Disordered but effective: short linear motifs as gene therapy targets for hyperexcitability disorders.

Multiple approaches have targeted voltage-gated sodium (Nav) channels for analgesia. In this issue of the JCI, Shin et al. identified a peptide aptamer, NaViPA1, carrying a short polybasic motif flanked by serine residues in a structurally disordered region of loop 1 in tetrodotoxin-sensitive (TTX-S) but not tetrodotoxin-resistant (TTX-R) channels. NaViPA1h inhibited TTX-S NaV channels and attenuated excitability of sensory neurons. Delivery of NaViPA1 in vivo via adeno-associated virions restricted its expression to peripheral sensory neurons and induced analgesia in rats. Targeting of short linear motifs in this manner may provide a gene therapy modality, with minimal side effects due to its peripherally-restricted biodistribution, which opens up a therapeutic strategy for hyperexcitability disorders, including pain.

Lidocaine Inhibits Rat Prostate Cancer Cell Invasiveness and Voltage-Gated Sodium Channel Expression in Plasma Membrane.

There is increasing evidence, mostly from breast cancer, that use of local anaesthetics during surgery can inhibit disease recurrence by suppressing the motility of the cancer cells dependent on inherent voltage-gated sodium channels (VGSCs). Here, the possibility that lidocaine could affect cellular behaviours associated with metastasis was tested using the Dunning cell model of rat prostate cancer. Mostly, the strongly metastatic (VGSC-expressing) Mat-LyLu cells were used under both normoxic and hypoxic conditions. The weakly metastatic AT-2 cells served for comparison in some experiments. Lidocaine (1-500 µM) had no effect on cell viability or growth but suppressed Matrigel invasion dose dependently in both normoxia and hypoxia. Used as a control, tetrodotoxin produced similar effects. Exposure to hypoxia increased Nav1.7 mRNA expression but VGSCα protein level in plasma membrane was reduced. Lidocaine under both normoxia and hypoxia had no effect on Nav1.7 mRNA expression. VGSCα protein expression was suppressed by lidocaine under normoxia but no effect was seen in hypoxia. It is concluded that lidocaine can suppress prostate cancer invasiveness without effecting cellular growth or viability. Extended to the clinic, the results would suggest that use of lidocaine, and possibly other local anaesthetics, during surgery can suppress any tendency for post-operative progression of prostate cancer.

Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Detection of Voltage-Gated Sodium Channel (VGSC) Mutation in Deltamethrin-Resistant Blattella germanica L. from Indonesia.

<b>Background and Objective:</b> German cockroach (<i>Blattella germanica</i> L.) is one of the most common residential pests in Indonesia. Controlling the population face obstacles due to insecticide resistance, especially to deltamethrin. This research investigated the resistance status and the possibility of a Voltage-Gated Sodium Channel (VGSC) knockdown resistance mutation (L1014F) in two field strains of German cockroaches collected from two cities in Indonesia (Bukittinggi, named RMKN-BKT and Bandung, named KRSA-BDG) with VCRU-WHO as the standard strain. <b>Materials and Methods:</b> This study started with a bioassay test to determine the lethal dose of 50% (LD₅₀) for each strain and followed by a molecular test for mutation detection. <b>Results:</b> The results showed that the RMKN-BKT and KRSA-BDG strains were highly resistant to deltamethrin with RR50 values of 80,090 times and 73,272 times, respectively. Only the RMKN-BKT strain was shown to carry L1014F kdr mutation which lead to an amino acid replacement from leucine (TTG) to phenylalanine (TTC). Two silent mutations were also found in both field strains at codons 983 (TGC/cysteine→TGT/cysteine) and 984 (GGG/ glycine→GGA/glycine) which were suggested as polymorphism phenomena. The absence of the L1014F mutation in the Bandung strain does not exclude the possibility of the presence of the VGSC mutation at other points. <b>Conclusion:</b> It requires subsequent investigation in mutation detection at other points and the possible presence of other resistance mechanisms to get a precise solution in the population control. Bioinsecticides may stand as a breakthrough so that the strategy will no longer focus on insecticides.

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar.

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.

Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy.

Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.

