In silico prediction of hERG blockers using machine learning and deep learning approaches.

The human ether-à-go-go-related gene (hERG) is associated with drug cardiotoxicity. If the hERG channel is blocked, it will lead to prolonged QT interval and cause sudden death in severe cases. Therefore, it is important to evaluate the hERG-blocking property of compounds in early drug discovery. In this study, a dataset containing 4556 compounds with IC50 values determined by patch clamp techniques on mammalian lineage cells was collected, and hERG blockers and non-blockers were distinguished according to three single thresholds and two binary thresholds. Four machine learning (ML) algorithms combining four molecular fingerprints and molecular descriptors as well as graph convolutional neural networks (GCNs) were used to construct a series of binary classification models. The results showed that the best models varied for different thresholds. The ML models implemented by support vector machine and random forest performed well based on Morgan fingerprints and molecular descriptors, with AUCs ranging from 0.884 to 0.950. GCN showed superior prediction performance with AUCs above 0.952, which might be related to its direct extraction of molecular features from the original input. Meanwhile, the classification of binary threshold was better than that of single threshold, which could provide us with a more accurate prediction of hERG blockers. At last, the applicability domain for the model was defined, and seven structural alerts that might generate hERG blockage were identified by information gain and substructure frequency analysis. Our work would be beneficial for identifying hERG blockers in chemicals.

Suppression of delayed rectifier K+ channels by gentamicin induces membrane hyperexcitability through JNK and PKA signaling pathways in vestibular ganglion neurons.

Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.

Chronic intermittent hypoxia transiently increases hippocampal network activity in the gamma frequency band and 4-Aminopyridine-induced hyperexcitability in vitro.

Chronic intermittent hypoxia (CIH) is the most distinct feature of obstructive sleep apnea (OSA), a common breathing and sleep disorder that leads to several neuropathological consequences, including alterations in the hippocampal network and in seizure susceptibility. However, it is currently unknown whether these alterations are permanent or remit upon normal oxygenation. Here, we investigated the effects of CIH on hippocampal spontaneous network activity and hyperexcitability in vitro and explored whether these alterations endure or fade after normal oxygenation. Results showed that applying CIH for 21 days to adult rats increases gamma-band hippocampal network activity and aggravates 4-Aminopyridine-induced epileptiform activity in vitro. Interestingly, these CIH-induced alterations remit after 30 days of normal oxygenation. Our findings indicate that hippocampal network alterations and increased seizure susceptibility induced by CIH are not permanent and can be spontaneously reverted, suggesting that therapeutic interventions against OSA in patients with epilepsy, such as surgery or continuous positive airway pressure (CPAP), could be favorable for seizure control.

Single amyloid-beta injection exacerbates 4-aminopyridine-induced seizures and changes synaptic coupling in the hippocampus.

Accumulation of amyloid-beta (Aß) in temporal lobe structures, including the hippocampus, is related to a variety of Alzheimer's disease symptoms and seems to be involved in the induction of neural network hyperexcitability and even seizures. Still, a direct evaluation of the pro-epileptogenic effects of Aß in vivo, and of the underlying mechanisms, is missing. Thus, we tested whether the intracisternal injection of Aß modulates 4-aminopyridine (4AP)-induced epileptiform activity, hippocampal network function, and its synaptic coupling. When tested 3 weeks after its administration, Aß (but not its vehicle) reduces the latency for 4AP-induced seizures, increases the number of generalized seizures, exacerbates the time to fully recover from seizures, and favors seizure-induced death. These pro-epileptogenic effects of Aß correlate with a reduction in the power of the spontaneous hippocampal network activity, involving all frequency bands in vivo and only the theta band (4-10 Hz) in vitro. The pro-epileptogenic effects of Aß also correlate with a reduction of the Schaffer-collateral CA1 synaptic coupling in vitro, which is exacerbated by the sequential bath application of 4-AP and Aß. In summary, Aß produces long-lasting pro-epileptic effects that can be due to alterations in the hippocampal circuit, impacting its coordinated network activity and its synaptic efficiency. It is likely that normalizing synaptic coupling and/or coordinated neural network activity (i.e., theta activity) may contribute not only to improve cognitive function in Alzheimer's disease but also to avoid hyperexcitation in conditions of amyloidosis.

