Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.

Gene inactivation by creating in-frame deletion mutations in Fusobacterium nucleatum is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX and radD, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes bamA and ftsZ, which are vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and Fusobacterium periodonticum. With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

Mechanism of Ligand Binding to Theophylline RNA Aptamer.

Studying RNA-ligand interactions and quantifying their binding thermodynamics and kinetics are of particular relevance in the field of drug discovery. Here, we combined biochemical binding assays and accelerated molecular simulations to investigate ligand binding and dissociation in RNA using the theophylline-binding RNA as a model system. All-atom simulations using a Ligand Gaussian accelerated Molecular Dynamics method (LiGaMD) have captured repetitive binding and dissociation of theophylline and caffeine to RNA. Theophylline's binding free energy and kinetic rate constants align with our experimental data, while caffeine's binding affinity is over 10,000 times weaker, and its kinetics could not be determined. LiGaMD simulations allowed us to identify distinct low-energy conformations and multiple ligand binding pathways to RNA. Simulations revealed a "conformational selection" mechanism for ligand binding to the flexible RNA aptamer, which provides important mechanistic insights into ligand binding to the theophylline-binding model. Our findings suggest that compound docking using a structural ensemble of representative RNA conformations would be necessary for structure-based drug design of flexible RNA.

Production of 1-methylxanthine via the biodegradation of theophylline by an optimized Escherichia coli strain.

1-Methylxanthine is a high-value derivative of caffeine of limited natural availability with many potential pharmaceutical applications. Unfortunately, production of 1-methylxanthine through purely chemical methods of synthesis are unfavorable due to lengthy chemical processes and the requirement of hazardous chemicals, ultimately resulting in low yields. Here, we describe a novel biosynthetic process for the production of 1-methylxanthine from theophylline using engineered Escherichia coli whole-cell biocatalysts and reaction optimization. When scaled-up to 1590 mL, the simple biocatalytic reaction produced approximately 1188 mg 1-methylxanthine from 1444 mg theophylline, constituting gram-scale production of 1-methylxanthine in as little as 3 hours. Following HPLC purification and solvent evaporation, 1163 mg of dried 1-methylxanthine powder was collected, resulting in a 97.9 wt% product recovery at a purity of 97.8%. This is the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 1-methylxanthine from theophylline. This process is also the most robust methylxanthine N-demethylation process featuring engineered E. coli to date, capable of gram-scale production.

Creation of Architecturally Minimal Transcriptionally Activating Riboswitches Responsive to Theophylline Reveals an Unconventional Design Strategy.

Riboswitches are noncoding RNA switches that are largely utilized in bacteria and play a significant role in synthetic biology. Nonetheless, their natural counterparts possess lengthy sequences and intricate structures, posing challenges for their modular integration into complex gene circuits. Consequently, it is imperative to develop simplified synthetic riboswitches that can be effortlessly incorporated into gene circuits. The conventional approach to generate synthetic riboswitches entails tedious library construction and extensive screening, which frequently yields suboptimal performance. To overcome this obstacle, alternative methods are urgently needed. In this study, we created a novel approach to designing a diverse set of transcription-activating riboswitches that exhibit high performance and broad compatibility. The strategy involved starting with a synthetic theophylline RNA aptamer and designing an expression platform that forms a transcriptional terminator in its inactive state but switches to an antiterminator when it is activated. Several sequences were designed, constructed, and subjected to virtual screening, resulting in the identification of two transcription-activating riboswitches. These riboswitches were then engineered to reduce the basal leakage and increase the activation level through extending the hairpin region using a screened random sequence. These architecturally minimal synthetic riboswitches were highly adapted to different constitutive promoters in a modular manner, generating a differentially responsive output to theophylline. As a proof-of-principle, the synthetic riboswitches were applied to rewire a synthetic quorum-sensing circuit (QSC). The reprogrammed QSC successfully modulated the temporal responsive profile against the activation. This strategy is expected to expand the variety of high-performance riboswitches that are responsive to different ligands, thereby further facilitating the design of complex genetic circuits.

Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.

HicA Toxin-Based Counterselection Marker for Allelic Exchange Mutations in Fusobacterium nucleatum.

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin radD gene within the F. nucleatum rad operon and the tnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This hicA-based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. IMPORTANCE Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA-based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the hicA gene as a counterselection marker in a bacterial genetic study.

Massively Parallel Double-Selection Workflow for the Evolution of Molecular Switches Based on Surface-Display in Escherichia coli.

