Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

In vivo knockdown of SK3 channels using antisense oligonucleotides protects against atrial fibrillation in rats.

INTRODUCTION: GapmeRs are oligonucleotides that bind to a specific RNA sequence and thereby affecting posttranscriptional gene regulation. They therefore hold the potential to manipulate targets where current pharmacological modulators are inefficient or exhibit adverse side effects. Here, we show that a treatment with a GapmeR, mediating knockdown of small conductance Ca2+-activated K+ channels (SK3), has an in vivo protective effect against atrial fibrillation (AF) in rats. MATERIAL AND METHODS: A unique SK3-GapmeR design was selected after thorough in vitro evaluation. 22 rats were randomly assigned to receive either 50 mg/kg SK3-GapmeR or vehicle subcutaneously once a week for two weeks. Langendorff experiments were performed seven days after the last injection, where action potential duration (APD90), effective refractory period (ERP) and AF propensity were investigated. SK3 channel activity was evaluated using the SK channel blocker, ICA (N-(pyridin-2-yl)-4-(pyridine-2-yl)thiazol-2-amine). SK3 protein expression was assessed by Western Blot. RESULTS: The designed GapmeR effectively down-regulate the SK3 protein expression in the heart (48% downregulation, p = 0.0095) and did indeed protect against AF. Duration of AF episodes elicited by burst pacing in the rats treated with SK3-GapmeR was reduced 78% compared to controls (3.7 s vs. 16.8 s, p = 0.0353). The number of spontaneous AF episodes were decreased by 68% in the SK3-GapmeR group (39 episodes versus 123 in the control group, respectively) and were also significantly shorter in duration (7.2 s versus 29.7 s in the control group, p = 0.0327). Refractoriness was not altered at sinus rhythm, but ERP prolongation following ICA application was blunted in the SK3-GapmeR group. CONCLUSION: The selected GapmeR silenced the cardiac SK3 channels, thereby preventing AF in rats. Thus, GapmeR technology can be applied as an experimental tool of downregulation of cardiac proteins and could potentially offer a novel modality for treatment of cardiac diseases.

A Novel SCN5A Variant Associated with Abnormal Repolarization, Atrial Fibrillation, and Reversible Cardiomyopathy.

A variety of life-threating arrhythmias are caused by mutations in the cardiac voltage-gated sodium channel encoded by the SCN5A gene. In this study, we report a novel loss-of-function SCN5A variant, p.Ile1343Val (c.4027A>G), identified in a 42-year-old proband who presented with an unusual ECG with abnormal repolarization with biphasic T-waves in anteroseptal leads, persistent atrial fibrillation (AF), intermittent left bundle branch block (LBBB), and reversible cardiomyopathy. The patient did not meet the diagnostic criteria for Brugada syndrome, long QT syndrome, or any other known SCN5A-associated phenotype. Characterization of the biophysical properties of the variant by in vitro patch clamp experiments revealed a reduced Na+ current with no effect on the inactivation kinetics of the channel. This loss-of-function of Na+ current could explain the intermittent LBBB as well as the AF. In conclusion, we describe a unique combination of electrical and structural abnormalities associated with a novel SCN5A variant. Our findings broaden the spectrum of cardiac phenotypes associated with SCN5A channelopathy, underlining the complex clinical manifestations of genetic variations within this gene.

Seizure control by decanoic acid through direct AMPA receptor inhibition.

The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones ß-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet.

The GABAA antagonist DPP-4-PIOL selectively antagonises tonic over phasic GABAergic currents in dentate gyrus granule cells.

GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model.

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

De novo designed proteins neutralize lethal snake venom toxins.

Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more1,2. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage3 and inhibition of nicotinic acetylcholine receptors (nAChRs) resulting in life-threatening neurotoxicity4. Currently, the only available treatments for snakebite consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs5,6,7. Here, we use deep learning methods to de novo design proteins to bind short- and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtain protein designs with remarkable thermal stability, high binding affinity, and near-atomic level agreement with the computational models. The designed proteins effectively neutralize all three 3FTx sub-families in vitro and protect mice from a lethal neurotoxin challenge. Such potent, stable, and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective, and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our computational design methodology should help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for development of therapies to neglected tropical diseases.

