Scanning Ion-Conductance Microscopy for Studying ß-Amyloid Aggregate Formation on Living Cell Surfaces.

ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.

Mass spectrometric studies of the variety of beta-amyloid proteoforms in Alzheimer's disease.

This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta-amyloid (Aß) peptides in human samples. Since Aß is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aß proteoforms, differing in length and modifications. Approaches based on matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography with electrospray ionization tandem MS are most common in Aß studies. However, Aß forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aß species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aß studies; and considers the potential of MS techniques for further studies of Aß-peptides.

Role of Interaction between Zinc and Amyloid Beta in Pathogenesis of Alzheimer's Disease.

Progression of Alzheimer's disease is accompanied by the appearance of extracellular deposits in the brain tissues of patients with characteristic supramolecular morphology (amyloid plaques) the main components of which are ß-amyloid isoforms (Aß) and biometal ions (zinc, copper, iron). For nearly 40 years and up to the present time, the vast majority of experimental data indicate critical role of formation and accumulation of amyloid plaques (cerebral amyloidogenesis) in pathogenesis of Alzheimer's disease, however, nature of the molecular agents that initiate cerebral amyloidogenesis, as well as causes of aggregation of the native Aß molecules in vivo remained unknown for a long time. This review discusses the current level of fundamental knowledge about the molecular mechanisms of interactions of zinc ions with a number of Aß isoforms present in amyloid plaques of the patients with Alzheimer's disease, and also shows how this knowledge made it possible to identify driving forces of the cerebral amyloidogenesis in Alzheimer's disease and made it possible to determine fundamentally new biomarkers and drug targets as part of development of innovative strategy for diagnosis and treatment of Alzheimer's disease.

Electrochemical Analysis in Studying ß-Amyloid Aggregation.

ß-amyloid (Aß) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aß aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aß aggregation have intensively being investigated using synthetic Aß peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aß aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aß (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aß oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aß, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aß are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aß aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Switching On/Off Amyloid Plaque Formation in Transgenic Animal Models of Alzheimer's Disease.

A hallmark of Alzheimer's disease (AD) are the proteinaceous aggregates formed by the amyloid-beta peptide (Aß) that is deposited inside the brain as amyloid plaques. The accumulation of aggregated Aß may initiate or enhance pathologic processes in AD. According to the amyloid hypothesis, any agent that has the capability to inhibit Aß aggregation and/or destroy amyloid plaques represents a potential disease-modifying drug. In 2023, a humanized IgG1 monoclonal antibody (lecanemab) against the Aß-soluble protofibrils was approved by the US FDA for AD therapy, thus providing compelling support to the amyloid hypothesis. To acquire a deeper insight on the in vivo Aß aggregation, various animal models, including aged herbivores and carnivores, non-human primates, transgenic rodents, fish and worms were widely exploited. This review is based on the recent data obtained using transgenic animal AD models and presents experimental verification of the critical role in Aß aggregation seeding of the interactions between zinc ions, Aß with the isomerized Asp7 (isoD7-Aß) and the α4ß2 nicotinic acetylcholine receptor.

Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Tetrapeptide Ac-HAEE-NH2 Protects α4ß2 nAChR from Inhibition by Aß.

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aß peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aß peptide mediates its interaction with α4ß2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aß-α4ß2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4ß2 nAChR. Indeed, we discovered a 35HAEE38 site in α4ß2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aß42-α4ß2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aß via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aß42-induced inhibition of α4ß2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aß on α4ß2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4ß2 nAChR-dependent cholinergic dysfunction in AD.

Application of MALDI-TOF/TOF-MS for relative quantitation of α- and ß-Asp7 isoforms of amyloid-ß peptide.

It is known that aspartic acid isomerization process plays a role in aging processes and may be used as a marker for aging of natural materials. As for Alzheimer's disease, the most abundant modification in the peptide profile is the aspartate isomerization of amyloid-ß. Liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry-based approaches with Collision Induced Dissociation (CID) or Electron Capture Dissociation (ECD) fragmentation provide a good and precise method for the relative quantitation of iso- to normal amyloid-ß peptides but require additional time consuming steps. In this study, MALDI-TOF/TOF-matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MS) method was developed as a high-throughput approach for the relative quantitation of the isomerized form of the amyloid-ß peptide.

A Binuclear Zinc Interaction Fold Discovered in the Homodimer of Alzheimer's Amyloid-ß Fragment with Taiwanese Mutation D7H.

Zinc-induced oligomerization of amyloid-ß peptide (Aß) produces potentially pathogenic agents of Alzheimer's disease. Mutations and modifications in the metal binding domain 1-16 of Aß peptide crucially affect its zinc-induced oligomerization by changing intermolecular zinc mediated interface. The 3D structure of this interface appearing in a range of Aß species is a prospective drug target for disease modifying therapy. Using NMR spectroscopy, EXAFS spectroscopy, mass spectrometry, and isothermal titration calorimetry the interaction of zinc ions with Aß fragments 1-7 and 1-10 carrying familial Taiwanese mutation D7H was studied. Zinc ions induce formation of a stable homodimer formed by the two peptide chains fastened by two zinc ions and stacking interactions of imidazole rings. A binuclear zinc interaction fold in the dimer structure was discovered. It can be used for designing zinc-regulated proteins and zinc-mediated self-assembling peptides.

