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Aim The study aimed to profile the volatile phytocomposition of snow mountain garlic (SMG) compared to normal garlic and investigate the anti-Candida efficacy against clinically relevant multi-drug resistant isolates of Candida species. Methods and Results Herein, SMG has shown significantly superior fungicidal power at 2x-MIC dose against C. albicans and C. glabrata in killing kinetic evaluation unlike the fungistatic effect of normal garlic. GC–MS headspace-based profiling of SMG showed 5 unique volatile compounds and a 5-fold higher content of saponins than normal garlic. In an in-silico analysis, cholesta-4,6-dien-3-ol,(3-beta) was uniquely identified in SMG as a potential inhibitor with high binding affinity to the active site of exo-1,3-betaglucan synthase, an established anti-candida drug target crucial for the biofilm matrix formation, thus suggesting a plausible anti-Candida mechanism. Conclusion The in-vitro and in-silico studies have demonstrated the Candida-cidal and anti-biofilm activities of SMG, distinguishing it from the Candida-static efficacy of normal garlic. Significance and Impact of the study This is the first report that identifies several phytochemical signatures of SMG along with a potential anti-Candida compound, that is cholesta-4,6-dien-3-ol,(3-beta)-, which appears worthy of detailed studies in the future to explore the utility of SMG as a fungal phytotherapy agent, especially against drug-resistant Candida sp.

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In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8–10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10–900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.

SCN8Aepileptic encephalopathy is a devastating epilepsy syndrome caused by mutantSCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model ofSCN8Aepileptic encephalopathy, we found that selective expression of the R1872WSCN8Amutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark ofSCN8Amutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only inSCN8Aepileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8Aepileptic encephalopathy is a devastating neurological disorder that results fromde novomutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation inSCN8Aepileptic encephalopathy has not been determined. We show that mice expressing a human-derivedSCN8Avariant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show thatSCN8Amutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only inSCN8Aepileptic encephalopathy, but for epilepsy broadly.

Voltage-gated sodium channels (VGSCs) are foundational to excitable cell function: Their coordinated passage of sodium ions into the cell is critical for the generation and propagation of action potentials throughout the nervous system. The classical paradigm of action potential physiology states that sodium passes through the membrane only transiently (1-2 milliseconds), before the channels inactivate and cease to conduct sodium ions. However, in reality, a small fraction of the total sodium current (1%-2%) remains at steady state despite prolonged depolarization. While this persistent sodium current (INaP) contributes to normal physiological functioning of neurons, accumulating evidence indicates a particularly pathogenic role for an elevated INaP in epilepsy (reviewed previously 1 ). Due to significant advances over the past decade of epilepsy research concerning the importance of INaP in sodium channelopathies, this review seeks to summarize recent evidence and highlight promising novel anti-seizure medication strategies through preferentially targeting INaP.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8–17% of patients with epilepsy. Although the mechanisms of SUDEP are unknown, one proposed mechanism is abnormal control of the heart by the autonomic nervous system (ANS). Our objective was to determine whether the broad changes in ictal heart rate experienced by mouse models of SUDEP are (1) due to the ANS and (2) contribute to seizure-induced death. Seizures were induced by electrical stimulation of the hippocampus of a mouse carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; “D/+ mice”). Using standard autonomic pharmacology, the relative roles of the parasympathetic and sympathetic nervous systems on heart rate changes associated with seizures were determined. All induced seizures had pronounced ictal bradycardia and postictal tachycardia. Seizure susceptibility or severity were unchanged by the pharmacological agents. Administration of Atropine, a muscarinic antagonist, eliminated ictal bradycardia, while carbachol, a muscarinic agonist, had no effect on ictal bradycardia, but reduced postictal tachycardia. Sotalol, an adrenergic β-receptor antagonist, had no effect on ictal bradycardia, but did suppress postictal tachycardia. Isoproterenol, a β-receptor agonist, had no effect on either ictal bradycardia or postictal tachycardia. Administration of the α1-receptor antagonist prazosin increases the incidence of seizure-induced death in D/+ mice. Although postictal heart rate was lower for these fatal seizures in the presence of prazosin, rates were not as low as that recorded for carbachol treated mice, which all survived. Both ictal bradycardia and postictal tachycardia are manifestations of the ANS. Bradycardia is mediated by a maximal activation of the parasympathetic arm of the ANS, and tachycardia is mediated by parasympathetic inactivation and sympathetic activation. While the changes in heart rate during seizures are profound, suppression of postictal heart rate did not increase seizure mortality.