From squid giant axon to automated patch-clamp: electrophysiology in venom and antivenom research.

Ion channels play a crucial role in diverse physiological processes, including neurotransmission and muscle contraction. Venomous creatures exploit the vital function of ion channels by producing toxins in their venoms that specifically target these ion channels to facilitate prey capture upon a bite or a sting. Envenoming can therefore lead to ion channel dysregulation, which for humans can result in severe medical complications that often necessitate interventions such as antivenom administration. Conversely, the discovery of highly potent and selective venom toxins with the capability of distinguishing between different isoforms and subtypes of ion channels has led to the development of beneficial therapeutics that are now in the clinic. This review encompasses the historical evolution of electrophysiology methodologies, highlighting their contributions to venom and antivenom research, including venom-based drug discovery and evaluation of antivenom efficacy. By discussing the applications and advancements in patch-clamp techniques, this review underscores the profound impact of electrophysiology in unravelling the intricate interplay between ion channels and venom toxins, ultimately leading to the development of drugs for envenoming and ion channel-related pathologies.

A single-chain variable fragment selected against a conformational epitope of a recombinantly produced snake toxin using phage display.

Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.

Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes.

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin - the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom.

The monocled cobra (Naja kaouthia) is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments. Here, we describe the discovery and subsequent optimization of a recombinant human monoclonal immunoglobulin G antibody against α-cobratoxin using phage display technology. Affinity maturation by light chain-shuffling resulted in a significant increase in in vitro neutralization potency and in vivo efficacy. The optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.

Perforated Whole-Cell Recordings in Automated Patch Clamp Electrophysiology.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca2+), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

Optogenetics and Optical Tools in Automated Patch Clamping.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.

Impact of arrhythmogenic calmodulin variants on small conductance Ca2+ -activated K+ (SK3) channels.

Calmodulin (CaM) is a ubiquitous Ca2+ -sensing protein regulating many important cellular processes. Several CaM-associated variants have been identified in a small group of patients with cardiac arrhythmias. The mechanism remains largely unknown, even though a number of ion channels, including the ryanodine receptors and the L-type calcium channels have been shown to be functionally affected by the presence of mutant CaM. CaM is constitutively bound to the SK channel, which underlies the calcium-gated ISK contributing to cardiac repolarization. The CaM binding to SK channels is essential for gating, correct assembly, and membrane expression. To elucidate the effect of nine different arrhythmogenic CaM variants on SK3 channel function, HEK293 cells stably expressing SK3 were transiently co-transfected with CaMWT or variant and whole-cell patch-clamp recordings were performed with a calculated free Ca2+ concentration of 400 nmol/L. MDCK cells were transiently transfected with SK3 and/or CaMWT or variant to address SK3 and CaM localization by immunocytochemistry. The LQTS-associated variants CaMD96V , CaMD130G , and CaMF142L reduced ISK,Ca compared with CaMWT (P < 0.01, P < 0.001, and P < 0.05, respectively). The CPVT associated variant CaMN54I also reduced the ISK,Ca (P < 0.05), which was linked to an accumulation of SK3/CaMN54I channel complexes in intracellular compartments (P < 0.05). The CPVT associated variants, CaMA103V and CaMD132E only revealed a tendency toward reduced current, while the variants CaMF90L and CaMN98S , causing LQTS syndrome, did not have any impact on ISK,Ca . In conclusion, we found that the arrhythmogenic CaM variants CaMN54I , CaMD96V , CaMD130G , and CaMF142L significantly down-regulate the SK3 channel current, but with distinct mechanism.

Pharmacological rescue of mutated Kv3.1 ion-channel linked to progressive myoclonus epilepsies.