Microbial surface display of proteins is a versatile method for a wide range of biotechnological applications. Herein, the use of a surface display system in E. coli for the evolution of a riboswitch from an RNA aptamer is presented. To this end, a streptavidin-binding peptide (SBP) is displayed at the bacterial surface, which can be used for massively parallel selection using a magnetic separation system. Coupling gene expression from a riboswitch library to the display of SBP hence allows selection of library members that express strongly in the presence of a ligand. As excessive SBP expression leads to bacterial growth inhibition, it can be used to negatively select against leaky riboswitches expressing in the absence of ligand. Based on this principle, we devise a double selection workflow that enables quick selection of functional riboswitches with a comparatively low screening workload. The efficiency of our protocol by re-discovering a previously isolated theophylline riboswitch from a library was demonstrated, as well as a new riboswitch that is similar in performance, but slightly more responsive at low theophylline concentrations. Our workflow is massively parallel and can be applied to the screening or pre-screening of large molecular libraries.

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

Urine caffeine metabolites are positively associated with cognitive performance in older adults: An analysis of US National Health and Nutrition Examination Survey (NHANES) 2011 to 2014.

The aim of this study was to explore urine caffeine metabolites in relation to cognitive performance among 2011-2014 National Health and Nutrition Examination Survey participants aged ≥60 years. We hypothesized that urine caffeine metabolites were positively associated with cognition in older adults. Caffeine and 14 of its metabolites were quantified in urine by use of high-performance liquid chromatography-electrospray ionization-tandem quadruple mass spectrometry with stable isotope labeled internal standards. Cognitive assessment was based on scores from the word learning and recall modules. Participants were categorized based on the quartiles of caffeine and its metabolites level. The association between caffeine metabolites and each cognitive dimension was analyzed using multiple logistic regression analysis in adjusted models. Stratification analyses by gender were also performed. For CERAD test, there was a significant association between 1-methyluric acid (OR=0.62, 95% CI: 0.42 to 0.92), 7-methylxanthine(OR=0.49, 95% CI: 0.27 to 0.89), theophylline (OR=0.52, 95% CI: 0.29 to 0.92), as well as paraxanthine (OR=0.49, 95% CI: 0.27 to 0.88) and cognitive function. For animal fluency test, there was a positive association between theophylline (TP) (OR=0.44, 95% CI: 0.22 to 0.89) and cognitive function. The trend that the risk of low cognitive function decreased with increasing concentration of 1-methylxanthine (P trend=0.0229) was also observed. Furthermore, the same trend existed for 3-methylxanthine (p trend = 0.0375) in men. In conclusion, there was a significant positive association between urine caffeine metabolites and cognitive performance in older adults, particularly for theophylline, paraxanthine and caffeine; and the association might be dependent on gender.

Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

Circadian oscillator NPAS2 regulates diurnal expression and activity of CYP1A2 in mouse liver.

We aimed to investigate the potential role of NPAS2 in controlling diurnal expression and activity of hepatic CYP1A2 and to determine the underlying mechanisms. Regulatory effects of NPAS2 on CYP1A2 were determined using Npas2 knockout (Npas2-/-) mice as well as AML-12, Hepa1-6 and HepG2 cells. mRNA and protein levels were detected by reverse transcription-quantitative real-time PCR and western blotting, respectively. In vitro and in vivo CYP1A2 activities were respectively evaluated using the probe substrates phenacetin and theophylline. Transcriptional regulation was investigated using luciferase reporter assays and ChIP-Seq analysis. Loss of Npas2 in mice decreased CYP1A2 expression (at both mRNA and protein levels) and blunted its rhythmicity in the liver. Likewise, Npas2 ablation down-regulated the enzymatic activity of CYP1A2 (probed by metabolism of phenacetin and theophylline) and abrogated its time-dependency. Cell-based assays confirmed that NPAS2 positively regulated CYP1A2 expression. Mechanistic study indicated that NPAS2 trans-activated Cyp1a2 through its specific binding to the -416 bp E-box-like element within the gene promoter. In conclusion, NPAS2 was identified as a key transcriptional regulator of diurnal expression of hepatic CYP1A2 in mice. Our findings have implications for improved understanding of circadian metabolism and chronopharmacokinetics.

Modulating myoblast differentiation with RNA-based controllers.

Tunable genetic controllers play a critical role in the engineering of biological systems that respond to environmental and cellular signals. RNA devices, a class of engineered RNA-based controllers, enable tunable gene expression control of target genes in response to molecular effectors. RNA devices have been demonstrated in a number of systems showing proof-of-concept of applying ligand-responsive control over therapeutic activities, including regulation of cell fate decisions such as T cell proliferation and apoptosis. Here, we describe the application of a theophylline-responsive RNA device in a muscle progenitor cell system to control myogenic differentiation. Ribozyme-based RNA switches responsive to theophylline control fluorescent reporter expression in C2C12 myoblasts in a ligand dependent manner. HRAS and JAK1, both anti-differentiation proteins, were incorporated into RNA devices. Finally, we demonstrate that the regulation of HRAS expression via theophylline-responsive RNA devices results in the modulation of myoblast differentiation in a theophylline-dependent manner. Our work highlights the potential for RNA devices to exert drug-responsive, tunable control over cell fate decisions with applications in stem cell therapy and basic stem cell biology research.