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Snow Mountain Garlic (SMG) (Allium ampeloprasum L.) is a wild trans-Himalayan member of the genus Allium, valued for its anti-inflammatory and anti-arthritic properties in the mountain folk medicinal system (Sowa-Rigpa). Despite its age-old medicinal usage by traditional therapists and the native population for various ailments including rheumatism, there is no scientific validation of its phyto-pharmaceutical merits.The present pre-clinical study compared the in-vivo anti-arthritic effects of SMG with reported efficacy doses of normal garlic (Allium sativum L.) extract and dexamethasone in a complete Freund's adjuvant (CFA)-induced arthritis rat model.The female Wistar rats were immunized by the subplannter injection of CFA into the right hind footpad. Aqueous extracts of SMG and normal garlic were administered orally at a dose of 250 mg/kg and 500 mg/kg for 28 days. Dexamethasone was used as positive control drug. Behavioral parameters including paw markers, arthritis index, joint stiffness, body weight change, etc. were measured. Also, the changes in histopathological indices, hematological profile, inflammatory mediators, and serum cytokines level was determined.Treatment of rats with SMG extracts significantly (p 0.001) prevented the reduction in body weight and hematological changes as well as ameliorated clinical symptoms such as arthritic index, joint stiffness, arthritis score, edema, hyperalgesia, and histopathological indices. This was associated with a significant reduction in the serum levels of RF, CRP, anti-CCP, and proinflammatory cytokines exhibiting strong anti-arthritic potential. SMG extracts could also significantly down regulate the NF-κB, COX-2, and iNOS expression in the ankle joint tissues.The present study is the first attempt to validate the phyto-pharmaceutical efficacy of this folk garlic variety from the trans-Himalayan region. Overall, SMG extract showed remarkable preventive anti-inflammatory and anti-arthritic activities which were closely comparable to therapeutic effects of dexamethasone and at par or even better than normal garlic w.r.t. several study parameters.

Variant-Specific Changes in Persistent or Resurgent Sodium Current in SCN8A-Related Epilepsy Patient-Derived Neurons Tidball AM, Lopez-Santiago LF, Yuan Y, et al. Brain. 2020;143(10):3025-3040. doi:10.1093/brain/awaa247Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from 3 patients with missense variants in SCN8A: p.R1872>L (patient 1); p.V1592>L (patient 2); and p.N1759>S (patient 3). Using small-molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all 3 patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while patient 3 had increased resurgent current compared to controls. Neurons from all 3 patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (patient 1) and the patient with increased resurgent current (patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small-molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient-induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modeling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca(2+) channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca(2+) channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca(2+) channel antagonist, TTA-P2 (1 μM), was applied to determine the role of T-type Ca(2+) channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca(2+) channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 μM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP’s in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Ca(v)3.1 mRNA compared to controls. Ca(v)3.2 or Ca(v)3.3 levels were unchanged. These findings support a role for T-type Ca(2+) channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Ca(v)3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy typically caused by loss-of-function de novo mutations in the SCN1A gene which encodes the voltage-gated sodium channel isoform NaV1.1. Decreased NaV1.1 expression results in impaired excitability of inhibitory interneurons and seizure onset. To date, there are no clinically available treatments for DS that directly address the core mechanism of disease; reduced NaV1.1 expression levels in interneurons. Recently, Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A by the antisense oligonucleotide (ASO) STK-001, was shown to increase Scn1a mRNA levels, increase NaV1.1 protein expression, reduce seizures, and improve survival in the Scn1a+/- mouse model of DS. However, it remains unknown whether STK-001 treatment rescues the reduced intrinsic excitability of parvalbumin-positive (PV) inhibitory interneurons associated with DS. In this study, we demonstrate that STK-001 treatment reduces seizures, prolongs survival, and rescues PV interneuron excitability in Scn1a+/- mice to levels observed in WT littermates. Together, these results support the notion that TANGO-mediated augmentation of NaV1.1 levels directly targets and rescues one of the core disease mechanisms of DS.