Genomic analyses reveal SCN7A is associated with the prognosis of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has a poor prognosis and occurs with high frequency in China. In particular, Fujian is one of the high-incidence areas of ESCC in China and the somatic mutation profile of ESCC there remains unclear. PATIENTS AND METHODS: Whole-exome sequencing (WES) was performed in 49 matched ESCC tumor-normal specimens to examine the somatic mutation profiles. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between mutational profile and survival were derived from Cox regression model. RESULTS: We constructed a preliminary somatic mutation profiling of ESCC in Fujian. Exome sequencing data showed that the main base substitutions in ESCC were C > T transformation (close to 50%), C > A and T > C transversion. The study identified 21 significantly mutated genes, including 8 driver genes and 11 predicted driver genes. Among the 19 driver or predicted driver genes, 9 are novel (OBSCN, PKHD1L1, FSIP2, HRNR, CUBN, CELSR3, SCN7A, TULP4, SRRM2) and 10 have been previously reported. Three mutational signatures were identified to be prevalent in ESCC including Signature_15, Signature_4 and Signature_6, of which Signature_15 was related to prognosis of ESCC (HR 2.81, 95% CI 1.30-6.05; p = 0.008). Survival analysis showed that SCN7A was correlated to overall survival with an HR of 2.76 (95% CI 0.96-7.90, p = 0.058). After controlling for confounding factors such as age, gender, stage and location, the correlation between SCN7A and survival was statistically significant based on multivariate COX regression analysis (HR 4.76, 95% CI 1.20-18.85; p = 0.026, padjust = 0.053). The tumor vascular invasion was associated with SCN7A of ESCC patients (p = 0.028). CONCLUSION: In summary, this study provided comprehensive analysis of the somatic mutation profiles of ESCC, and identified SCN7A and Signature_15 for the prognosis of ESCC for the first time. The findings might serve as a conceptual basis for molecular diagnosis and prevention of ESCC.

Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China.

The two-spotted spider mite Tetranychus urticate is an important agricultural pest worldwide. It is extremely polyphagous and has developed resistance to many pesticides. Here, we assessed the pesticide resistance of seven field populations of T. urticae in China, their target site mutations and the activities of their detoxification enzymes. The results showed that abamectin and the traditional pesticides pyridaben, profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr, spinetoram, cyflumetofen, cyenopyrafen, bifenazate and B-azolemiteacrylic. The frequency of point mutations related to abamectin resistance, G314D in the glutamate-gated chloride channel 1 (GluCl1) and G326E in GluCl3, ranged 47%-70% and 0%-97%, respectively. The frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene (VGSC), which may increase resistance to pyrethroids, ranged 88%-100% and 10%-100%, respectively. For target sites related to organophosphate resistance, mutation frequencies ranged 25%-92% for G119S and 0%-23% for A201S in the acetycholinesterase gene (Ace). Mutation G126S in the bifenazate resistance-related cytochrome b gene (Cytb) was observed in three of the seven T. urticae populations. Higher activities of detoxification enzymes (P450, GST, CarEs and UGTs) were observed in two T. urticae populations, with significant difference in the XY-SX population. These results provide useful information on the status of pesticide resistance of T. urticae in China and suggest that T. urticae field populations may have multiple resistance mechanisms.

Perioperative Management and Considerations for Patients With Voltage-Gated Sodium Channel Mutations: A Pediatric Case Report.

A 13-year-old girl with a voltage-gated sodium channel mutation (SCN8A)-associated intractable epilepsy presented for bilateral mastectomy for painful juvenile fibroadenomatosis. Sodium channel mutations are more frequently diagnosed with continued advances in genetic testing. Understanding the effects of sodium channel mutations is important to provide safe anesthetic care to these patients. In this article, we discuss what is known regarding the physiology of SCN8A channels and the anesthetic considerations when caring for patients with an SCN8A mutation.

Phosphoinositide regulates dynamic movement of the S4 voltage sensor in the second repeat in two-pore channel 3.

The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second S4 helix, showing that the PI-binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs and was potentiated by PI. In conclusion, this regulation of the second S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.

Purification and characterization of peptides Ap2, Ap3 and Ap5 (ω-toxins) from the venom of the Brazilian tarantula Acanthoscurria paulensis.

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels - NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 - Ap2, Ap3 and Ap5 inhibited 20-30 % of CaV2.1 current at 1 µM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.

Investigation of binding modes of spider toxin-human voltage-gated sodium channel subtybe 1.7.

