Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Double blocking gap-filling-ligation coupled with cascade isothermal amplification for ultrasensitive quantification of N6-methyladenosine.

We developed a method for quantifying N6-methyladenosine at one-nucleotide resolution based on double blocking gap-filling-ligation and cascade isothermal amplification. This proposed method can detect as low as 1 fM target RNA, achieving selectivity up to approximately 100-fold between m6A and A, and has been successfully applied to the analysis of m6A at specific sites in cell samples.

A novel photoelectrochemical strategy for sequence-spot bispecific analysis of N6-methyladenosine modification based on proximity ligation-triggered cascade amplification.

N6-methyladenosine (m6A) modification as the most prevalent mammalian RNA internal modification has been considered as the invasive biomarkers in clinical diagnosis and biological mechanism researches. It is still challenged to explore m6A functions due to technical limitations on base- and location-resolved m6A modification. Herein, we firstly proposed a sequence-spot bispecific photoelectrochemical (PEC) strategy based on in situ hybridization mediated proximity ligation assay for m6A RNA characterization with high sensitivity and accuracy. Firstly, the target m6A methylated RNA could be transferred to the exposed cohesive terminus of H1 based on the special self-designed auxiliary proximity ligation assay (PLA) with sequence-spot bispecific recognition. The exposed cohesive terminus of H1 could furtherly trigger the next catalytic hairpin assembly (CHA) amplification and in situ exponential nonlinear hyperbranched hybridization chain reaction for highly sensitive monitoring of m6A methylated RNA. Compared with conventional technologies, the proposed sequence-spot bispecific PEC strategy for m6A methylation of special RNA based on proximity ligation-triggered in situ nHCR performed improved sensitivity and selectivity with a detection limit of 53 fM, providing new insights into highly sensitive monitoring m6A methylation of RNA in bioassay, disease diagnosis and RNA mechanism.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

METTL3 inhibition reduces N6 -methyladenosine levels and prevents allogeneic CD4+ T-cell responses.

Alloreactive CD4+ T cells play a central role in allograft rejection. However, the post-transcriptional regulation of the effector program in alloreactive CD4+ T cells remains unclear. N6 -methyladenosine (m6 A) RNA modification is involved in various physiological and pathological processes. Herein, we investigated whether m6 A methylation plays a role in the allogeneic T-cell effector program. m6 A levels of CD4+ T cells from spleens, draining lymph nodes and skin allografts were determined in a skin transplantation model. The effects of a METTL3 inhibitor (STM2457) on CD4+ T-cell characteristics including proliferation, cell cycle, cell apoptosis and effector differentiation were determined after stimulation of polyclonal and alloantigen-specific (TEa; CD4+ T cells specific for I-Eα52-68 ) CD4+ T cells with α-CD3/α-CD28 monoclonal antibodies and cognate CB6F1 alloantigen, respectively. We found that graft-infiltrating CD4+ T cells expressed high m6 A levels. Administration of STM2457 reduced m6 A levels, inhibited T-cell proliferation and suppressed effector differentiation of polyclonal CD4+ T cells. Alloreactive TEa cells challenged with 40 µm STM2457 exhibited deficits in T-cell proliferation and T helper type 1 cell differentiation, a cell cycle arrest in the G0 phase and elevated cell apoptosis. Moreover, these impaired T-cell responses were associated with the diminished expression levels of transcription factors Ki-67, c-Myc and T-bet. Therefore, METTL3 inhibition reduces the expression of several key transcriptional factors for the T-cell effector program and suppresses alloreactive CD4+ T-cell effector function and differentiation. Targeting m6 A-related enzymes and molecular machinery in CD4+ T cells represents an attractive therapeutic approach to prevent allograft rejection.

Ultrasensitive detection of small biomolecules using aptamer-based molecular recognition and nanoparticle counting.