DNA methylation is an important regulatory mechanism in the control of neuronal function. Both during development and following exposure to salient stimuli, plasticity in the methylation of cytosine residues leads to a change in neuron excitability that subsequently sculpts animal behavior. However, although the response of DNA methyltransferase enzymes in adult neurons to stimuli such as drugs of abuse have been described, less is known about how these enzymes regulate methylation at specific loci to change the drive to ingest natural rewards. Specifically, we do not understand how changes in methylation within important brain areas known to regulate palatable food intake can affect ingestion, while a detailed investigation of the neurophysiological and genomic effects of perturbing methyltransferase function has not been pursued. By deleting DNA methyltransferase 1 and 3a in the mouse prefrontal cortex, we observed the requirement for these enzymes in the regulation of nutrient rich food consumption in the absence of any effect on the intake of low fat and low sugar chow. We also determined that the deletion profoundly affected neuron excitability within pyramidal cells resident in superficial layers II/III of the cortex but had little effect in deep layer V neurons. Finally, reduced representation bisulfite sequencing revealed both hypo and hypermethylation in response to methyltransferase deletion, an effect that was observed in binding sites for retinoic acid receptor beta (RARβ) located within regulatory regions of genes known to affect neuronal function. Together, our data suggest that alterations in the actions of RARβ could shift neuronal activity to reduce palatable food intake.

Objective Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. Methods We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. Results The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. Interpretation We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.

SCN8Aepileptic encephalopathy is a devastating epilepsy syndrome caused by mutantSCN8Awhich encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. We found that selective expression of the R1872W mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of wild-type SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark ofSCN8Amutations, were observed and were found to contribute directly to aberrant SST physiology in computational and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only inSCN8Aencephalopathy, but epilepsy in general.

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.

We present here the correlation of picomolar affinities between surface-plasmon and electrochemical immunoassays for the binding of serum glutamic acid decarboxylase 65 autoantibody (GADA), a biomarker of type 1 diabetes (T1D), to its antigen GAD-65. Carboxylated (∼5.0%)-graphene-modified immunoassembly on a gold surface-plasmon chip or on an electrochemical array provided significantly larger binding affinity, higher sensitivity, and lower detection limits than a self-assembled monolayer surface of mercaptopropionic acid (MPA). Estimation of the relative surface -COOH groups by covalent tagging of an electroactive aminoferrocene showed that the graphenyl surface displayed a greater number of -COOH groups (9-fold) than the MPA surface. X-ray-photoelectron-spectroscopy analysis showed more C-O and C═O functionalities on the graphene-COOH surface than on the MPA surface. The graphene-COOH coating on gold exhibited ∼5.5-fold enhancement of plasmon signals compared with a similar coating on a plain glass surface. In summary, this article provides a quantitative comparison of carboxylated graphene with a mercapto-monolayer immunoassembly. Additionally, we propose that the binding-constant value can be useful as a quality-control checkpoint for reproducible and reliable production of large-scale biosensors for clinical bioassays.

The putative cache (Ca2+channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENTThis is the first study to characterize the Ca2+channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

