolium salts reacted with carbon disulfide and cesium carbonate under mild, aerobic conditions to afford imidazolium-dithiocarboxylate zwitterions bearing 2,6-diisopropylphenyl or mesityl substituents on their nitrogen atoms. The addition of CS2 took place regioselectively at the exocyclic C2α position of the starting materials and four new compounds were isolated in satisfactory to high yields. Two of them featured a chiral center and were obtained as racemic mixtures. Compared to previous strategies that required a strong base to deprotonate the imidazolium salts, followed by the nucleophilic addition of the intermediate N-heterocyclic olefins (NHOs) onto CS2, our method involved only one step and relied on a weak, innocuous base. Moreover, it did not necessitate the use of dry and degassed solvents under an inert atmosphere, making it more environmentally friendly and easier to implement. The four NHO·CS2 zwitterions served as ligands to prepare a small library of heteroleptic ruthenium-arene complexes with the generic formula [RuCl(p-cymene)(S2C·NHO)](PF6) and homoleptic dicationic complexes of the type [Ru(S2C·NHO)3](PF6)2. These chelates are the first examples of coordination compounds based on NHO·CS2 inner salts. They were fully characterized using various analytical techniques and the molecular structures of three of them were determined. We also synthesized the IMes(?)CH2-d2·CS2 betaine in three steps from 1,3-dimesitylimidazolium chloride (IMes·HCl) using a straightforward procedure, and we used it as an isotopically labeled ligand to form the stable [RuCl(p-cymene)(S2C·CH2(?)IMes-d2)](PF6) complex in 95% yield. This experiment served as a proof of concept to demonstrate the potential of our methodology for the design of new deuterated metallodrugs. Lastly, the catalytic activity of the eight Ru(S2C·NHO) chelates was probed in the transfer hydrogenation of acetophenone with isopropanol and potassium hydroxide. Gratifyingly, they proved superior to their Ru(S2C·NHC) cousins featuring xanthinium-8-dithiocarboxylate ligands derived from caffeine and theophylline.

Green tea supplements (GTSs) are a favorite among the general population and, due to their methylxanthine (caffeine, theobromine, theophylline) content, might represent a potential ergogenic source in athletes' performance. The aim of this study was to develop and validate a method that quantifies the methylxanthine content of commercially available GTSs and to evaluate their potential relevance for ergogenic use. An ultra-high-performance liquid chromatography coupled with photodiode array detection and a QDa mass detector (UHPLC-PDA-QDa) method was developed, validated and applied to determine the content of methylxanthines in seven commercially available GTSs. Method validation parameters demonstrated satisfactory performance, with rapid and efficient separation and quantification of the three methylxanthines. Considerable variability was observed among the analyzed products, with caffeine, theobromine, and theophylline contents ranging from 0.37 to 12.13, 0.07 to 3.59, and 0.05 to 0.83 mg/capsule, respectively. However, the methylxanthine amounts measured in the analyzed products were below the doses commonly associated with ergogenic effects. Overall, the developed UHPLC-PDA-QDa method provided a reliable approach for methylxanthine quantification, while the seven products analyzed did not represent meaningful ergogenic sources of these compounds.

Molecular docking is increasingly used to infer aptamer-target interactions, yet most studies rely on computationally predicted aptamer structures rather than experimentally determined ones. Using a benchmark set of aptamers with known high-resolution structures, we show that commonly used modeling approaches, including RNAComposer and AlphaFold3, fail to reliably reproduce aptamer conformations, particularly at the binding sites critical for molecular recognition. Key limitations include the use of A-form RNA models to represent B-form DNA structures, the prediction of ligand-free rather than ligand-bound conformations, and the scarcity of experimentally determined aptamer structures for training machine-learning models. Using the theophylline aptamer, for which high-resolution structures are available in both DNA and RNA forms, we systematically evaluated each step of the standard docking workflow. We found that structure-prediction errors generate incorrect binding pockets, docking scores fail to distinguish theophylline from caffeine despite a 250,000-fold difference in affinity, and molecular dynamics simulations do not overcome these shortcomings. Together, these results reveal fundamental weaknesses in current aptamer docking workflows and caution against using docking-derived models to infer binding mechanisms in the absence of experimental structural data.

A metal-free radical tandem carbamoylation/cyclization strategy for the divergent synthesis of benzimidazole-fused heterocycles has been described. Using commercially available oxamic acids and imidazoles tethered with unactivated alkenes under mild conditions, this method provides the first efficient access to stable [6,5,7]- and [6,5,8]-fused frameworks along with [6,5,6] systems. It exhibits excellent functional group tolerance and broad substrate scope, including 2-heteroarylbenzimidazoles. Preliminary studies also revealed that monoalkyl oxalates can serve as effective radical precursors in this transformation, offering potential for further derivatization of the products. The method is also applicable to the late-stage diversification of bioactive molecules, such as theophylline.

