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Global sensitivity analysis combined with quantitative high-throughput screening (GSA-qHTS) uses random starting points of the trajectories in mixture design, which may lead to potential contingency and a lack of representativeness. Moreover, a scenario in which all factor levels were at stimulatory effects was not considered, thereby hindering a comprehensive understanding of GSA-qHTS. Accordingly, this study innovatively introduced an optimised experimental design, uniform design (UD), to generate non-random and representative sample points with smaller uniformity deviation as starting points of multiple trajectories. By combining UD with the previously optimised one-factor-at-a-time (OAT) method, a novel mixture design method was developed (UD-OAT). The single toxicity tests showed that three pyridinium and five imidazolium ionic liquids (ILs) exerted stimulatory effects on Vibrio qinghaiensis sp.-Q67; thus, four stimulatory effective concentrations of each IL were selected as factor levels. The UD-OAT generated 108 mixture samples with equal frequency and without repetition. High-throughput microplate toxicity analysis revealed that all 108 mixtures exhibited inhibitory effects. Among these, type B mixtures exhibited increasing toxicities that subsequently decreased, unlike type C mixtures, which consistently increased over time. GSA successfully identified three of the eight ILs as important factors influencing the toxicities of the mixtures. When individual ILs produced stimulatory effects, mixtures containing two to three ILs exhibited either stimulatory effects or none. In contrast, mixtures containing five to eight ILs exhibited inhibitory effects, while those containing four ILs showed a transition from stimulatory to inhibitory effects. This study provides a novel mixture design method for studying mixture toxicity and fills the application gap of GSA-qHTS. The phenomenon of individuals being beneficial while mixtures can be harmful challenges traditional mixture risk assessments.

Mitral cells (MCs) in the main olfactory bulb relay odor information to higher-order olfactory centers by encoding the information in the form of action potentials. The firing patterns of these cells are influenced by both their intrinsic properties and their synaptic connections within the neural network. However, reports on MC firing patterns have been inconsistent, and the mechanisms underlying these patterns remain unclear. Using whole-cell patch-clamp recordings in mouse brain slices, we discovered that MCs exhibit two types of integrative behavior: regular/rhythmic firing and bursts of action potentials. These firing patterns could be transformed both spontaneously and chemically. MCs with regular firing maintained their pattern even in the presence of blockers of fast synaptic transmission, indicating this was an intrinsic property. However, regular firing could be transformed into bursting by applying GABAA receptor antagonists to block inhibitory synaptic transmission. Burst firing could be reverted to regular firing by blocking ionotropic glutamate receptors, rather than applying a GABAA receptor agonist, indicating that ionotropic glutamatergic transmission mediated this transformation. Further experiments on long-lasting currents (LLCs), which generated burst firing, also supported this mechanism. In addition, cytoplasmic Ca2+ in MCs was involved in the transformation of firing patterns mediated by glutamatergic transmission. Metabotropic glutamate receptors also played a role in LLCs in MCs. These pieces of evidence indicate that odor information can be encoded on a mitral cell (MC) platform, where it can be relayed to higher-order olfactory centers through intrinsic and dendrodendritic mechanisms in MCs.

As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit systemic and mucosal humoral immunity to limit the emergence of new variants. Using the Chinese Hu191 measles virus (MeV-hu191) vaccine strain as a backbone, we developed MeV chimeras stably expressing the prefusion forms of either membrane-anchored, full-length spike (rMeV-preFS), or its soluble secreted spike trimers with the help of the SP-D trimerization tag (rMeV-S+SPD) of SARS-CoV-2 Omicron BA.2. The two vaccine candidates were administrated in golden Syrian hamsters through the intranasal or subcutaneous routes to determine the optimal immunization route for challenge. The intranasal delivery of rMeV-S+SPD induced a more robust mucosal IgA antibody response than the subcutaneous route. The mucosal IgA antibody induced by rMeV-preFS through the intranasal routine was slightly higher than the subcutaneous route, but there was no significant difference. The rMeV-preFS vaccine stimulated higher mucosal IgA than the rMeV-S+SPD vaccine through intranasal or subcutaneous administration. In hamsters, intranasal administration of the rMeV-preFS vaccine elicited high levels of NAbs, protecting against the SARS-CoV-2 Omicron BA.2 variant challenge by reducing virus loads and diminishing pathological changes in vaccinated animals. Encouragingly, sera collected from the rMeV-preFS group consistently showed robust and significantly high neutralizing titers against the latest variant XBB.1.16. These data suggest that rMeV-preFS is a highly promising COVID-19 candidate vaccine that has great potential to be developed into bivalent vaccines (MeV/SARS-CoV-2).