Protein interactomics based on direct molecular fishing on paramagnetic particles: practical realization and further SPR validation.

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity-based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC-MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR-based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 µM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein-protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-ß binding proteins: relevance to Alzheimer's disease?

The amyloid-ß peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-ß accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-ß binding proteins and 0.1 mM isatin decreased the number of identified amyloid-ß binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-ß binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-ß oligomers described in the literature for some isatin derivatives.

The radiopharmaceutical radium-223 has immunomodulatory effects in patients and facilitates anti-programmed death receptor-1 therapy in murine models of bone metastatic prostate cancer.

BACKGROUND & PURPOSE: Radium-223 (Ra223) improves survival in metastatic prostate cancer (mPC), but its impact on systemic immunity is unclear, and biomarkers of response are lacking. We examined markers of immunomodulatory activity during standard clinical Ra223 and studied the impact of Ra223 on response to immune checkpoint inhibition (ICI) in preclinical models. MATERIALS & METHODS: We conducted a single-arm biomarker study of Ra223 in 22 bone mPC patients. We measured circulating immune cell subsets and a panel of cytokines before and during Ra223 therapy and correlated them with overall survival (OS). Using two murine mPC models-orthotopic PtenSmad4-null and TRAMP-C1 grafts in syngeneic immunocompetent mice-we tested the efficacy of combining Ra223 with ICI. RESULTS: Above-median level of IL-6 at baseline was associated with a median OS of 358 versus 947 days for below levels; p = 0.044, from the log-rank test. Baseline PlGF and PSA inversely correlated with OS (p = 0.018 and p = 0.037, respectively, from the Cox model). Ra223 treatment was associated with a mild decrease in some peripheral immune cell populations and a shift in the proportion of MDSCs from granulocytic to myeloid. In mice, Ra223 increased the proliferation of CD8+ and CD4+ helper T cells without leading to CD8+ T cell exhaustion in the mPC lesions. In one of the models, combining Ra223 and anti-PD-1 antibody significantly prolonged survival, which correlated with increased CD8+ T cell infiltration in tumor tissue. CONCLUSION: The inflammatory cytokine IL-6 and the angiogenic biomarker PlGF at baseline were promising outcome biomarkers after standard Ra223 treatment. In mouse models, Ra223 increased intratumoral CD8+ T cell infiltration and proliferation and could improve OS when combined with anti-PD-1 ICI.

LC-MS/MS determination of GTS-201, a dipeptide mimetic of the brain-derived neurotrophic factor, and neurotransmitter metabolites with application to a pharmacokinetic study in rats.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family with diverse psychopharmacological effects including antidepressant and anxiolytic actions. However, the clinical use of BDNF is limited due to its poor pharmacokinetic properties. The development of low-molecular-weight BDNF mimetics passing through the blood-brain barrier is an emerging strategy for improved managing psychiatric diseases. The present study characterizes a novel dipeptide mimetic of the 2nd BDNF loop named GTS-201, which exhibits psychotropic properties in experimental animal models of anxiety and alcohol dependence. The aim of this work was to study the pharmacokinetics of GTS-201 in rats at a saturating dosage of 5 mg/kg applied by the intraperitoneal route and to characterize the effects on neurotransmitter levels in the blood and brain. The maximum concentration (Cmax) of GTS-201 in the plasma (867 ± 69 ng/ml) was recorded at 35 ± 7.7 min after administration (Tmax) with a half-elimination period (T1/2) of 19.5 ± 1.8 min, while in the brain tissue Cmax was 14.92 ± 3.11 ng/ml, Tmax was 40.0 ± 7.7 min and T1/2 were 87.5 ± 12.7 min. The relative tissue availability of the GTS-201 for the brain reached 2.9%. At the dose applied, GTS-201 induced a significant increase of serotonin (5-fold) and dopamine levels in the brain tissue (8-fold) along with a decrease in cortisol content in blood plasma 45 min after acute administration. In summary, GTS-201 crosses the blood-brain barrier after acute administration and affects the activity of serotonergic and dopaminergic systems, which may underlie its neuropsychotropic effects described previously.

Zn-dependent ß-amyloid Aggregation and its Reversal by the Tetrapeptide HAEE.