Riboswitches are compact RNA-based regulatory elements capable of modulating gene expression in response to small molecules, without the need for additional proteins. Various synthetic riboswitches have been engineered using in vitro-generated tetracycline and theophylline aptamers. However, many of these constructs exhibit suboptimal switching efficiency and background expression. Moreover, efforts to enhance their performance often involve time-consuming and costly screening processes. Here we report that artificial riboswitches can be efficiently optimized by engineering fusion aptamers that contain two binding pockets (apdimers). Following this rational approach, we generated cooperativity between both binding pockets, resulting in the improved performance of splicing-based and ribozyme-based synthetic riboswitches. We finally combined optimized tetracycline switches, yielding dynamic ranges exceeding 1000-fold with minimal background expression in the OFF state. In addition, we show that the optimized tetracycline riboswitches can be used to efficiently induce AAV-mediated transgene expression in mice. The presented strategy offers a straightforward and effective approach for the optimization of existing synthetic riboswitches and the design of novel riboswitches.

Pre-analytical errors such as sample mix-ups can compromise the integrity of pharmacokinetic (PK) data. We simulated vancomycin PK data and introduced two error types: intra-individual time point swap (TS) errors and inter-individual concentration swap (CS) errors. This study developed and validated two automated detection approaches-a run-test-based non-parametric method and a Mahalanobis distance-based method with leave-one-out cross-validation (LOOCV)-for identifying pre-analytical errors in PK datasets. Performance was evaluated using 12,500 simulated profiles comparing the two methods. The distance-based method demonstrated significantly higher error detection rate (79.7% vs. 59.6%, p < 0.001) and greater area under the receiver operating characteristic curve (0.856; 95% confidence interval [CI], 0.816-0.889 vs. 0.574; 95% CI, 0.541-0.610) compared to the run-test, while maintaining reasonable specificity (76.4% vs. 84.0%). For TS errors, the distance-based method achieved higher recall (80.4% vs. 71.9%) and modestly higher F1-score (0.530 vs. 0.515). For CS errors, the distance-based method demonstrated superior precision (86.1% vs. 59.8%) with comparable F1-score (0.572 vs. 0.564). Detection was robust across alternative PopPK models, a structurally different drug (theophylline), training-set contamination (with the minimum covariance determinant estimator recommended for non-curated training), missing data (with sub-vector marginal handling), and sampling-time jitter. Overall, the distance-based method with LOOCV outperformed the conventional run-test across key performance metrics and can serve as a practical quality control tool for ensuring PK data integrity in clinical pharmacology research.

Indomethacin (IND), a Biopharmaceutics Classification System (BCS) Class II drug, exhibits poor aqueous solubility and low dissolution rate, which may limit its oral absorption. In this study, IND cocrystals with benzoic acid (AcBz), theophylline (THP), and caffeine (CAF) were prepared to improve their in vitro dissolution performance. Saturated solutions of IND and each coformer in methanol (MeOH) or ethanol (EtOH) were subjected to dropwise addition of water as antisolvent at solvent/antisolvent volume ratios of 1:0.5, 1:1, and 1:2. The resulting solids were collected by vacuum filtration, dried for 24 h, and characterised by PXRD, FT-IR, DSC, and SEM. Dissolution data were statistically analysed using three-way ANOVA (p < 0.05). Solid-state characterisation supported the formation of new crystalline phases in the IND-AcBz, IND-THP, and IND-CAF systems. PXRD patterns showed distinctive reflections within the 2θ range of 6-15°, while FT-IR spectra revealed shifts in the 1600-1700 cm[-1] region, consistent with changes in intermolecular hydrogen bonds. DSC thermograms further supported the formation of new solid phases through melting endotherms distinct from those of the parent compounds. Three-way ANOVA showed that solvent type, coformer selection, and solvent/antisolvent ratio significantly influenced the dissolution profiles. The 1:1 solvent/antisolvent ratio produced the most favourable systems, with IND-AcBz prepared in MeOH showing the best release profile and significantly outperforming pure IND, with a 53.7% increase in cumulative drug release at 1 h Overall, these results indicate that antisolvent cocrystallisation is an effective strategy for improving the solid-state properties and in vitro dissolution behaviour of IND. In particular, the 1:1 solvent/antisolvent was associated with improved crystalline quality and enhanced dissolution performance, highlighting the potential of engineered cocrystals as a formulation strategy to address solubility-related limitations in poorly water-soluble drugs.

The tendency for cocrystal formation between alkaloids and flavonoids is found to be quite common (around 30%) and based on the optimization of hydrogen-bond formation between the acceptor-rich alkaloids and donor-rich flavonoids. Within each molecular family there is substantial variation in cocrystal tendency, leading to considerable scope for selective cocrystal precipitation to assist in the separation of complex mixtures of these natural product families. Theophylline (C7H8N4O2) follows caffeine and theacrine in forming a substantial number of flavonoid cocrystals - six in total, of which three were reported previously. By contrast, the 8-halo-substituted analogues 8-X-Tph [X = Cl (C7H7ClN4O2), Br (C7H7BrN4O2)] form cocrystals with just myricetin (C15H10O8) and kaempferol (C15H10O6). Weakening of the acceptor capability of the alkaloid N9 atom upon proximal halo substitution may play a role in the reduced tendency of 8-X-Tph towards cocrystal formation, since improved packing efficiency is not a key factor in the formation of these cocrystals. Most of the cocrystal phases may be conveniently prepared with good yield and purity through either liquid-assisted grinding (LAG) or microwave-assisted cocrystallization from 1-butanol.