ABSTRACTCoxsackievirus A10 (CV-A10) has become one of the major pathogens of hand, foot and mouth disease (HFMD), and studies on the vaccine and animal model of CV-A10 are still far from complete. Our study used a mouse-adapted CV-A10 strain, which was lethal for 14-day-old mice, to develop an infected mouse model. Then this model was employed to establish an actively immunized-challenged mouse model to evaluate the efficacy of a formaldehyde-inactivated CV-A10 vaccine, which was prepared from a Vero cell-adapted strain. CV-A10 vaccine at a dose of 0.5 or 2.0 μg was inoculated intraperitoneally in neonatal Kunming mice on the third and ninth day. Then the mice were challenged on day 14. The survival rate of mice immunized with 0.5 or 2.0 μg vaccine were 90% and 100%, respectively, while all Alum-inoculated mice died. Compared to those in the two vaccinated groups, the Alum-inoculated mice showed severe pathological damage, strong viral protein expression and high viral loads. The antisera from vaccinated mice showed high level of neutralizing antibodies against CV-A10. Meanwhile, three potential T cell epitopes located at the carboxyl-terminal regions of the VP1 and VP3 were identified and exhibited CV-A10 serotype-specific. The humoral and cellular immunogenicity analysis showed that immunization with two doses of the vaccine elicited CV-A10 specific neutralizing antibody and T cell response in BALB/c mice. Collectively, these findings indicated that this actively immunized-challenged mouse model will be invaluable in future studies on CV-A10 pathogenesis and evaluation of vaccine candidates.

The research framework combining global sensitivity analysis (GSA) with quantitative high-throughput screening (qHTS), called GSA-qHTS, provides a potentially feasible way to screen for important factors that induce toxicities of complex mixtures. Despite its value, the mixture samples designed using the GSA-qHTS technique still have a shortage of unequal factor levels, which leads to an asymmetry in the importance of elementary effects (EEs). In this study, we developed a novel method for mixture design that enables equal frequency sampling of factor levels (called EFSFL) by optimizing both the trajectory number and the design and expansion of the starting points for the trajectory. The EFSFL has been successfully employed to design 168 mixtures of 13 factors (12 chemicals and time) that each have three levels. By means of high-throughput microplate toxicity analysis, the toxicity change rules of the mixtures are revealed. Based on EE analysis, the important factors affecting the toxicities of the mixtures are screened. It was found that erythromycin is the dominant factor and time is an important non-chemical factor in mixture toxicities. The mixtures can be classified into types A, B, and C mixtures according to their toxicities at 12 h, and all the types B and C mixtures contain erythromycin at the maximum concentration. The toxicities of the type B mixtures increase firstly over time (0.25 ∼ 9 h) and then decrease (12 h), while those of the type C mixtures consistently increase over time. Some type A mixtures produce stimulation that increases with time. With the present new approach to mixture design, the frequency of factor levels in mixture samples is equal. Consequently, the accuracy of screening important factors is improved based on the EE method, providing a new method for the study of mixture toxicity.

The purple non-sulfur bacterium Rhodobacter sphaeroides was selected as a biological model to investigate its response to the toxicity of 1-alkyl-3-methylimidazolium bromide ([Cnmim]Br), a type of ionic liquid (IL), with different alkyl chain lengths (n describes the number of carbon atoms in the alkyl chain). The inhibition of bacterial growth by [Cnmim]Br was positively correlated with n. Morphological characterization revealed that [Cnmim]Br caused cell membrane perforation. The signal amplitude of the electrochromic absorption band shift of endogenous carotenoids showed a negatively linear correlation with n, and the amplitude of the blue-shift of the B850 band in light-harvesting complex 2 showed a positively linear correlation with n. Furthermore, an increase in blocked ATP synthesis and increase in antioxidant enzyme activity were observed in chromatophores treated with ILs containing longer alkyl chains. In summary, the purple bacterium can be developed as a model to monitor ecotoxicity and examine the mechanism of IL toxicity.