With the overwhelmingly rapid advancement in the field of nanotechnology, the engineered nanomaterials (ENMs) have been extensively used in various areas of the plant system, including quality improvement, growth and nutritional value enhancement, gene preservation etc. There are several recent reports on the ENMs' influence on growth enhancements, growth inhibition as well as certain toxic impacts on plant. However, translocation, growth responses and stress modulation mechanisms of ENMs in the plant systems call for better and in-depth understanding. Herein, we are presenting a comprehensive and critical account of different types of ENMs, their applications and their positive, negative and null impacts on physiological and molecular aspects of plant growth, development and stress responses. Recent reports revealed mixed effects on plants, ranging from enhanced crop yield, epi/genetic alterations, and phytotoxicity, resulting from the ENMs' exposure. Creditable research in recent years has revealed that the effects of ENMs on plants are species specific and are variable among plant species. ENM exposures are reported to trigger free radical formation, responsive scavenging, and antioxidant armories in the exposed plants. The ENMs are also reported to induce aberrant expressions of microRNAs, the key post-transcriptional regulators of plant growth, development and stress-responses of plants. However, these modulations, if judiciously done, may lead to improved plant growth and yield. A better understanding of the interactions between ENMs and plant responses, including their uptake transport, internalization, and activity, could revolutionize crop production through increased disease resistance, nutrient utilization, and crop yield. Therefore, in this review, we are presenting a critical account of the different selected ENMs, their uptake by the plants, their positive/negative impacts on plant growth and development, along with the resultant ENM-responsive post-transcriptional modifications, especially, aberrant miRNA expressions. In addition, underlying mechanisms of various ENM-plant cell interactions have been discussed.

Variants in the neuronal sodium channel gene SCN8A have been implicated in several neurological disorders. Early infantile epileptic encephalopathy type 13 results from de novo gain-of-function mutations that alter the biophysical properties of the channel. Complete loss-of-function variants of SCN8A have been identified in cases of isolated intellectual disability. We now report a novel heterozygous SCN8A variant, p.Pro1719Arg, in a small pedigree with five family members affected with autosomal dominant upper limb isolated myoclonus without seizures or cognitive impairment. Functional analysis of the p.Pro1719Arg variant in transfected neuron-derived cells demonstrated greatly reduced Nav 1.6 channel activity without altered gating properties. Hypomorphic alleles of Scn8a in the mouse are known to result in similar movement disorders. This study expands the phenotypic and functional spectrum of SCN8A variants to include inherited nonepileptic isolated myoclonus. SCN8A can be considered as a candidate gene for isolated movement disorders without seizures.

Objective: Monoallelic de novo gain-of-function variants in the voltage-gated sodium channel SCN8A are one of the recurrent causes of severe developmental and epileptic encephalopathy (DEE). In addition, a small number of de novo or inherited monoallelic loss-of-function variants have been found in patients with intellectual disability, autism spectrum disorder, or movement disorders. Inherited monoallelic variants causing either gain or loss-of-function are also associated with less severe conditions such as benign familial infantile seizures and isolated movement disorders. In all three categories, the affected individuals are heterozygous for a SCN8A variant in combination with a wild-type allele. In the present study, we describe two unusual families with severely affected individuals who inherited biallelic variants of SCN8A. Methods: We identified two families with biallelic SCN8A variants by diagnostic gene panel sequencing. Functional analysis of the variants was performed using voltage clamp recordings from transfected ND7/23 cells. Results: We identified three probands from two unrelated families with DEE due to biallelic SCN8A variants. Each parent of an affected individual carried a single heterozygous SCN8A variant and exhibited mild cognitive impairment without seizures. In both families, functional analysis demonstrated segregation of one allele with complete loss-of-function, and one allele with altered biophysical properties consistent with partial loss-of-function. Significance: These studies demonstrate that SCN8A DEE may, in rare cases, result from inheritance of two variants, both of which exhibit reduced channel activity. In these families, heterozygosity for the dominant variants results in less severe disease than biallelic inheritance of two variant alleles. The clinical consequences of variants with partial and complete loss of SCN8A function are variable and likely to be influenced by genetic background.

SummaryObjective SCN8A encephalopathy (early infantile epileptic encephalopathy; EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0 and 18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard antiepileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. Methods The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 μm) on mutant and wild-type (WT) channels were compared. Results Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, whereas inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 μm) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use-dependent block of I1327V mutant channels relative to WT. Significance SCN8A – I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A.