Progressive myoclonus epilepsies (PMEs) constitute a cluster of inherent, genetically diverse, rare seizure disorders characterized by ataxia, tonic-clonic seizures, and action myoclonus. Recently, a mutation in the KCNC1 gene (Arg320His) was described in a group of PME patients. The KCNC1 gene encodes the Kv3.1 potassium ion channel responsible for the rapid repolarization of the membrane potential following action potential firing in fast spiking GABAergic interneurons (FSI), thereby enabling high firing frequency. In the present study, we demonstrate that the Arg320His mutation cause a reduction in the Kv3.1 current amplitude and acts in a dominantly negative fashion. The mutation profoundly affects channel activation and deactivation kinetics, and we further find that it impairs recruitment of the Kv3.1 channel to the plasma membrane. The Kv3 activating compound, RE01, partly rescues the electrophysiological deficit, suggesting that pharmacological activation of Kv3.1 activity might be a feasible approach for treatment of this cohort of PME patients.

Inhibition of Small Conductance Calcium-Activated Potassium (SK) Channels Prevents Arrhythmias in Rat Atria During ß-Adrenergic and Muscarinic Receptor Activation.

Sympathetic and vagal activation is linked to atrial arrhythmogenesis. Here we investigated the small conductance Ca2+-activated K+ (SK)-channel pore-blocker N-(pyridin-2-yl)-4-(pyridine-2-yl)thiazol-2-amine (ICA) on action potential (AP) and atrial fibrillation (AF) parameters in isolated rat atria during ß-adrenergic [isoprenaline (ISO)] and muscarinic M2 [carbachol (CCh)] activation. Furthermore, antiarrhythmic efficacy of ICA was benchmarked toward the class-IC antiarrhythmic drug flecainide (Fleca). ISO increased the spontaneous beating frequency but did not affect other AP parameters. As expected, CCh hyperpolarized resting membrane potential (-6.2 ± 0.9 mV), shortened APD90 (24.2 ± 1.6 vs. 17.7 ± 1.1 ms), and effective refractory period (ERP; 20.0 ± 1.3 vs. 15.8 ± 1.3 ms). The duration of burst pacing triggered AF was unchanged in the presence of CCh compared to control atria (12.8 ± 5.3 vs. 11.2 ± 3.6 s), while ß-adrenergic activation resulted in shorter AF durations (3.3 ± 1.7 s) and lower AF-frequency compared to CCh. Treatment with ICA (10 µM) in ISO -stimulated atria prolonged APD90 and ERP, while the AF burden was reduced (7.1 ± 5.5 vs. 0.1 ± 0.1 s). In CCh-stimulated atria, ICA treatment also resulted in APD90 and ERP prolongation and shorter AF durations. Fleca treatment in CCh-stimulated atria prolonged APD90 and ERP and abbreviated the AF duration to a similar extent as with ICA. Muscarinic activated atria constitutes a more arrhythmogenic substrate than ß-adrenoceptor activated atria. Pharmacological inhibition of SK channels by ICA is effective under both conditions and equally efficacious to Fleca.

Human P2Y11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.

Anticonvulsive evaluation of THIP in the murine pentylenetetrazole kindling model: lack of anticonvulsive effect of THIP despite functional δ-subunit-containing GABAA receptors in dentate gyrus granule cells.

THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a GABAA receptor agonist with varying potencies and efficacies at γ-subunit-containing receptors. More importantly, THIP acts as a selective superagonist at δ-subunit-containing receptors (δ-GABAA Rs) at clinically relevant concentrations. Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated with several animal models of epilepsy, we first investigated the presence of functional δ-GABAA receptors. Both immunohistochemistry and Western blot data demonstrated that δ-GABAA R expression is not only present in the dentate gyrus, but also the expression level was enhanced in the early phase after PTZ kindling. Whole-cell patch-clamp studies in acute hippocampal brain slices revealed that THIP was indeed able to induce a tonic inhibition in dentate gyrus granule cells. However, THIP induced a tonic current of similar magnitude in the PTZ-kindled mice compared to saline-treated animals despite the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties.

Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.

BACKGROUND AND AIM: The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. METHODS: Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). RESULTS: Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. CONCLUSION: This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.