The pathogenesis of Alzheimer's disease (AD) is associated with the formation of cerebral amyloid plaques, the main components of which are the modified Aß molecules as well as the metal ions. Aß isomerized at Asp7 residue (isoD7-Aß) is the most abundant isoform in amyloid plaques. We hypothesized that the pathogenic effect of isoD7-Aß is due to the formation of zinc-dependent oligomers, and that this interaction can be disrupted by the rationally designed tetrapeptide (HAEE). Here, we utilized surface plasmon resonance, nuclear magnetic resonance, and molecular dynamics simulation to demonstrate Zn2+-dependent oligomerization of isoD7-Aß and the formation of a stable isoD7-Aß:Zn2+:HAEE complex incapable of forming oligomers. To demonstrate the physiological importance of zinc-dependent isoD7-Aß oligomerization and the ability of HAEE to interfere with this process at the organismal level, we employed transgenic nematodes overexpressing human Aß. We show that the presence of isoD7-Aß in the medium triggers extensive amyloidosis that occurs in a Zn2+-dependent manner, enhances paralysis, and shortens the animals' lifespan. Exogenous HAEE completely reverses these pathological effects of isoD7-Aß. We conclude that the synergistic action of isoD7-Aß and Zn2+ promotes Aß aggregation and that the selected small molecules capable of interrupting this process, such as HAEE, can potentially serve as anti-amyloid therapeutics.

Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

NMR solution structure of rat aß(1-16): toward understanding the mechanism of rats' resistance to Alzheimer's disease.

In an attempt to reveal the mechanism of rats' resistance to Alzheimer's disease, we determined the structure of the metal-binding domain 1-16 of rat ß-amyloid (rat Aß(1-16)) in solution in the absence and presence of zinc ions. A zinc-induced dimerization of the domain was detected. The zinc coordination site was found to involve residues His-6 and His-14 of both peptide chains. We used experimental restraints obtained from analyses of NMR and isothermal titration calorimetry data to perform structure calculations. The calculations employed an explicit water environment and a simulated annealing molecular-dynamics protocol followed by quantum-mechanical/molecular-mechanical optimization. We found that the C-tails of the two polypeptide chains of the rat Aß(1-16) dimer are oriented in opposite directions to each other, which hinders the assembly of rat Aß dimers into oligomeric aggregates. Thus, the differences in the structure of zinc-binding sites of human and rat Aß(1-16), their ability to form regular cross-monomer bonds, and the orientation of their hydrophobic C-tails could be responsible for the resistance of rats to Alzheimer's disease.

Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.

The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a "normalization window"--a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.

Vascular damage in tumors: a key player in stereotactic radiation therapy?

The use of stereotactic radiation therapy (SRT) for cancer treatment has grown in recent years, showing excellent results for some tumors. The greatly increased doses per fraction in SRT compared to conventional radiotherapy suggest a 'new biology' that determines treatment outcome. Proposed mechanisms include significant damage to tumor blood vessels and enhanced antitumor immune responses, which are also vasculature-dependent. These ideas are mostly based on the results of radiation studies in animal models because direct observations in humans are limited. However, even preclinical findings are somewhat incomplete and result in ambiguous conclusions. Current evidence of vasculature-related mechanisms of SRT is reviewed. Understanding them could result in better optimization of SRT alone or in combination with immune or other cancer therapies.

Development of Peptide Biopharmaceuticals in Russia.

Peptides are low-molecular-weight substances that participate in numerous important physiological functions, such as human growth and development, stress, regulation of the emotional state, sexual behavior, and immune responses. Their mechanisms of action are based on receptor-ligand interactions, which result in highly selective effects. These properties and low toxicity enable them to be considered potent drugs. Peptide preparations became possible at the beginning of the 20th century after a method was developed for selectively synthesizing peptides; however, after synthesis of the first peptide drugs, several issues related to increasing the stability, bioavailability, half-life, and ability to move across cell membranes remain unresolved. Here, we briefly review the history of peptide production and development in the biochemical industry and outline potential areas of peptide biopharmaceutical applications and modern approaches for creating pharmaceuticals based on synthetic peptides and their analogs. We also focus on original peptide drugs and the approaches used for their development by the Russian Federation.

Insights into the Cardiotoxic Effects of Veratrum Lobelianum Alkaloids: Pilot Study.

Jervine, protoveratrine A (proA), and protoveratrine B (proB) are Veratrum alkaloids that are presented in some remedies obtained from Veratrum lobelianum, such as Veratrum aqua. This paper reports on a single-center pilot cardiotoxic mechanism study of jervine, proA, and proB in case series. The molecular aspects were studied via molecular dynamic simulation, molecular docking with cardiac sodium channel NaV1.5, and machine learning-based structure-activity relationship modeling. HPLC-MS/MS method in combination with clinical events were used to analyze Veratrum alkaloid cardiotoxicity in patients. Jervine demonstrates the highest docking score (-10.8 kcal/mol), logP value (4.188), and pKa value (9.64) compared with proA and proB. Also, this compound is characterized by the lowest calculated IC50. In general, all three analyzed alkaloids show the affinity to NaV1.5 that highly likely results in cardiotoxic action. The clinical data of seven cases of intoxication by Veratrum aqua confirms the results of molecular modeling. Patients exhibited nausea, muscle weakness, bradycardia, and arterial hypotension. The association between alkaloid concentrations in blood and urine and severity of patient condition is described. These experiments, while primary, confirmed that jervine, proA, and proB contribute to cardiotoxicity by NaV1.5 inhibition.