On April 21, 2015, the first SCN8A Encephalopathy Research Group convened in Washington, DC, to assess current research into clinical and pathogenic features of the disorder and prepare an agenda for future research collaborations. The group comprised clinical and basic scientists and representatives of patient advocacy groups. SCN8A encephalopathy is a rare disorder caused by de novo missense mutations of the sodium channel gene SCN8A, which encodes the neuronal sodium channel Nav 1.6. Since the initial description in 2012, approximately 140 affected individuals have been reported in publications or by SCN8A family groups. As a result, an understanding of the severe impact of SCN8A mutations is beginning to emerge. Defining a genetic epilepsy syndrome goes beyond identification of molecular etiology. Topics discussed at this meeting included (1) comparison between mutations of SCN8A and the SCN1A mutations in Dravet syndrome, (2) biophysical properties of the Nav 1.6 channel, (3) electrophysiologic effects of patient mutations on channel properties, (4) cell and animal models of SCN8A encephalopathy, (5) drug screening strategies, (6) the phenotypic spectrum of SCN8A encephalopathy, and (7) efforts to develop a bioregistry. A panel discussion of gaps in bioregistry, biobanking, and clinical outcomes data was followed by a planning session for improved integration of clinical and basic science research. Although SCN8A encephalopathy was identified only recently, there has been rapid progress in functional analysis and phenotypic classification. The focus is now shifting from identification of the underlying molecular cause to the development of strategies for drug screening and prioritized patient care.

SCN8A epileptic encephalopathy is a severe genetic epilepsy syndrome caused by de novo gain-of-function mutations of SCN8A encoding the voltage-gated sodium (Na) channel (VGSC) Na(V)1.6. Therapeutic management is difficult in many patients, leading to uncontrolled seizures and risk of sudden unexpected death in epilepsy (SUDEP). There is a need to develop novel anticonvulsants that can specifically target aberrant Na channel activity associated with SCN8A gain-of-function mutations. In this study, we investigate the effects of Prax330, a novel Na channel inhibitor, on the biophysical properties of WT Na(V)1.6 and the patient mutation p.Asn1768Asp (N1768D) in ND7/23 cells. The effects of Prax330 on persistent (I(NaP)) and resurgent (I(NaR)) Na currents and neuronal excitability in subiculum neurons from a knock-in mouse model of the Scn8a-N1768D mutation (Scn8a(D/+)) were also examined. In ND7/23 cells, Prax330 reduced I(NaP) currents recorded from cells expressing Scn8a-N1768D and hyperpolarized steady-state inactivation curves. Recordings from brain slices demonstrated elevated I(NaP) and I(NaR) in subiculum neurons from Scn8a(D/+) mutant mice and abnormally large action potential (AP) burst-firing events in a subset of neurons. Prax330 (1 μM) reduced both I(NaP) and I(NaR) and suppressed AP bursts, with smaller effect on AP waveforms that had similar morphology to WT neurons. Prax330 (1 μM) also reduced synaptically-evoked APs in Scn8a(D/+) subiculum neurons but not in WT neurons. Our results highlight the efficacy of targeting I(NaP) and I(NaR) and inactivation parameters in controlling subiculum excitability and suggest Prax330 as a promising novel therapy for SCN8A epileptic encephalopathy.

Optogenetic and chemogenetic actuators are critical for deconstructing the neural correlates of behavior. However, these tools have several limitations, including invasive modes of stimulation or slow on/off kinetics. We have overcome these disadvantages by synthesizing a single-component, magnetically sensitive actuator, "Magneto," comprising the cation channel TRPV4 fused to the paramagnetic protein ferritin. We validated noninvasive magnetic control over neuronal activity by demonstrating remote stimulation of cells using in vitro calcium imaging assays, electrophysiological recordings in brain slices, in vivo electrophysiological recordings in the brains of freely moving mice, and behavioral outputs in zebrafish and mice. As proof of concept, we used Magneto to delineate a causal role of striatal dopamine receptor 1 neurons in mediating reward behavior in mice. Together our results present Magneto as an actuator capable of remotely controlling circuits associated with complex animal behaviors.