Detection of small biomolecules is critical for understanding molecular mechanisms in biological systems and performing in vitro diagnosis in clinics. Current antibody based detection methods face large challenges in detecting small biomolecules at low concentrations. We report a new method for detecting small biomolecules based on molecular recognition and nanoparticle (NP) counting. Aptamer-functionalized NPs are attached to complementary sequence (CS)-conjugated microparticle (MP) carriers. In the presence of target small biomolecules at ultra low concentrations, NPs would be released from the MP carriers. Coupled with a resistive pulse sensor (RPS) using a micropore that counts the released NPs, this method can measure the concentrations of target biomolecules at low concentrations with high sensitivity and high throughput. Adenosine was used as a model to demonstrate the feasibility of this method. It is demonstrated that this method can detect a wide range of adenosine concentrations with a low detection limit of 0.168 nM, which is 10 times lower than that of the ELISA kit. With its simple structure, high sensitivity, and high reproducibility, this detection method holds great potential for the ultrasensitive detection of low abundance small biomolecules.

Detection of N6-methyladenosine in SARS-CoV-2 RNA by methylated RNA immunoprecipitation sequencing.

N 6 -methylation of adenosine (m6A) is the most abundant internal mRNA modification and is an important post-transcriptional regulator of gene expression. Here, we describe a protocol for methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to detect and quantify m6A modifications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The protocol is optimized for low viral RNA levels and is readily adaptable for other applications. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

METTL3 Intensifies the Progress of Oral Squamous Cell Carcinoma via Modulating the m6A Amount of PRMT5 and PD-L1.

N6-Methyladenosine (m6A) modification is one of the commonest chemical modifications in eukaryotic mRNAs, which has essential effects on mRNA translation, splicing, and stability. Currently, there is a rising concern on the regulatory role of m6A in tumorigenesis. As a known component in the m6A methyltransferase complex, METTL3 (methyltransferase-like 3) plays an essential role in m6A methylation. Till now, the functions of METTL3 in oral squamous cell carcinoma (OSCC) and its relative mechanism remain to be explored. In this research, through the GEPIA database, we found that high METTL3 expression has a correlation with poor prognosis of squamous cell carcinoma of head and neck. qRT-PCR displayed that METTL3 was highly expressed in OSCC cells. Functionally, METTL3 knockdown reduced the invasion, migration, and proliferation competence of OSCC cells and attenuated the activation of CD8+ T cells. In contrast, METTL3 overexpression resulted in opposite results. GEPIA, UALCAN, and SRAMP databases, PCR, western blot, and m6A RNA methylation assay confirmed the m6A modification of PRMT5 and PD-L1 mediated by METTL3. In conclusion, our results displayed that METTL3 intensified the metastasis and proliferation of OSCC by modulating the m6A amounts of PRMT5 and PD-L1, suggesting that METTL3 may be a therapeutic target for OSCC patients.

m6A-seq analysis of microRNAs reveals that the N6-methyladenosine modification of miR-21-5p affects its target expression.

In eukaryotes, N6-methyladenosine (m6A) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most m6A researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of m6A in small RNAs. Nevertheless, current knowledge about m6A prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of m6A methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific m6A profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA m6A immunoprecipitation sequencing and constructed the consensus motif in m6A-enriched miRNAs de novo. We found that miR-21-5p, an oncogenic miRNA, showed the highest m6A enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21-5p, but altered the m6A abundance of miR-21-5p, thereby changing the expression levels of its target gene. We further synthesized m6A modified miR-21-5p mimics in vitro and demonstrated that in NSCLC cells, m6A marks in mature miR-21-5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of m6A modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.

Microarray analysis of genes with differential expression of m6A methylation in lung cancer.

PURPOSE: N6-methyladenosine (m6A) is among the most abundant mRNA modifications in eukaryote. The aim of the present study was to investigate function of m6A mRNA methylation in lung cancer and the underlying mechanism. METHODS: Microarray analysis was performed to detect the differences in RNA expression between cancerous and adjacent non-cancerous tissue samples. The target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hierarchical clustering of RNAs was conducted to identify distinct m6A methylation or expression patterns between the samples. RESULTS: In the present study, some differentially expressed genes (DEGs) of mRNAs were identified, including up-regulated secret phosphoprotein 1 (SPP1) and down-regulated pRB. Functional enrichment analysis revealed that while differential hypermethylation was related to cell cycle, intracellular part and protein binding, the main pathway involved herpes simplex virus 1 infection related to down-regulated AKT, Araf1 and BCL2A1. In the meantime, sexual reproduction, cohesin complex and protein C-terminus binding was functionally linked to differential hypomethylation, while fluid shear stress and atherosclerosis were identified as the main pathways related to up-regulated GST and CNP. CONCLUSIONS: We showed that lung cancer development involved differential expression of SPP1 and pRB mRNA, as well as m6A mRNA methylation in AKT, APAF1, BCL2A1, GST and CNP genes.