The Catch-22 of Predicting hERG Blockade Using Publicly Accessible Bioactivity Data.

Drug-induced inhibition of the human ether-à-go-go-related gene (hERG)-encoded potassium ion channels can lead to fatal cardiotoxicity. Several marketed drugs and promising drug candidates were recalled because of this concern. Diverse modeling methods ranging from molecular similarity assessment to quantitative structure-activity relationship analysis employing machine learning techniques have been applied to data sets of varying size and composition (number of blockers and nonblockers). In this study, we highlight the challenges involved in the development of a robust classifier for predicting the hERG end point using bioactivity data extracted from the public domain. To this end, three different modeling methods, nearest neighbors, random forests, and support vector machines, were employed to develop predictive models using different molecular descriptors, activity thresholds, and training set compositions. Our models demonstrated superior performance in external validations in comparison with those reported in the previous studies from which the data sets were extracted. The choice of descriptors had little influence on the model performance, with minor exceptions. The criteria used to filter bioactivity data, the activity threshold settings used to separate blockers from nonblockers, and the structural diversity of blockers in training data set were found to be the crucial indicators of model performance. Training sets based on a binary threshold of 1 µM/10 µM to separate blockers (IC50/ Ki ≤ 1 µM) from nonblockers (IC50/ Ki > 10 µM) provided superior performance in comparison with those defined using a single threshold (1 µM or 10 µM). A major limitation in using the public domain hERG activity data is the abundance of blockers in comparison with nonblockers at usual activity thresholds, since not many studies report the latter.

Development of a Focused Library of Triazole-Linked Privileged-Structure-Based Conjugates Leading to the Discovery of Novel Phenotypic Hits against Protozoan Parasitic Infections.

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9 µg mL-1 against T. cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.

Novel Kunitz-like Peptides Discovered in the Zoanthid Palythoa caribaeorum through Transcriptome Sequencing.

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.

N'-mono- and N, N'-diacyl derivatives of benzyl and arylhydrazines as contact insecticides against adult Anopheles gambiae.

New public health insecticides are urgently required to prevent the spread of vector-borne disease. With the goal of identifying new K+-channel-directed mosquitocides, analogs of the RH-5849 family of diacyl t-butylhydrazines were synthesized and tested for topical toxicity against adult Anopheles gambiae, the African vector of malaria. In total, 80N'-monoacyl and N, N'-diacyl derivatives of benzyl- and arylhydrazines were prepared. Three compounds (2bo, 2kb, 3ab) were identified that were more toxic than RH-5849 and RH-1266. The potencies of these compounds to block K+ currents in An. gambiae and human Kv2.1 channels were assessed to address their possible mechanism of mosquitocidal action. Selectivity for inhibition of An. gambiae Kv2.1 vs human Kv2.1 did not exceed 3-fold. Furthermore, no correlation was seen between the potency of insecticidal action and K+ channel blocking potency. These observations, combined with the minimal knockdown seen with 2bo near its LD50 value, suggests a mode of action outside of the nervous system.

3D-SDAR modeling of hERG potassium channel affinity: A case study in model design and toxicophore identification.

A dataset of 237 human Ether-à-go-go Related Gene (hERG) potassium channel inhibitors (180 of which were used for model building and validation, whereas 57 constituted the "true" external prediction set) collected from 22 literature sources was modeled by 3D-SDAR. To produce reliable and reproducible classification models for hERG blocking, the initial set of 180 chemicals was split into two subsets: a balanced modeling set consisting of 118 compounds and an unbalanced validation set comprised of 62 compounds. A PLS bagging-like algorithm written in Matlab was used to process the data and assign each compound to one of the two (hERG+ or hERG-) activity classes. The best predictive model evaluated on the basis of a fully randomized hold-out test set (comprising 20% of the modeling set) used 4 latent variables and a grid of 6ppm×6ppm×1Å in the C-C region, 6ppm×30ppm×1Å in the C-N region, and 30ppm×30ppm×1Å in the N-N region. An overall accuracy of 0.84 was obtained for both the hold-out test set and the validation set. Further, an external prediction set consisting of 57 drugs and drug derivatives was used to estimate the true predictive power of the reported 3D-SDAR model - a slight reduction of the overall accuracy down to 0.77 was observed. 3D-SDAR map of the most frequently occurring bins and their projection on the standard coordinate space of the chemical structures allowed identification of a three-center toxicophore composed of two aromatic rings and an amino group. A U test along the distance axis of the most frequently occurring 3D-SDAR bins was used to set the distance limits of the toxicophore. This toxicophore was found to be similar to an earlier reported phospholipidosis (PLD) toxicophore.