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

For persistent organic pollutants, a concern of environmental supervision, predicted no-effect concentrations (PNECs) are often used in ecological risk assessment, which is commonly derived from the hazardous concentration of 5% (HC5) of the species sensitivity distribution (SSD). To address the problem of a lack of toxicity data, the objectives of this study are to propose and apply two improvement ideas for SSD application, taking polycyclic aromatic hydrocarbons (PAHs) as an example: whether the chronic PNEC can be derived from the acute SSD curve; whether the PNEC may be calculated by HC10 to avoid solely statistical extrapolation. In this study, the acute SSD curves for eight PAHs and the chronic SSD curves for three PAHs were constructed. The quantity relationship of HC5s between the acute and chronic SSD curves was explored, and the value of the assessment factor when using HC10 to calculate PNEC was derived. The results showed that, for PAHs, the chronic PNEC can be estimated by multiplying the acute PNEC by 0.1, and the value of the assessment factor corresponding to HC10 is 10. For acenaphthene, anthracene, benzo[a]pyrene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene, the chronic PNECs based on the acute HC10s were 0.8120, 0.008925, 0.005202, 0.07602, 2.328, 12.75, 0.5731, and 0.05360 μg/L, respectively.

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C. By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23PILPGF28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A. It may be useful for the development of a cost-effective and pan-Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

ABSTRACTThe ongoing COVID-19 pandemic is causing huge impact on health, life, and global economy, which is characterized by rapid spreading of SARS-CoV-2, high number of confirmed cases and a fatality/case rate worldwide reported by WHO. The most effective intervention measure will be to develop safe and effective vaccines to protect the population from the disease and limit the spread of the virus. An inactivated, whole virus vaccine candidate of SARS-CoV-2 has been developed by Wuhan Institute of Biological Products and Wuhan Institute of Virology. The low toxicity, immunogenicity, and immune persistence were investigated in preclinical studies using seven different species of animals. The results showed that the vaccine candidate was well tolerated and stimulated high levels of specific IgG and neutralizing antibodies. Low or no toxicity in three species of animals was also demonstrated in preclinical study of the vaccine candidate. Biochemical analysis of structural proteins and purity analysis were performed. The inactivated, whole virion vaccine was characterized with safe double-inactivation, no use of DNases and high purity. Dosages, boosting times, adjuvants, and immunization schedules were shown to be important for stimulating a strong humoral immune response in animals tested. Preliminary observation in ongoing phase I and II clinical trials of the vaccine candidate in Wuzhi County, Henan Province, showed that the vaccine is well tolerant. The results were characterized by very low proportion and low degree of side effects, high levels of neutralizing antibodies, and seroconversion. These results consistent with the results obtained from preclinical data on the safety.

ABSTRACTIn the face of COVID-19 pandemic caused by the newly emerged SARS-CoV-2, an inactivated, Vero cell-based, whole virion vaccine candidate has been developed and entered into phase III clinical trials within six months. Biochemical and immunogenic characterization of structural proteins and their post-translational modifications in virions, the end-products of the vaccine candidate, would be essential for the quality control and process development of vaccine products and for studying the immunogenicity and pathogenesis of SARS-CoV-2. By using a panel of rabbit antisera against virions and five structural proteins together with a convalescent serum, the spike (S) glycoprotein was shown to be N-linked glycosylated, PNGase F-sensitive, endoglycosidase H-resistant and cleaved by Furin-like proteases into S1 and S2 subunits. The full-length S and S1/S2 subunits could form homodimers/trimers. The membrane (M) protein was partially N-linked glycosylated; the accessory protein 3a existed in three different forms, indicative of cleavage and dimerization. Furthermore, analysis of the antigenicity of these proteins and their post-translationally modified forms demonstrated that S protein induced the strongest antibody response in both convalescent and immunized animal sera. Interestingly, immunization with the inactivated vaccine did not elicit antibody response against the S2 subunit, whereas strong antibody response against both S1 and S2 subunits was detected in the convalescent serum. Moreover, vaccination stimulated stronger antibody response against S multimers than did the natural infection. This study revealed that the native S glycoprotein stimulated neutralizing antibodies, while bacterially-expressed S fragments did not. The study on S modifications would facilitate design of S-based anti-SARS-CoV-2 vaccines.