End-labeling-based electrochemical strategy for detection of adenine methylation in nucleic acid by differential pulse voltammetry.

A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein.

Multiplexing neurochemical detection with carbon fiber multielectrode arrays using fast-scan cyclic voltammetry.

Carbon fiber microelectrodes (CFMEs) have been extensively used to measure neurotransmitters with fast-scan cyclic voltammetry (FSCV) due to their ability to adsorb cationic monoamine neurotransmitters. Although FSCV, in tandem with CFMEs, provides high temporal and spatial resolution, only single-channel potentiostats and electrodes have been primarily utilized. More recently, the need and use of carbon fiber multielectrode arrays has risen to target multiple brain regions. Previous studies have shown the ability to detect dopamine using multielectrode arrays; however, they are not readily available to the scientific community. In this work, we interfaced a carbon fiber multielectrode array (MEA or multielectrode array), to a commercially available four-channel potentiostat for multiplexing neurochemical measurements. The MEA's relative performance was compared to single CFMEs where dopamine detection was found to be adsorption controlled to the electrode's surface. Multiple waveforms were applied to each fiber of the multielectrode array simultaneously to detect different analytes on each electrode of the array. A proof of concept ex vivo experiment showed that the multielectrode array could record redox activity in different areas within the mouse caudate putamen and detect dopamine in a 3-mm2 area. To our knowledge, this is the first use of the multielectrode array paired with a commercially available multichannel potentiostat for multi-waveform application and neurotransmitter co-detection. This novel array may aid in future studies to better understand complex brain heterogeneity, the dynamic neurochemical environment, and how disease states or drugs affect separate brain areas concurrently. Graphical abstract.

M6A-BiNP: predicting N6-methyladenosine sites based on bidirectional position-specific propensities of polynucleotides and pointwise joint mutual information.

N6-methyladenosine (m6A) plays an important role in various biological processes. Identifying m6A site is a key step in exploring its biological functions. One of the biggest challenges in identifying m6A sites is how to extract features comprising rich categorical information to distinguish m6A and non-m6A sites. To address this challenge, we propose bidirectional dinucleotide and trinucleotide position-specific propensities, respectively, in this paper. Based on this, we propose two feature-encoding algorithms: Position-Specific Propensities and Pointwise Mutual Information (PSP-PMI) and Position-Specific Propensities and Pointwise Joint Mutual Information (PSP-PJMI). PSP-PMI is based on the bidirectional dinucleotide propensity and the pointwise mutual information, while PSP-PJMI is based on the bidirectional trinucleotide position-specific propensity and the proposed pointwise joint mutual information in this paper. We introduce parameters α and ß in PSP-PMI and PSP-PJMI, respectively, to represent the distance from the nucleotide to its forward or backward adjacent nucleotide or dinucleotide, so as to extract features containing local and global classification information. Finally, we propose the M6A-BiNP predictor based on PSP-PMI or PSP-PJMI and SVM classifier. The 10-fold cross-validation experimental results on the benchmark datasets of non-single-base resolution and single-base resolution demonstrate that PSP-PMI and PSP-PJMI can extract features with strong capabilities to identify m6A and non-m6A sites. The M6A-BiNP predictor based on our proposed feature encoding algorithm PSP-PJMI is better than the state-of-the-art predictors, and it is so far the best model to identify m6A and non-m6A sites.

Exploration of the potential roles of m6A regulators in the uterus in pregnancy and infertility.