Contains fulltext : 168024.pdf (Publisher’s version ) (Open Access) OBJECTIVE: The early infantile epileptic encephalopathy type 13 (EIEE13, OMIM #614558) results from de novo missense mutations of SCN8A encoding the voltage-gated sodium channel Nav1.6. More than 20% of patients have recurrent mutations in residues Arg1617 or Arg1872. Our goal was to determine the functional effects of these mutations on channel properties. METHODS: Clinical exome sequencing was carried out on patients with early-onset seizures, developmental delay, and cognitive impairment. Two mutations identified here, p.Arg1872Leu and p.Arg1872Gln, and two previously identified mutations, p.Arg1872Trp and p.Arg1617Gln, were introduced into Nav1.6 cDNA, and effects on electrophysiological properties were characterized in transfected ND7/23 cells. Interactions with FGF14, G-protein subunit Gbetagamma, and sodium channel subunit beta1 were assessed by coimmunoprecipitation. RESULTS: We identified two patients with the novel mutation p.Arg1872Leu and one patient with the recurrent mutation p.Arg1872Gln. The three mutations of Arg1872 and the mutation of Arg1617 all impaired the sodium channel transition from open state to inactivated state, resulting in channel hyperactivity. Other observed abnormalities contributing to elevated channel activity were increased persistent current, increased peak current density, hyperpolarizing shift in voltage dependence of activation, and depolarizing shift in steady-state inactivation. Protein interactions were not affected. INTERPRETATION: Recurrent mutations at Arg1617 and Arg1872 lead to elevated Nav1.6 channel activity by impairing channel inactivation. Channel hyperactivity is the major pathogenic mechanism for gain-of-function mutations of SCN8A. EIEE13 differs mechanistically from Dravet syndrome, which is caused by loss-of-function mutations of SCN1A. This distinction has important consequences for selection of antiepileptic drugs and the development of gene- and mutation-specific treatments.

Ischemic stroke is a leading cause of morbidity and mortality in the US; however, there currently exists only one effective acute pharmacological therapeutic intervention. Purinergic signaling has been shown to regulate vascular function and pathological processes, including inflammation and arterial myogenic reactivity, and plays a role in ischemic stroke outcome. Purinergic signaling requires extracellular purines; however, the mechanism of purine release from cells is unclear. Pannexin1 (Panx1) channels are potentially novel purine release channels expressed throughout the vascular tree that couples regulated purine release with purinergic signaling. Therefore, we examined the role of smooth muscle and endothelial cell Panx1, using conditional cell type-specific transgenic mice, in cerebral ischemia/reperfusion injury outcomes. Deletion of endothelial cell Panx1, but not smooth muscle cell Panx1, significantly reduced cerebral infarct volume after ischemia/reperfusion. Infiltration of leukocytes into brain tissue and development of cerebral myogenic tone were both significantly reduced when mice lacked endothelial Panx1. Panx1 regulation of myogenic tone was unique to the cerebral circulation, as mesenteric myogenic reactivity and blood pressure were independent of endothelial Panx1. Overall, deletion of endothelial Panx1 mitigated cerebral ischemic injury by reducing inflammation and myogenic tone development, indicating that endothelial Panx1 is a possible novel target for therapeutic intervention of ischemic stroke.

Variants in the neuronal sodium channel gene SCN8A have been implicated in several neurological disorders. Early infantile epileptic encephalopathy type 13 results from de novo gain‐of‐function mutations that alter the biophysical properties of the channel. Complete loss‐of‐function variants of SCN8A have been identified in cases of isolated intellectual disability. We now report a novel heterozygous SCN8A variant, p.Pro1719Arg, in a small pedigree with five family members affected with autosomal dominant upper limb isolated myoclonus without seizures or cognitive impairment. Functional analysis of the p.Pro1719Arg variant in transfected neuron‐derived cells demonstrated greatly reduced Nav1.6 channel activity without altered gating properties. Hypomorphic alleles of Scn8a in the mouse are known to result in similar movement disorders. This study expands the phenotypic and functional spectrum of SCN8A variants to include inherited nonepileptic isolated myoclonus. SCN8A can be considered as a candidate gene for isolated movement disorders without seizures.