In the hazard assessment of mixtures, the mixture predicted no-effect concentration (mPNEC) is always derived by the concentration addition (CA) model (mPNECCA) to assess the risk of mixtures combined with exposure assessment. However, the independent action (IA) model, which is also widely used as the CA model in the prediction and evaluation of mixture toxicity, is always used to calculate the population fraction showing a predefined effect, not mPNEC, and this limits the application of IA model in the mixture risk assessment. In this study, we explored the process of mPNEC derived by the IA method (mPNECIA) based on the species sensitivity distribution (SSD) and compared mPNECIA with mPNECCA. Taking two common pesticides, dimethoate (DIM) and dichlorvos (DIC), exposed in the actual water environment as an example, their SSD models were constructed separately using nine distribution functions after toxicity data screening and quality testing. For both DIC and DIM, all different nine models had passed the Kolmogorov-Smirnov test. Then, the PNECs of two pesticides were derived based on SSD models. Finally, mPNECIA with different concentration ratios was derived and compared to mPNECCA based on 81 combinations of nine SSD models. Most mPNEC values derived by IA model were more conservative than those by CA. It is worth noting that the mPNECIA is more conservative than mPNECCA for the commonly used log-logit distribution (function 7), log-normal distribution (8), and log-Weibull distribution (9). This study provides a new direction for the application of IA in the risk assessment and enriches the framework of mixture risk assessment.

Abstract Background KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development. Methods Here we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders. Results Using the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 μM. Conclusion Our results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.

Current Chinese surface water environmental quality standard GB3838-2002 for ammonia fails to take water quality factors and native organism distributions in different basins into consideration. In this study, ammonia toxicity tests were performed using three aquatic organisms native to the Shaying River Basin (China). Published ammonia toxicity data with pH and temperature, and toxicity data acquired in this study were used to establish water quality criteria. The final criterion maximum concentration (CMC) and criterion continuous concentration (CCC) for the Shaying River Basin were 5.09 and 1.36 (mg total ammonia nitrogen (TAN))/L (pH 7 and 20 °C), respectively. In addition, based on the corresponding relationship between ammonia toxicity and temperature and pH, the ecological risk assessment of ammonia was conducted in different seasons for the Shaying River using a tiered approach of both hazard quotient (HQ) and the joint probability (JPC) methods. Two methods gave consistent results: the ecological risks of ammonia to aquatic species in the Shaying River Basin were severe and the risk could be ranked as wet season > flat season > dry season. It is therefore indicating that monitoring, evaluation, and early warning of ammonia pollution need to be taken to prevent and control the risks posed by ammonia pollution, especially for wet season (because of high temperatures and pH) or flat season (because of high pH values). We hope the present work could provide valuable information to manage and control ammonia pollution in the Shaying River Basin.

This data article presents 1) the acute toxicity (LC50 or EC50 (μg⋅L−1)) values of various chemicals for ten species, which were used to develop ten robust quantitative structure–activity relationship (QSAR) models, 2) the values of the various descriptors in the ten QSAR models, and 3) the acute toxicity values of six pesticides (acetochlor, chlorpyrifos, dimethoate, glyphosate, malathion, and paraquat) for various species, which were applied to establish species sensitivity distribution (SSD) models. The provided LC50 or EC50 (μg⋅L−1) data were collected from the PAN pesticide database and the United States Environmental Protection Agency ecotoxicology database and/or were predicted by the QSAR models. The values of the descriptors in the ten QSAR models were based on the optimal descriptors computed by the DRAGON software (version 7) and subsequently optimized by partial least squares modeling. All the data included in this manuscript are related to the research titled, “Conlecs: A novel procedure for deriving the concentration limits of chemicals outside the criteria of human drinking water using existing criteria and species sensitivity distribution based on quantitative structure–activity relationship prediction” [1]. Keywords: Water quality criteria, Non-linear curve fitting, Toxicity prediction, Variable selection and modeling, Variable importance in projection

