"Acquired" Brugada syndrome in a cardiac allograft.

INTRODUCTION: Brugada syndrome is an inherited channelopathy characterized by arrhythmia and an increased risk of sudden cardiac death (SCD). Implantation of a defibrillator for primary or secondary prevention is the only effective strategy to decrease the risk of SCD in Brugada syndrome. We present a case in which a cardiac donor had a pathogenic variant for Brugada syndrome, discovered on genetic testing after transplantation. CASE REPORT: A young child with dilated cardiomyopathy underwent orthotopic heart transplantation from a donor with in-hospital cardiac arrest in the context of fever and a normal ECG. Approximately 1 month after transplant, the donor's post mortem genetic testing revealed a pathogenic loss-of-function SCN5A variant associated with Brugada syndrome, which was confirmed on genetic testing on a post-transplant endomyocardial biopsy from the recipient. The recipient's post-transplant electrocardiographic monitoring revealed persistent right bundle branch block and progressive, asymptomatic sinus node dysfunction. The recipient was managed with precautionary measures including aggressive fever management, avoidance of drugs that increase arrhythmia risk in Brugada syndrome, and increased frequency of arrhythmia surveillance. The recipient remains asymptomatic at over 3 years post-transplant with preserved graft function and no documented ventricular arrhythmias. CONCLUSION: We describe the clinical course of "acquired" Brugada syndrome in a cardiac allograft recipient, which has not been previously reported. The time-sensitive nature of donor organ selection, especially in critically ill recipients, combined with the growing use of molecular autopsies in patients with unexplained etiologies for death may increasingly result in important donor genetic information being made available after transplantation.

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship.

Technological advances promise unprecedented opportunities for whole exome sequencing and proteomic analyses of populations. Currently, data from genome and exome sequencing or proteomic studies are searched against reference genome annotations. This provides the foundation for research and clinical screening for genetic causes of pathologies. However, current genome annotations substantially underestimate the proteomic information encoded within a gene. Numerous studies have now demonstrated the expression and function of alternative (mainly small, sometimes overlapping) ORFs within mature gene transcripts. This has important consequences for the correlation of phenotypes and genotypes. Most alternative ORFs are not yet annotated because of a lack of evidence, and this absence from databases precludes their detection by standard proteomic methods, such as mass spectrometry. Here, we demonstrate how current approaches tend to overlook alternative ORFs, hindering the discovery of new genetic drivers and fundamental research. We discuss available tools and techniques to improve identification of proteins from alternative ORFs and finally suggest a novel annotation system to permit a more complete representation of the transcriptomic and proteomic information contained within a gene. Given the crucial challenge of distinguishing functional ORFs from random ones, the suggested pipeline emphasizes both experimental data and conservation signatures. The addition of alternative ORFs in databases will render identification less serendipitous and advance the pace of research and genomic knowledge. This review highlights the urgent medical and research need to incorporate alternative ORFs in current genome annotations and thus permit their inclusion in hypotheses and models, which relate phenotypes and genotypes.

Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging.

Quantitative-phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative-phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a digital holographic microscope (DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how quantitative-phase digital holographic microscopy (QP-DHM) can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-DHM can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM.

IL-1ß enables CNS access to CCR2hi monocytes and the generation of pathogenic cells through GM-CSF released by CNS endothelial cells.

Molecular interventions that limit pathogenic CNS inflammation are used to treat autoimmune conditions such as multiple sclerosis (MS). Remarkably, IL-1ß-knockout mice are highly resistant to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we show that interfering with the IL-1ß/IL-1R1 axis severely impairs the transmigration of myeloid cells across central nervous system (CNS) endothelial cells (ECs). Notably, we report that IL-1ß expression by inflammatory CCR2hi monocytes favors their entry into the spinal cord before EAE onset. Following activation with IL-1ß, CNS ECs release GM-CSF, which in turn converts monocytes into antigen-presenting cells (APCs). Accordingly, spinal cord-infiltrated monocyte-derived APCs are associated with dividing CD4+ T cells. Factors released from the interaction between IL-1ß-competent myeloid cells and CD4+ T cells are highly toxic to neurons. Together, our results suggest that IL-1ß signaling is an entry point for targeting both the initiation and exacerbation of neuroinflammation.

Involvement of the IL-1 system in experimental autoimmune encephalomyelitis and multiple sclerosis: Breaking the vicious cycle between IL-1ß and GM-CSF.