Glutamate-Mediated Blood-Brain Barrier Opening: Implications for Neuroprotection and Drug Delivery.

UNLABELLED: The blood-brain barrier is a highly selective anatomical and functional interface allowing a unique environment for neuro-glia networks. Blood-brain barrier dysfunction is common in most brain disorders and is associated with disease course and delayed complications. However, the mechanisms underlying blood-brain barrier opening are poorly understood. Here we demonstrate the role of the neurotransmitter glutamate in modulating early barrier permeability in vivo Using intravital microscopy, we show that recurrent seizures and the associated excessive glutamate release lead to increased vascular permeability in the rat cerebral cortex, through activation of NMDA receptors. NMDA receptor antagonists reduce barrier permeability in the peri-ischemic brain, whereas neuronal activation using high-intensity magnetic stimulation increases barrier permeability and facilitates drug delivery. Finally, we conducted a double-blind clinical trial in patients with malignant glial tumors, using contrast-enhanced magnetic resonance imaging to quantitatively assess blood-brain barrier permeability. We demonstrate the safety of stimulation that efficiently increased blood-brain barrier permeability in 10 of 15 patients with malignant glial tumors. We suggest a novel mechanism for the bidirectional modulation of brain vascular permeability toward increased drug delivery and prevention of delayed complications in brain disorders. SIGNIFICANCE STATEMENT: In this study, we reveal a new mechanism that governs blood-brain barrier (BBB) function in the rat cerebral cortex, and, by using the discovered mechanism, we demonstrate bidirectional control over brain endothelial permeability. Obviously, the clinical potential of manipulating BBB permeability for neuroprotection and drug delivery is immense, as we show in preclinical and proof-of-concept clinical studies. This study addresses an unmet need to induce transient BBB opening for drug delivery in patients with malignant brain tumors and effectively facilitate BBB closure in neurological disorders.

Synthesis and pharmacological evaluation of piperidine (piperazine)-substituted benzoxazole derivatives as multi-target antipsychotics.

The present study describes the optimization of a series of novel benzoxazole-piperidine (piperazine) derivatives combining high dopamine D2 and serotonin 5-HT1A, 5-HT2A receptor affinities. Of these derivatives, the pharmacological features of compound 29 exhibited high affinities for the DA D2, 5-HT1A and 5-HT2A receptors, but low affinities for the 5-HT2C and histamine H1 receptors and human ether-a-go-go-related gene (hERG) channels. Furthermore, compound 29 reduced apomorphine-induced climbing and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head twitching without observable catalepsy, even at the highest dose tested. Thus, compound 29 is a promising candidate as a multi-target antipsychotic treatment.

Modelling of drug-induced QT-interval prolongation: estimation approaches and translational opportunities.

Safety pharmacology studies are performed to assess whether compounds may provoke severe arrhythmias (e.g. Torsades de Pointes, TdP) and sudden death in man. Although there is strong evidence that drugs inducing TdP in man prolong the QT interval in vivo and block the human ether-a-go-go-related gene (hERG) ion channel in vitro, not all drugs affecting the QT interval or the hERG will induce TdP. Nevertheless, QT-interval prolongation and hERG blockade currently represent the most accepted early risk biomarkers to deselect drugs. An extensive pharmacokinetic/pharmacodynamic (PK/PD) analysis is developed to understand moxifloxacin's-induced effects on the QT interval by comparing the relationship between results of an in vitro patch-clamp model to in vivo models. The frequentist and the fully Bayesian estimation procedures were compared and provided similar performances when the best model selected in NONMEM is subsequently implemented in WinBUGS, which guarantees a straightforward calculation of the probability of QT-interval prolongation greater than 2.5 % (10 ms). The use of the percent threshold to account for the intrinsic differences between species and a new calculation of the probability curve are introduced. The concentration providing the 50 % probability indicates that dogs are more sensitive than humans to QT-interval prolongation. However, based on the drug effect, a clear distinction between species cannot be made. An operational PK/PD model of agonism was used to investigate the relationship between effects on the hERG and QT-interval prolongation in dogs. The proposed analysis contributes to establish a translational relationship that could potentially reduce the need for thorough QT studies.