Bioluminescent bacteria are mainly found in marine habitats. Vibrio qinghaiensis sp.-Q67 (Q67), a nonpathogenic freshwater bacterium, has been a focus due to its wide use in the monitoring of environmental pollution and the assessment of toxicity. However, the lack of available crystal structures limits the elucidation of the structures of the functional proteins of the quorum-sensing (QS) system that regulates bacterial luminescence in Q67. In this study, 19 functional proteins were built through monomer and oligomer modeling based on their coding proteins in the QS system of Q67 using MODELLER. Except for the failure to construct LuxM due to the lack of a suitable template, 18 functional proteins were successfully constructed. Furthermore, the relationships between the function and predicted structures of 19 functional proteins were explored one by one according to the three functional classifications: autoinducer synthases and receptors, signal transmission proteins (phosphotransferases, an RNA chaperone, and a transcriptional regulator), and enzymes involved in bacterial bioluminescence reactions. This is the first analysis of the whole process of bioluminescence regulation from the perspective of nonpathogenic freshwater bacteria at the molecular level. It provides a theoretical basis for the explanation of applications of Q67 in which luminescent inhibition is used as the endpoint.

In an ongoing effort to identify novel drugs that can be used as neurotherapeutic compounds, we have focused on anilino enaminones as potential anticonvulsant agents. Enaminones are organic compounds containing a conjugated system of an amine, an alkene and a ketone. Here, we review the effects of a small library of anilino enaminones on neuronal activity. Our experimental approach employs an olfactory bulb brain slice preparation using whole-cell patch-clamp recording from mitral cells in the main olfactory bulb. The main olfactory bulb is a key integrative center in the olfactory pathway. Mitral cells are the principal output neurons of the main olfactory bulb, receiving olfactory receptor neuron input at their dendrites within glomeruli, and projecting glutamatergic axons through the lateral olfactory tract to the olfactory cortex. The compounds tested are known to be effective in attenuating pentylenetetrazol (PTZ) induced convulsions in rodent models. One compound in particular, KRS-5Me-4-OCF3, evokes potent inhibition of mitral cell activity. Experiments aimed at understanding the cellular mechanism underlying the inhibitory effect revealed that KRS-5Me-4-OCF3 shifts the concentration-response curve for GABA to the left. KRS-5Me-4-OCF3 enhances GABA affinity and acts as a positive allosteric modulator of GABAA receptors. Application of a benzodiazepine site antagonist blocks the effect of KRS-5Me-4-OCF3 indicating that KRS-5Me-4-OCF3 binds at the classical benzodiazepine site to exert its pharmacological action. This anilino enaminone KRS-5Me-4-OCF3 emerges as a candidate for clinical use as an anticonvulsant agent in the battle against epileptic seizures.

Essential oils and the constituents in them exhibit different pharmacological activities, such as antinociceptive, anxiolytic-like, and anticonvulsant effects. They are widely applied as a complementary therapy for people with anxiety, insomnia, convulsion, pain, and cognitive deficit symptoms through inhalation, oral administration, and aromatherapy. Recent studies show that essential oils are emerging as a promising source for modulation of the GABAergic system and sodium ion channels. This review summarizes the recent findings regarding the pharmacological properties of essential oils and compounds from the oils and the mechanisms underlying their effects. Specifically, the review focuses on the essential oils and their constituents targeting the GABAergic system and sodium channels, and their antinociceptive, anxiolytic, and anticonvulsant properties. Some constituents target transient receptor potential (TRP) channels to exert analgesic effects. Some components could interact with multiple therapeutic target proteins, for example, inhibit the function of sodium channels and, at the same time, activate GABAA receptors. The review concentrates on perspective compounds that could be better candidates for new drug development in the control of pain and anxiety syndromes.

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia. We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na+/K+-ATPase. The direct action of bufadienolide on brain Na+ channels was tested by recordings from stably Nav1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Nav1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation. Our results suggest that inhibition of Na+/K+-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an Nterminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels. [ABSTRACT FROM AUTHOR]