Multiple sclerosis (MS) is an autoimmune disease that affects hundreds of thousands of people worldwide. Given the autoimmune nature of the disease, a large part of the research has focused on autoreactive T and B cells. However, research on the involvement of myeloid cells in the pathophysiology of MS has received a strong and renewed attention over the recent years. Despite the multitude of inflammatory mediators involved in innate immunity, only a select group of cytokines are absolutely critical to the development of CNS autoimmunity, among which is interleukin (IL)-1. While the importance of the IL-1 system in experimental autoimmune encephalomyelitis (EAE) and MS has been recognized for about 20years, it is only recently that we have begun to understand that IL-1 plays multifaceted roles in disease initiation, development, amplification and chronicity. Here, we review the recent findings showing an implication of the IL-1 system in EAE and MS, and introduce a model that highlights how IL-1ß and granulocyte-macrophage colony-stimulating factor (GM-CSF) are interacting together to create a vicious feedback cycle of CNS inflammation that ultimately leads to myelin and neuronal damage.

Myeloid cell transmigration across the CNS vasculature triggers IL-1ß-driven neuroinflammation during autoimmune encephalomyelitis in mice.

Growing evidence supports a role for IL-1 in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), but how it impacts neuroinflammation is poorly understood. We show that susceptibility to EAE requires activation of IL-1R1 on radiation-resistant cells via IL-1ß secreted by bone marrow-derived cells. Neutrophils and monocyte-derived macrophages (MDMs) are the main source of IL-1ß and produce this cytokine as a result of their transmigration across the inflamed blood-spinal cord barrier. IL-1R1 expression in the spinal cord is found in endothelial cells (ECs) of the pial venous plexus. Accordingly, leukocyte infiltration at EAE onset is restricted to IL-1R1(+) subpial and subarachnoid vessels. In response to IL-1ß, primary cultures of central nervous system ECs produce GM-CSF, G-CSF, IL-6, Cxcl1, and Cxcl2. Initiation of EAE or subdural injection of IL-1ß induces a similar cytokine/chemokine signature in spinal cord vessels. Furthermore, the transfer of Gr1(+) cells on the spinal cord is sufficient to induce illness in EAE-resistant IL-1ß knockout (KO) mice. Notably, transfer of Gr1(+) cells isolated from C57BL/6 mice induce massive recruitment of recipient myeloid cells compared with cells from IL-1ß KO donors, and this recruitment translates into more severe paralysis. These findings suggest that an IL-1ß-dependent paracrine loop between infiltrated neutrophils/MDMs and ECs drives neuroinflammation.

The P2X7/P2X4 interaction shapes the purinergic response in murine macrophages.

The ATP-gated P2X4 and P2X7 receptors are cation channels, co-expressed in excitable and non-excitable cells and play important roles in pain, bone development, cytokine release and cell death. Although these receptors interact the interacting domains are unknown and the functional consequences of this interaction remain unclear. Here we show by co-immunoprecipitation that P2X4 interacts with the C-terminus of P2X7 and by fluorescence resonance energy transfer experiments that this receptor-receptor interaction is driven by ATP. Furthermore, disrupting the ATP-driven interaction by knocking-out P2X4R provoked an attenuation of P2X7-induced cell death, dye uptake and IL-1ß release in macrophages. Thus, P2X7 interacts with P2X4 via its C-terminus and disrupting the P2X7/P2X4 interaction hinders physiological responses in immune cells.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases.

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

Central canal ependymal cells proliferate extensively in response to traumatic spinal cord injury but not demyelinating lesions.

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9-10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.

A highly sensitive capillary electrophoresis method using p-nitrophenyl 5'-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities.

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5µm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80µA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K(m) values for NPP1 and NPP3, K(i) values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.

NTPDase1 controls IL-8 production by human neutrophils.

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.

Impact of ectoenzymes on p2 and p1 receptor signaling.

P2 receptors that are activated by extracellular nucleotides (e.g., ATP, ADP, UTP, UDP, Ap(n)A) and P1 receptors activated by adenosine control a diversity of biological processes. The activation of these receptors is tightly regulated by ectoenzymes that metabolize their ligands. This review presents these enzymes as well as their roles in the regulation of P2 and P1 receptor activation. We focus specifically on the role of ectoenzymes in processes of our interest, that is, inflammation, vascular tone, and neurotransmission. An update on the development of ectonucleotidase inhibitors is also presented.