We present a highly sensitive and selective electrochemical sandwich immunosensor (the analyte is “sandwiched” between two antibodies) based on chitosan (CH) modified nickel oxide (NiO) nanoparticles for the detection of Vibrio cholerae (Vc). The primary monoclonal antibodies specific to Vibrio cholerae (Ab-Vc) and bovine serum albumin (BSA) were co-immobilized on a CH–NiO surface deposited onto an indium tin oxide (ITO) coated glass electrode. The specific binding of Ab-Vc towards Vc was confirmed by interaction of secondary antibodies conjugated with protein [horse radish peroxidase (HRP)], with varying concentrations of hydrogen peroxide (H2O2), via electrochemical as well as optical techniques. The CH–NiO/ITO and Ab-Vc/CH–NiO/ITO electrodes have been characterized using X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, high-resolution transmission electron microscopy and electrochemical techniques. This immunoelectrode (BSA/Ab-Vc/CH–NiO/ITO) exhibits a detection range of 20–700 ng mL−1 with a sensitivity of 0.644 μA ng mL−1 cm−2 and a low detection range of 0.108 ng mL−1 to Vc concentration. Besides this, the electrochemical response of the sandwich immunosensor (HRP–Ab-Vc/Vc/BSA/Ab-Vc/CH–NiO/ITO) towards H2O2 concentration is found to be linear in the range of 10–50 mM with excellent sensitivity (2.95 mA mM−1 cm−2).

Temporal lobe epilepsy (TLE) is a common form of adult epilepsy involving the limbic structures of the temporal lobe. Subiculum neurons act to provide a major output from the hippocampus and consist of a large population of endogenously bursting excitatory neurons. In TLE, subiculum neurons are largely spared, become hyperexcitable and show spontaneous epileptiform activity. The basis for this hyperexcitability is unclear, but is likely to involve alterations in the expression levels and function of various ion channels. In this study, we sought to determine the importance of sodium channel currents in facilitating neuronal hyperexcitability of subiculum neurons in the continuous hippocampal stimulation (CHS) rat model of TLE. Subiculum neurons from TLE rats were hyperexcitable, firing a higher frequency of action potentials after somatic current injection and action potential (AP) bursts after synaptic stimulation. Voltage clamp recordings revealed increases in resurgent (INaR) and persistent (INaP) sodium channel currents and pro-excitatory shifts in sodium channel activation and inactivation parameters that would facilitate increases in AP generation. Attenuation of INaR and INaP currents with 4,9-anhydro-tetrodotoxin (4,9-ah TTX; 100 nM), a toxin with increased potency against Nav1.6 channels, suppressed neuronal firing frequency and inhibited AP bursting induced by synaptic stimulation in TLE neurons. These findings support an important role of sodium channels, particularly Nav1.6, in facilitating subiculum neuron hyperexcitability in TLE and provide further support for the importance of INaR and INaP currents in establishing epileptiform activity of subiculum neurons.

SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Nav1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8aD/+), homozygous (Scn8aD/D)), and WT (Scn8a+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Nav1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Nav1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.

We present analyses of new optical photometric observations of three W UMa-type contact binaries FZ Ori, V407 Peg and LP UMa. Results from the first polarimetric observations of the FZ Ori and V407 Peg are also presented. The periods of FZ Ori, V407 Peg and LP UMa are derived to be 0.399986, 0.636884 and 0.309898 d, respectively. The O−C analyses indicate that the orbital periods of FZ Ori and LP UMa have increased with the rate of 2.28×10−8 and 1.25×10−6 d yr−1, respectively and which is explained by transfer of mass between the components. In addition to the secularly increasing rate of orbital period, it was found that the period of FZ Ori has varied in sinusoidal way with oscillation period of ∼30.1 yr. The period of oscillations are most likely to be explained by the light-time effect due to the presence of a tertiary companion. Small asymmetries have been seen around the primary and secondary maxima of light curves of all three systems, which is probably due to the presence of cool/hot spots on the components. The light curves of all three systems are analysed by using Wilson-Devinney code (WD) and the fundamental parameters of these systems have been derived. The present analyses show that FZ Ori is a W-subtype, and V407 Peg and LP UMa are A-subtype of the W UMa-type contact binary systems. The polarimetric observations in B, V, R and I bands, yield average values of polarization to be 0.26±0.03, 0.22±0.02, 0.22±0.03 and 0.22±0.05 per cent for FZ Ori and 0.21±0.02, 0.29±0.03, 0.31±0.01 and 0.31±0.04 per cent for V407 Peg, respectively.