The enhancement of cardiac toxicity by concomitant administration of Berberine and macrolides.

As is well-known, hERG plays an essential role in phase III repolarization of cardiac action potentials. Blocking of hERG channels can lead to LQTS. Inhibition of the metabolism of CYPs activities may elevate plasma levels, to further increase accumulation of drug on cardiac. The elevated serum levels may however elicit unexpected toxicities. Therefore, the inhibition tests of hERG and CYP are central to the preclinical studies because they may lead to severe cardiac toxicity. Berberine is widely used as an antibacterial agent and often combined with macrolides to treat gastropathy. Our objective was to assess cardiac toxicity during the combined use of Berberine with macrolides. (1) Azithromycin reduced hERG currents by accelerated channel inactivation. (2) The combination of Berberine with Azithromycin reduced hERG currents, producing an inhibitive effect stronger than use of a single drug alone, due to the high binding affinity for the onset of inactivation. (3) When cells were perfused concomitantly with Berberine and Clarithromycin, they showed a stronger inhibitive effect on hERG currents by decreasing the time constant for the onset of inactivation. (4) The combined administration of Berberine with Clarithromycin had a powerful inhibitive effect on CYP3A activities than use of a single drug alone. Collectively, these results demonstrated that concomitant use of Berberine with macrolides may require close monitoring because of potential drug toxicities, especially cardiac toxicity.

Role of mutation and pharmacologic block of human KCNH2 in vasculogenesis and fetal mortality: partial rescue by transforming growth factor-ß.

BACKGROUND: N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis. METHODS AND RESULTS: N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-ß treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-ß treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-ß treatment also rescued the dofetilide-induced phenotype toward normal. CONCLUSIONS: Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.

Targeting a mitochondrial potassium channel to fight cancer.

Although chemotherapy is able to cure many patients with malignancies, it still also often fails. Therefore, novel approaches and targets for chemotherapeutic treatment of malignancies are urgently required. Recent studies demonstrated the expression of several potassium channels in the inner mitochondrial membrane. Among them the voltage gated potassium channel Kv1.3 and the big-potassium (BK) channel were shown to directly function in cell death by serving as target for pro-apoptotic Bax and Bak proteins. Here, we discuss the role of mitochondrial potassium channel Kv1.3 (mitoKv1.3) in cell death and its potential function in treatment of solid tumors, leukemia and lymphoma. Bax and Bak inhibit mitoKv1.3 by directly binding into the pore of the channel, by a toxin-like mechanism. Inhibition of mitoKv1.3 results in an initial hyperpolarization of the inner mitochondrial membrane that triggers the production of reactive oxygen species (ROS). ROS in turn induce a release of cytochrome c from the cristae of the inner mitochondrial membrane and an activation of the permeability transition pore, resulting in opening of the intrinsic apoptotic cell death. Since mitoKv1.3 functions downstream of pro-apoptotic Bax and Bak, compounds that directly inhibit mitoKv1.3 may serve as a new class of drugs for treatment of tumors, even with an altered expression of either pro- or anti-apoptotic Bcl-2 protein family members. This was successfully proven by the in vivo treatment of mouse melanoma and ex vivo human chronic leukemia B cells with inhibitors of mitoKv1.3.

Up-regulation of miR-21 and miR-23a Contributes to As2 O3 -induced hERG Channel Deficiency.

