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.
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).
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.
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.
Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters ...that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na
concentrations and a consequent increase in astrocytic Ca
through Na
/Ca
exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.
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.
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.
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.
The hypocretin/orexin neuropeptides (hcrt) are key players in the control of sleep and wakefulness evidenced by the fact that lack of hcrt leads to the sleep disorder Narcolepsy Type 1. Sleep ...disturbances are common in mood disorders, and hcrt has been suggested to be poorly regulated in depressed subjects. To study seasonal variation in hcrt levels, we obtained data on hcrt-1 levels in the cerebrospinal fluid (CSF) from 227 human individuals evaluated for central hypersomnias at a Danish sleep center. The samples were taken over a 4 year timespan, and obtained in the morning hours, thus avoiding impact of the diurnal hcrt variation. Hcrt-1 concentration was determined in a standardized radioimmunoassay. Using biometric data and sleep parameters, a multivariate regression analysis was performed. We found that the average monthly CSF hcrt-1 levels varied significantly across the seasons following a sine wave with its peak in the summer (June-July). The amplitude was 19.9 pg hcrt/mL 12.8-26.9 corresponding to a 10.6% increase in midsummer compared to winter. Factors found to significantly predict the hcrt-1 values were day length, presence of snow, and proximity to the Christmas holiday season. The hcrt-1 values from January were much higher than predicted from the model, suggestive of additional factors influencing the CSF hcrt-1 levels such as social interaction. This study provides evidence that human CSF hcrt-1 levels vary with season, correlating with day length. This finding could have implications for the understanding of winter tiredness, fatigue, and seasonal affective disorder. This is the first time a seasonal variation of hcrt-1 levels has been shown, demonstrating that the hcrt system is, like other neurotransmitter systems, subjected to long term modulation.
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.
Sympathetic and vagal activation is linked to atrial arrhythmogenesis. Here we investigated the small conductance Ca
-activated K
(SK)-channel pore-blocker
...-(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 β-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 APD
(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 β-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 APD
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 APD
and ERP prolongation and shorter AF durations. Fleca treatment in CCh-stimulated atria prolonged APD
and ERP and abbreviated the AF duration to a similar extent as with ICA. Muscarinic activated atria constitutes a more arrhythmogenic substrate than β-adrenoceptor activated atria. Pharmacological inhibition of SK channels by ICA is effective under both conditions and equally efficacious to Fleca.
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.
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 P2Y
11
receptor. In a recombinant expression system, the coexpression of the human P2Y
11
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 P2Y
11
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 P2Y
11
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 P2Y
11
receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.
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