ABSTRACTSThe human voltage-gated sodium channel subtype 1.7 (hNaV1.7) is an attractive target for the development of potent and selective novel analgesics. HwTx-IV, a spider derived peptide toxin with 35-residue, inhibits hNaV1.7 with high potency by influencing the kinetics and gating behaviors of the channel, kinetics refers to the control of ion channels on and off by binding to a voltage sensor domains, so it is classified as gating modifier toxins (GMTs). In this study, we study how HwTx-IV and its variant exert its inhibitory potency on hNav1.7 using a range of biophysical techniques including homology modelling, molecular docking, molecular dynamics simulation, and umbrella sampling. The results show that the binding free energy of HwTx-IV and m3-HwTx-IV to hNaV1.7 is -15.00 kJ/mol and -16.2 kJ/mol, respectively, which are consistent with the experiential results；hydrophobic and electrostatic interaction both are important concerns about toxin blocking ion channels：the interactions of m3-HwTx-IV-hNaV1.7 are enhanced by mutating several residues in HwTx-IV. In comparison with the other peptide toxins of NaSpTx-F1, it is found that NaSpTx-F1 also had a similar binding characteristic. Combined above results, it was concluded that K32, W30 and F6, K7 and A8 in N-groove were critical for the interaction strength；G1, L3, G4, I5 in the N terminus and W33, I35 in the C terminus together can determine the peptide binding orientation relative to the channel and ultimately altered the inhibitory effect. This conclusion would be useful for designing high potency peptide inhibitor for hNav1.7.Communicated by Ramaswamy H. Sarma.

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I.

The role of voltage-gated sodium (NaV) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms NaV1.8 and NaV1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat NaV1.8 and mouse NaV1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant NaV isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human NaV1.8 and NaV1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism.

Prokaryotic NaV channels are tetramers and eukaryotic NaV channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of NaVMs, a prokaryotic NaV channel, suggests that the S4-S5 linker (S4-S5L) interacts with the C-terminus of S6 (S6T) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5L-mimicking peptides, we previously demonstrated that S4-S5L/S6T interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic NaV channel, NaVSp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A NaVSp1-specific S4-S5L peptide, containing the residues supposed to interact with S6T according to the NaVMs structure, induced both an increase in NaVSp1 current density and a negative shift in the activation curve, consistent with S4-S5L stabilizing the open state. Using this approach on a human NaV channel, hNaV1.4, and testing 12 hNaV1.4 S4-S5L peptides, we identified four activating S4-S5L peptides. These results suggest that, in eukaryotic NaV channels, the S4-S5L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state.

Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels.

Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

Knockdown of sodium channel Nax reduces dermatitis symptoms in rabbit skin.

The skin plays a critical role in maintenance of water homeostasis. Dysfunction of the skin barrier causes not only delayed wound healing and hypertrophic scarring, but it also contributes to the development of various skin diseases. Dermatitis is a chronic inflammatory skin disorder that has several different subtypes. Skin of contact dermatitis and atopic dermatitis (AD) show epidermal barrier dysfunction. Nax is a sodium channel that regulates inflammatory gene expression in response to perturbation of barrier function of the skin. We found that in vivo knockdown of Nax using RNAi reduced hyperkeratosis and keratinocyte hyperproliferation in rabbit ear dermatitic skin. Increased infiltration of inflammatory cells (mast cells, eosinophils, T cells, and macrophages), a characteristic of dermatitis, was reduced by Nax knockdown. Upregulation of PAR-2 and thymic stromal lymphopoietin (TSLP), which induce Th2-mediated allergic responses, was inhibited by Nax knockdown. In addition, expression of COX-2, IL-1ß, IL-8, and S100A9, which are downstream genes of Nax and are involved in dermatitis pathogenesis, were also decreased by Nax knockdown. Our data show that knockdown of Nax relieved dermatitis symptoms in vivo and indicate that Nax is a novel therapeutic target for dermatitis, which currently has limited therapeutic options.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Voltage gated sodium channels in cancer and their potential mechanisms of action.

Voltage gated sodium channels (VGSC) are implicated in cancer cell invasion and metastasis. However, the mechanism by which VGSC increase cell invasiveness and probability of metastasis is still unknown. In this review we outline lesser known functions of VGSC outside of action potential propagation, and the current understanding of the effects of VGSC in cancer. Finally, we discuss possible downstream effects of VGSC activation in cancer cells. After extensive review of the literature, the most likely role of VGSC in cancer is in the invadopodia, the leading edge of metastatic cancer cells. Sodium gradients are used to drive many biological processes in the body, and invadopodia may be similar. The function of the sodium hydrogen exchanger (NHE) and sodium calcium exchanger (NCX) are driven by sodium gradients. Voltage gated calcium channels, activated by membrane depolarization, are also capable of becoming activated in response to VGSC activity. Changes to hydrogen ion exchange or calcium handling have functional consequences for invadopodia and would explain the relationship between VGSC expression and invasiveness of cancer cells.