A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer.

Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 µM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 µM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.

Anti-melanogenic effects of glucosylceramides and elasticamide derived from rice oil by-products in melanoma cells, melanocytes, and human skin.

Glucosylceramides (GlcCer), which are present in many edible plants, suppress melanin production in mouse melanocytes. Rice GlcCer consist of multiple molecules that comprise different types of sphingoid bases as well as diverse lengths and stereotypes of free fatty acids. Adjacent to the GlcCer fraction, there are free ceramides (Cer) as minor constituents. However, the anti-melanogenic activities of individual GlcCer and Cer remain unknown. Therefore, we herein isolated 13 GlcCer and elasticamide, a Cer [AP] from the gummy by-products of rice bran oil, and examined their anti-melanogenic activities. In theophylline-induced melanogenesis in B16 melanoma cells, GlcCer [d18:2(4E,8Z)/18:0], GlcCer [d18:2(4E,8Z)/20:0], and elasticamide significantly suppressed melanin production with IC50 values of 6.6, 5.2, and 3.9 µM, respectively. Elasticamide, but not GlcCer [d18:2 (4E,8Z)/20:0], suppressed melanogenesis in human 3D-cultured melanocytes and the expression of tyrosinase-related protein 1 in normal human melanocytes. Based on these results, we conducted a clinical trial on the effects of rice ceramide extract (Oryza ceramide®), containing 1.2 mg/day of GlcCer and 56 µg/day of elasticamide, on UV-B-induced skin pigmentation. The ingestion of Oryza ceramide® for 8 weeks significantly suppressed the accumulation of melanin 7 days after UV irradiation (1288 and 1546 mJ/cm2 ·S). Rice-derived GlcCer and elasticamide, which exhibited anti-melanogenic activities, were suggested to contribute to the suppressive effects of Oryza ceramide® on UV-induced skin pigmentation. Although the mechanisms underlying the anti-melanogenic activities of GlcCer remain unclear, elasticamide was identified as a promising Cer that exhibits anti-melanogenic activity. PRACTICAL APPLICATIONS: The anti-melanogenic activities of rice-derived GlcCer and elasticamide currently remain unclear. We herein demonstrated the inhibitory effects of individual GlcCer and elasticamide on melanogenesis in melanoma cells, melanocytes, and human skin.

Urinary caffeine and caffeine metabolites, asthma, and lung function in a nationwide study of U.S. adults.

OBJECTIVE: Coffee intake has been inversely associated with asthma in adults. We examined the relation between urinary levels of caffeine or caffeine metabolites and asthma, lung function, and fractional exhaled nitric oxide (FeNO) in adults. METHODS: Cross-sectional study of 2,832 adults aged 18-79 years in the US National Health and Nutrition Examination Survey (NHANES). Multivariable logistic or linear regression was used for the analysis of urinary levels of caffeine or each of its three major metabolites (paraxanthine, theobromine, and theophylline) and current asthma, lung function, and FeNO. RESULTS: Subjects with urinary paraxanthine levels in the fourth quartile (Q4) had 53% lower odds of current asthma than those whose urinary paraxanthine levels were in the first quartile (Q1; 95% confidence = 0.22 to 1.00). Among never and former smokers, subjects with urinary theophylline levels above Q1 had 49% lower odds of current asthma than those whose urinary theophylline level was in Q1 (95% CI = 0.31 to 0.85). Among subjects without current asthma, each log10-unit increment in paraxanthine level was associated with a 0.83% increment in percent predicted (%pred) FEV1 and a 1.27% increment in %pred FVC, while each log10-unit in theophylline was associated with a 1.24% increment in %pred FVC. Neither urinary caffeine nor any urinary caffeine metabolite was associated with bronchodilator response or FeNO. CONCLUSIONS: Our findings suggest that two caffeine metabolites (theophylline and paraxanthine) may contribute to the previously reported inverse association between coffee intake and asthma in adults.

Finger sweat analysis enables short interval metabolic biomonitoring in humans.