Diadenosine 5',5''-(boranated)polyphosphonate analogues as selective nucleotide pyrophosphatase/phosphodiesterase inhibitors.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinucleotides and thus control purinergic signaling. Enhanced NPP activity is implicated in health disorders such as osteoarthritis and cancer. We designed novel diadenosine polyphosphonate derivatives as potential NPP inhibitors. Analogues 1-4 bear a phosphonate and/or boranophosphate group and/or a 2'-H atom instead of a 2'-OH group. In comparison to ATP, analogues 1-4 were barely hydrolyzed by human NTPDase1, -2, -3, and -8 (<5% hydrolysis) and NPP1 and -3 (≤ 13%) and were not hydrolyzed by ecto-5'-nucleotidase, unlike AMP. These derivatives did not affect NTPDase activity, and analogues 1 and 2 did not inhibit ecto-5'-nucleotidase. All analogues blocked ∼80% of the NPP2-dependent hydrolysis of pnp-TMP, a specific NPP substrate, and inhibited the catabolism of pnp-TMP (K(i) and IC50 both found to be between 10 and 60 µM), Ap5A, and ATP by NPP1. The activity of NPP3 was inhibited to a lesser extent by the new analogues, with compounds 1 and 4 being the most effective in that respect. The analogues dramatically reduced the level of hydrolysis of pnp-TMP at the cell surface of both osteocarcinoma and colon cancer cells. Importantly, analogues 1-4 exhibited significantly reduced agonistic activity toward human P2Y1,11) receptors (except for analogue 1) and no activity with human P2Y2 receptor. Our data provide strong evidence that analogue 2 is the first specific NPP inhibitor to be described.

NTPDase1 governs P2X7-dependent functions in murine macrophages.

P2X7 receptor is an adenosine triphosphate (ATP)-gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X7 effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X7-dependent responses in these cells. Macrophages isolated from NTPDase1-null mice (Entpd1(-/-)) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1(+/+)). Entpd1(-/-) macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL-1beta and IL-18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo-Pro-1 more efficiently (suggestive of increased pore formation) than Entpd1(+/+) cells. Consistent with these observations, NTPDase1 regulated P2X7-associated IL-1beta release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase-1. NTPDase1 also inhibited IL-1beta release in vivo in the air pouch inflammatory model. Exudates of LPS-injected Entpd1(-/-) mice had significantly higher IL-1beta levels when compared with Entpd1(+/+) mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X7-dependent macrophage responses.

The P2 receptor antagonist PPADS abrogates LPS-induced neutrophil migration in the murine air pouch via inhibition of MIP-2 and KC production.

In this work, we show that P2 nucleotide receptors control lipopolysaccharide (LPS)-induced neutrophil migration in the mouse air pouch model. Neutrophil infiltration in LPS-treated air pouches was reduced by the intravenous (iv) administration of the non-selective P2 receptor antagonist PPADS but not by suramin and RB-2. In addition, the iv administration of a P2 receptor ligand, UTP, enhanced LPS-induced neutrophil migration. In contrast, the iv injection of UDP had no effect on neutrophil migration. These data suggest that LPS-induced neutrophil migration in the air pouch could involve P2Y(4) receptor which is antagonized by PPADS, activated by UTP, but not UDP, and insensitive to suramin. The inhibition of neutrophil migration by PPADS correlated with a diminished secretion of chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC) in the air pouch exudates. As determined in vitro, PPADS did not affect MIP-2 and KC release from air pouch resident cells nor from accumulated neutrophils. MIP-2 and KC production in the LPS-treated air pouches correlated with an early neutrophil migration (1h after LPS injection), and both of these effects were significantly reduced in mice administered with PPADS. Altogether, these data suggest that P2Y(4) receptor expressed in circulating leukocytes and/or endothelium controls LPS-induced acute neutrophil recruitment in mouse air pouch.

Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration.

The chemokine interleukin 8 (IL-8) is a major chemoattractant for human neutrophils. Here, we demonstrate novel evidence that IL-8-induced neutrophil chemotaxis requires a concurrent activation of P2 receptors, most likely the P2Y(2) which is dominantly expressed in these cells. Indeed, the migration of human neutrophils towards IL-8 was significantly inhibited by the P2Y receptor antagonists, suramin and reactive blue 2 (RB-2) and potentiated by a P2Y(2) ligand, ATP, but insensitive to specific antagonists of P2Y(1), P2Y(6) and P2Y(11) receptors. Adenosine had no effect on neutrophil migration towards IL-8 which contrasted with the stimulatory effect of this molecule on neutrophil chemotaxis caused by formyl-Met-Leu-Phe (fMLP or fMLF). Taken together, these data suggest that extracellular ATP is necessary for IL-8 to exert its chemotactic effect on neutrophils.

A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions.

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 microA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.