Infertility is a prevalent female reproductive disease worldwide. Currently, there are many unknown etiologies of infertility. N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic mRNA. This study intended to investigate the implications of m6A regulators in the uterus for pregnancy and infertility. Pregnant ICR mice on days (D) 0, 4, 6, 10, and 15 were used to monitor m6A methylation in the uterus by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then m6A methylation regulators were detected by real-time quantitative PCR (qPCR), western blot and immunohistochemistry (IHC). We found that m6A levels increased and that m6A regulators were expressed differently in the uterus during pregnancy. Then, we acquired expression data from endometrial tissue from women with infertility and recurrent pregnancy loss from the Gene Expression Omnibus (GEO) database. The expression of m6A regulators in infertility was significantly dysregulated according to the data mining technique. Specifically, the mRNA levels of METTL16 (p = 0.0147) and WTAP (p = 0.028) were lower and those of ALKBH5 (p = 0.0432) and IGF2BP2 (p = 0.0016) were higher in the endometrium of infertile patients. Meanwhile, many immunity-related pathways are abnormal in infertility, such as cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity and leukocyte transendothelial migration. In conclusion, we found that the m6A levels in the uterus increased as pregnancy progressed, and these regulators were dysregulated in the endometrium of infertility patients. These results suggest that m6A methylation may be very important in the establishment of implantation and maintenance of pregnancy and may become a new direction for research on infertility.

Simultaneous determination of cellular adenosine nucleotides, malondialdehyde, and uric acid using HPLC.

Adenine nucleotides and malondialdehyde (MDA) are key components involved in energy metabolism and reactive oxygen species (ROS) production. Measuring the levels of these components at the same time would be critical in studying mitochondrial functions. We have established a HPLC method to simultaneously measure adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, MDA, and uric acid (UA). The samples were treated with perchloric acid followed by centrifugation. After neutralization, the supernatant was subjected to HPLC determination. HPLC was performed using a C18 chromatographic column, isocratic elusion, and UV detection. The detection and quantification limits for these components were determined with standard solutions. The precision, repeatability, and 24-h stability were evaluated using cellular samples, and their relative standard deviations were all within 2%. The reproducibility and efficiency were confirmed with sample recovery tests and the observed oxidative effects of H2 O2 on Jurkat cells. With this method, we discovered the dependence of energy and oxidative states on the density of Jurkat cells cultured in suspension. We also found a significant correlation between UA in serum and that in saliva. These results indicate that this method has good accuracy and applicability. It can be used in biological, pharmacological, and clinical studies, especially those involving mitochondria, ROS, and purinergic signaling.

Analysis of m6A-Related Signatures in the Tumor Immune Microenvironment and Identification of Clinical Prognostic Regulators in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high rate of mortality and recurrence. N6-methyladenosine methylation (m6A) is the most common modification to affect cancer development, but to date, the potential role of m6A regulators in ACC prognosis is not well understood. In this study, we systematically analyzed 21 m6A regulators in ACC samples from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. We identified three m6A modification patterns with different clinical outcomes and discovered a significant relationship between diverse m6A clusters and the tumor immune microenvironment (immune cell types and ESTIMATE algorithm). Additionally, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) revealed that the m6A clusters were strongly associated with immune infiltration in the ACC. Next, to further explore the m6A prognostic signatures in ACC, we implemented Lasso (Least Absolute Shrinkage and Selection Operator) Cox regression to establish an eight-m6A-regulator prognostic model in the TCGA dataset, and the results showed that the model-based high-risk group was closely correlated with poor overall survival (OS) compared with the low-risk group. Subsequently, we validated the key modifications in the GEO datasets and found that high HNRNPA2B1 expression resulted in poor OS and event-free survival (EFS) in ACC. Moreover, to further decipher the molecular mechanisms, we constructed a competing endogenous RNA (ceRNA) network based on HNRNPA2B1, which consists of 12 long noncoding RNAs (lncRNAs) and 1 microRNA (miRNA). In conclusion, our findings indicate the potential role of m6A modification in ACC, providing novel insights into ACC prognosis and guiding effective immunotherapy.

Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders.