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues, which are impeding time-course studies. Here, we show that the metabolic profiling of the minute amounts of sweat sampled from fingertips addresses this challenge. Sweat sampling from fingertips is non-invasive, robust and can be accomplished repeatedly by untrained personnel. The sweat matrix represents a rich source for metabolic phenotyping. We confirm the feasibility of short interval sampling of sweat from the fingertips in time-course studies involving the consumption of coffee or the ingestion of a caffeine capsule after a fasting interval, in which we successfully monitor all known caffeine metabolites as well as endogenous metabolic responses. Fluctuations in the rate of sweat production are accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. To conclude, metabotyping using sweat from fingertips combined with mathematical network modelling shows promise for broad applications in precision medicine by enabling the assessment of dynamic metabolic patterns, which may overcome the limitations of purely compositional biomarkers.

Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC-MS/MS.

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 µm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface. The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Investigating how amine structure influences drug-amine ion-pair formation and uptake via the polyamine transporter in A549 lung cells.

Transiently associating amines with therapeutic agents through the formation of ion-pairs has been established both in vitro and in vivo as an effective means to systemically direct drug delivery to the lung via the polyamine transport system (PTS). However, there remains a need to better understand the structural traits required for effective PTS uptake of drug ion-pairs. This study aimed to use a structurally related series of amine counterions to investigate how they influenced the stability of theophylline ion-pairs and their active uptake in A549 cells. Using ethylamine (mono-amine), ethylenediamine (di-amine), spermidine (tri-amine) and spermine (tetra-amine) as counterions the ion-pair affinity was shown to increase as the number of protonated amine groups in the counterion structure increased. The mono and diamines generated a single hydrogen bond and the weakest ion-pair affinities (pKFTIR: 1.32 ± 0.04 and 1.43 ± 0.02) whereas the polyamines produced two hydrogen bonds and thus the strongest ion-pair affinities (pKFTIR: 1.93 ± 0.05 and 1.96 ± 0.04). In A549 cells depleted of endogenous polyamines using α-difluoromethylornithine (DFMO), the spermine-theophylline uptake was significantly increased (p < 0.05) compared to non-amine depleted cells and this evidenced the active PTS sequestering of the ion-pair. The mono-amine and di-amine failed to enhance theophylline uptake in these A549 cells, but the tri-amine and tetra-amine both almost doubled the theophylline uptake into the cells when compared to the uptake of free drug. As the data indicated that polyamines with at least 3 amines were required to form ion-pairs that could enhance A549 cell uptake, it suggested that at least two amines were required to physically stabilise the ion-pair and one to interact with the PTS.

Theophylline-induced synergic activation of guide RNA to control CRISPR/Cas9 function.

Ligand-induced activation of CRISPR/Cas9 function is achieved based on a synergic approach through the integration of the theophylline aptamer into protein-unrecognized regions of guide RNA. This design of allosteric regulation opens a new window towards the broad involvement of RNA aptamers for conditional control of CRISPR/Cas9 function.

A Phase 1 Open-Label, Fixed-Sequence Pharmacokinetic Drug Interaction Trial to Investigate the Effect of Cannabidiol on the CYP1A2 Probe Caffeine in Healthy Subjects.

This pharmacokinetic (PK) drug-interaction trial investigated the effects of repeated dosing of a plant-derived pharmaceutical formulation of highly purified cannabidiol (CBD; Epidiolex in the United States and Epidyolex in Europe; 100 mg/mL oral solution) on caffeine clearance via modulation of cytochrome P450 (CYP) 1A2 activity in healthy adults. In this phase 1 open-label, fixed-sequence trial, all subjects received a single 200 mg caffeine dose and placebo on day 1. Subjects then titrated CBD from 250 mg once daily to 750 mg twice daily between days 3 and 11 and took 750 mg CBD twice daily between days 12 and 27. On day 26, subjects received a single 200-mg caffeine dose with their morning CBD dose. Plasma concentrations of caffeine and its CYP1A2-mediated metabolite, paraxanthine, were determined on days 1 and 26 and PK parameters derived using noncompartmental analysis. Safety was monitored throughout. Sixteen subjects enrolled, and 9 completed treatment. When caffeine was administered with steady-state CBD, caffeine exposure increased by 15% for Cmax and 95% for AUC0-∞ , tmax increased from 1.5 to 3.0 hours, and t1/2 increased from 5.4 to 10.9 hours compared with caffeine administered with placebo. Under the same conditions, paraxanthine exposure decreased by 22% for Cmax and increased by 18% for AUC0-∞ , tmax increased from 8.0 to 14.0 hours, and t1/2 increased from 7.2 to 13.7 hours. Overall, there were no unexpected adverse events; diarrhea was most common, and 6 subjects discontinued because of elevated liver transaminases. These data suggest that CBD is an inhibitor of CYP1A2.