Arsenic trioxide (As2O3) is used to treat acute pro-myelocytic leukaemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the hERG current via disturbing its trafficking to cellular membrane. Consistent with these findings, in this study, we reported that As2O3 inhibited hERG channel at both protein and mRNA levels and damaged hERG current but did not affect channel kinetics. Further, we demonstrated that As2O3 up-regulated miR-21 and miR-23a expression in hERG-HEK293 cells and neonatal cardiomyocytes. In addition, knock-down of miR-21 by its specific antisense molecules AMO-21 was able to rescue Sp1 and hERG inhibition caused by As2O3. Consistently, phosphorylation of NF-κB, the upstream regulatory factor of miR-21, was significantly up-regulated by As2O3 . This finding revealed that regulation of the NF-κB-miR-21-Sp1 signalling pathway is a novel mechanism for As2O3-induced hERG inhibition. Meanwhile, the expression of Hsp90 and hERG was rescued by transfection with AMO-23a. And the hERG channel inhibition induced by As2O3 was rescued after being transfected with AMO-23a, which may be a molecular mechanism for the role of As2O3 in hERG trafficking deficiency. In brief, our study revealed that miR-21 and miR-23a are involved in As2O3-induced hERG deficiency at transcriptional and transportational levels. This discovery may provide a novel mechanism of As2O3-induced hERG channel deficiency, and these miRNAs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

Emerging pattern mining to aid toxicological knowledge discovery.

Knowledge-based systems for toxicity prediction are typically based on rules, known as structural alerts, that describe relationships between structural features and different toxic effects. The identification of structural features associated with toxicological activity can be a time-consuming process and often requires significant input from domain experts. Here, we describe an emerging pattern mining method for the automated identification of activating structural features in toxicity data sets that is designed to help expedite the process of alert development. We apply the contrast pattern tree mining algorithm to generate a set of emerging patterns of structural fragment descriptors. Using the emerging patterns it is possible to form hierarchical clusters of compounds that are defined by the presence of common structural features and represent distinct chemical classes. The method has been tested on a large public in vitro mutagenicity data set and a public hERG channel inhibition data set and is shown to be effective at identifying common toxic features and recognizable classes of toxicants. We also describe how knowledge developers can use emerging patterns to improve the specificity and sensitivity of an existing expert system.

Experimental conversion of a defensin into a neurotoxin: implications for origin of toxic function.

Scorpion K(+) channel toxins and insect defensins share a conserved three-dimensional structure and related biological activities (defense against competitors or invasive microbes by disrupting their membrane functions), which provides an ideal system to study how functional evolution occurs in a conserved structural scaffold. Using an experimental approach, we show that the deletion of a small loop of a parasitoid venom defensin possessing the "scorpion toxin signature" (STS) can remove steric hindrance of peptide-channel interactions and result in a neurotoxin selectively inhibiting K(+) channels with high affinities. This insect defensin-derived toxin adopts a hallmark scorpion toxin fold with a common cysteine-stabilized α-helical and ß-sheet motif, as determined by nuclear magnetic resonance analysis. Mutations of two key residues located in STS completely diminish or significantly decrease the affinity of the toxin on the channels, demonstrating that this toxin binds to K(+) channels in the same manner as scorpion toxins. Taken together, these results provide new structural and functional evidence supporting the predictability of toxin evolution. The experimental strategy is the first employed to establish an evolutionary relationship of two distantly related protein families.

Photoacoustic and optical coherence tomography of epilepsy with high temporal and spatial resolution and dual optical contrasts.

Epilepsy mapping with high spatial and temporal resolution has great significance for both fundamental research on epileptic neurons and the clinical management of epilepsy. In this communication, we demonstrate for the first time in vivo epilepsy mapping with high spatial and temporal resolution and dual optical contrasts in an animal model. Through the variations of a depthresolved optical coherence tomography signal with optical scattering contrast, we observed that epileptic neuron activities modulated the optical refractive index of epileptic neurons and their surrounding tissue. Simultaneously, through neurovasculature coupling mechanisms and optical absorption contrast, we used photoacoustic signals to document the hemodynamic changes of the microvasculature surrounding the epileptic neurons. The epilepsy mapping results were confirmed by a simultaneously recorded electroencephalogram signal during epileptic seizure. Our new epilepsy mapping tool, with high temporal and spatial resolution and dual optical contrasts, may find many applications, such as drug development and epilepsy surgery.