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary vasomotor disorders (impaired vasodilatation and/or enhanced vasoconstriction/spasm) represent important functional causes for such a clinical presentation. Although impaired vasodilatation may be assessed with non-invasive techniques such as positron emission tomography or cardiac magnetic resonance imaging, there is currently no reliable non-invasive technique for the diagnosis of coronary spasm available. Thus, invasive diagnostic procedures (IDP) have been developed for the diagnosis of coronary vasomotor disorders including spasm testing as well as assessment of coronary vasodilatation. The identification of the underlying type of disorder (so called endotype) allows the initiation of targeted pharmacological treatments. Despite the fact that such an approach is recommended by the current European Society of Cardiology guidelines for the management of chronic coronary syndromes based on the CorMicA study, comparability of results as well as multicenter trials are currently hampered by major differences in institutional protocols for coronary functional testing. This article describes a comprehensive IDP protocol including intracoronary acetylcholine provocation testing for diagnosis of epicardial/microvascular spasm, followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) in search of coronary vasodilatory impairment.

Impact of aging on the effects of intracoronary adenosine, peak hyperemia and its duration during fractional flow reserve assessment.

INTRODUCTION: Functional assessment of coronary stenoses is crucial for determining the correct therapeutic strategy. Age-related modifications in cardiovascular function could alter the functional significance of an intermediate coronary lesion. Therefore, the aim of the present study was to investigate the impact of age on fractional flow reserve (FFR) measurements in patients with intermediate coronary artery disease. METHODS: We included patients undergoing coronary angiography at our Division of Cardiology from June 2008 to February 2019 for elective indication or recent acute coronary syndrome and receiving FFR assessment for an intermediate coronary stenosis (angiographic 40-70% stenoses). FFR measurement was performed by pressure-recording guidewire (Prime Wire; Volcano Imaging System Philips Healthcare, San Diego, California, USA), after induction of hyperemia with intracoronary boluses of adenosine (from 60 to 720 µg, with dose doubling at each step). RESULTS: We included in our study 276 patients, undergoing FFR evaluation on 314 lesions, that were divided according to age (< or ≥70 years). Elderly patients displayed a higher cardiovascular risk profile and received more often specific therapy. We found significantly higher FFR values and lower Delta FFR and time to recovery in patients with age ≥70 years old even with high-dose adenosine. Elderly patients showed a trend in lower percentage of positive FFRs, especially with high-dose (P = 0.09). Overall, any FFR ≤ 0.80 was observed in 33.5% of younger patients and 21.1% of patients ≥70 years (P = 0.02). Results were confirmed after correction for baseline differences [adjusted odds ratio (95% confidence interval) = 0.60 (0.33-1.09), P = 0.08]. CONCLUSION: This is one of the first studies investigating the impact of age on the measurement of FFR with high-dose adenosine. Patients with age >70 years old with intermediate CAD are more likely to have higher FFR values and lower duration of hyperemia after adenosine boluses, as compared with younger patients.

Biological insights from the direct measurement of purine release.

Although purinergic signalling has been a well-established and accepted mechanism of chemical communication for many years, it remains important to measure the extracellular concentration of ATP and adenosine in real time. In this review I summarize the reasons why such measurements are still needed, how they provide additional mechanistic insight and give an overview of the techniques currently available to make spatially localised measurements of ATP and adenosine in real time. To illustrate the impact of direct real-time measurements, I explore CO2 and nutrient sensing in the medulla oblongata and hypothalamus. In both of these examples, the sensing involves hemichannel mediated ATP release from glial cells. For CO2 the hemichannels involved, connexin26, are directly CO2-sensitive. This mechanism contributes to the chemosensory control of breathing. In the hypothamalus, specialised glial cells, tanycytes, directly contact the cerebrospinal fluid in the 3rd ventricle and sense nutrients via sweet and umami taste receptors. Nutrient sensing by tanycytes is likely to contribute to the control of body weight as their selective stimulation alters food intake. To illustrate the importance of direct adenosine measurements, I consider the complex and multiple mechanisms of activity-dependent adenosine release in different brain regions. This activity dependent release of adenosine is likely to mediate important feedback regulation and may also be involved in controlling the sleep-wake state. I finish by briefly considering the potential of whole blood purine measurements in clinical practice.