During epileptogenesis a series of molecular and cellular events occur, culminating in an increase in neuronal excitability, leading to seizure initiation. The entorhinal cortex has been implicated in the generation of epileptic seizures in both humans and animal models of temporal lobe epilepsy. This hyperexcitability is due, in part, to proexcitatory changes in ion channel activity. Sodium channels play an important role in controlling neuronal excitability, and alterations in their activity could facilitate seizure initiation. We sought to investigate whether medial entorhinal cortex (mEC) layer II neurons become hyperexcitable and display proexcitatory behavior of Na channels during epileptogenesis. Experiments were conducted 7 days after electrical induction of status epilepticus (SE), a time point during the latent period of epileptogenesis and before the onset of seizures. mEC layer II stellate neurons from post-SE animals were hyperexcitable, eliciting action potentials at higher frequencies compared with control neurons. Na channel currents recorded from post-SE neurons revealed increases in Na current amplitudes, particularly persistent and resurgent currents, as well as depolarized shifts in inactivation parameters. Immunocytochemical studies revealed increases in voltage-gated Na (Nav) 1.6 isoform levels. The toxin 4,9-anhydro-tetrodotoxin, which has greater selectivity for Nav1.6 over other Na channel isoforms, suppressed neuronal hyperexcitability, reduced macroscopic Na currents, persistent and resurgent Na current densities, and abolished depolarized shifts in inactivation parameters in post-SE neurons. These studies support a potential role for Nav1.6 in facilitating the hyperexcitability of mEC layer II neurons during epileptogenesis.

Objective: To determine the functional effect of SCN8A missense mutations in 2 children with intellectual disability and developmental delay but no seizures. Methods: Genomic DNA was analyzed by next-generation sequencing. SCN8A variants were introduced into the Nav1.6 complementary DNA by site-directed mutagenesis. Channel activity was measured electrophysiologically in transfected ND7/23 cells. The stability of the mutant channels was assessed by Western blot. Results: Both children were heterozygous for novel missense variants that altered conserved residues in transmembrane segments of Nav1.6, p.Gly964Arg in D2S6 and p.Glu1218Lys in D3S1. Both altered amino acids are evolutionarily conserved in vertebrate and invertebrate channels and are predicted to be deleterious. Neither was observed in the general population. Both variants completely prevented the generation of sodium currents in transfected cells. The abundance of Nav1.6 protein was reduced by the Glu1218Lys substitution. Conclusions: Haploinsufficiency of SCN8A is associated with cognitive impairment. These observations extend the phenotypic spectrum of SCN8A mutations beyond their established role in epileptic encephalopathy (OMIM#614558) and other seizure disorders. SCN8A should be considered as a candidate gene for intellectual disability, regardless of seizure status.

Ion channels are protein molecules that span across the cell membrane allowing the passage of ions from one side of the membrane to the other. They have an aqueous pore, which becomes accessible to ions after a conformational change in the protein structure that causes the ion channel to open. Ion channels are selective meaning that they only allow certain ions to pass through them, and they play critical roles in controlling neuronal excitability. Ion channels are divided into those that are opened by changes in membrane potential, voltage-gated ion channels, and ion channels that are opened by the binding of a ligand, such as a hormone or a neurotransmitter, ligand-gated ion channels. In this chapter we introduce both voltage-gated and ligand-gated ion channels that are abundantly expressed within the central nervous system and the peripheral nervous system. Here, we discuss their roles in neurological disorders and introduce some common clinically used drugs that target ion channels as a means of treatment.