Bioactive glasses incorporating less-common ions to improve biological and physical properties.

Bioactive glasses (BGs) have been a focus of research for over five decades for several biomedical applications. Although their use in bone substitution and bone tissue regeneration has gained important attention, recent developments have also seen the expansion of BG applications to the field of soft tissue engineering. Hard and soft tissue repair therapies can benefit from the biological activity of metallic ions released from BGs. These metallic ions are incorporated in the BG network not only for their biological therapeutic effects but also in many cases for influencing the structure and processability of the glass and to impart extra functional properties. The "classical" elements in silicate BG compositions are silicon (Si), phosphorous (P), calcium (Ca), sodium (Na), and potassium (K). In addition, other well-recognized biologically active ions have been incorporated in BGs to provide osteogenic, angiogenic, anti-inflammatory, and antibacterial effects such as zinc (Zn), magnesium (Mg), silver (Ag), strontium (Sr), gallium (Ga), fluorine (F), iron (Fe), cobalt (Co), boron (B), lithium (Li), titanium (Ti), and copper (Cu). More recently, rare earth and other elements considered less common or, some of them, even "exotic" for biomedical applications, have found room as doping elements in BGs to enhance their biological and physical properties. For example, barium (Ba), bismuth (Bi), chlorine (Cl), chromium (Cr), dysprosium (Dy), europium (Eu), gadolinium (Gd), ytterbium (Yb), thulium (Tm), germanium (Ge), gold (Au), holmium (Ho), iodine (I), lanthanum (La), manganese (Mn), molybdenum (Mo), nickel (Ni), niobium (Nb), nitrogen (N), palladium (Pd), rubidium (Rb), samarium (Sm), selenium (Se), tantalum (Ta), tellurium (Te), terbium (Tb), erbium (Er), tin (Sn), tungsten (W), vanadium (V), yttrium (Y) as well as zirconium (Zr) have been included in BGs. These ions have been found to be particularly interesting for enhancing the biological performance of doped BGs in novel compositions for tissue repair (both hard and soft tissue) and for providing, in some cases, extra functionalities to the BG, for example fluorescence, luminescence, radiation shielding, anti-inflammatory, and antibacterial properties. This review summarizes the influence of incorporating such less-common elements in BGs with focus on tissue engineering applications, usually exploiting the bioactivity of the BG in combination with other functional properties imparted by the presence of the added elements.

Pulmonary fibrosis distal airway epithelia are dynamically and structurally dysfunctional.

The airway epithelium serves as the interface between the host and external environment. In many chronic lung diseases, the airway is the site of substantial remodeling after injury. While, idiopathic pulmonary fibrosis (IPF) has traditionally been considered a disease of the alveolus and lung matrix, the dominant environmental (cigarette smoking) and genetic (gain of function MUC5B promoter variant) risk factor primarily affect the distal airway epithelium. Moreover, airway-specific pathogenic features of IPF include bronchiolization of the distal airspace with abnormal airway cell-types and honeycomb cystic terminal airway-like structures with concurrent loss of terminal bronchioles in regions of minimal fibrosis. However, the pathogenic role of the airway epithelium in IPF is unknown. Combining biophysical, genetic, and signaling analyses of primary airway epithelial cells, we demonstrate that healthy and IPF airway epithelia are biophysically distinct, identifying pathologic activation of the ERBB-YAP axis as a specific and modifiable driver of prolongation of the unjammed-to-jammed transition in IPF epithelia. Furthermore, we demonstrate that this biophysical state and signaling axis correlates with epithelial-driven activation of the underlying mesenchyme. Our data illustrate the active mechanisms regulating airway epithelial-driven fibrosis and identify targets to modulate disease progression.

Green Synthesis of Copper Oxide Nanoparticles using Cucumis Sativus (Cucumber) Extracts and their Bio-Physical and Biochemical Characterization for Cosmetic and Dermatologic Applications.

BACKGROUND & OBJECTIVE: Nanoparticles are used in cosmetic and dermatologic products, due to better skin penetration properties. Incorporation of natural products exhibiting medicinal properties in nano-preparations could significantly improve the efficacy of these products and improve the quality of life without the side effects of synthetic formulations. METHODS: We here report the green synthesis of Copper Oxide nanoparticles, using Cucumber extract, and their detailed bio-physical and bio-chemical characterization. RESULTS: These Copper Oxide-Cucumber nanoparticles exhibit significant anti-bacterial and anti-fungal properties, Ultra Violet-radiation protection ability and reactive-oxygen species inhibition properties. Importantly, these nanoparticles do not exhibit significant cellular toxicity and, when incorporated in skin cream, exhibit skin rejuvenating properties. CONCLUSION: Our findings have implications for nanoparticle-based cosmetics and dermatologic applications.

Modulation of membrane physical properties by natural insecticidal ketones.

The insecticidal activity of Mentha oil and its main components has been tested and established for various insects/pests. Several mint ketones have demonstrated to act on GABAA receptors (GABAA-R), a transmembrane channel target of several important insecticides whose activity can be modulated by surface-active compounds and by changes in the physical properties of the lipid membrane. In the present work, we analyze the capacity of monoterpenic ketones most commonly found in Mentha species, pulegone and menthone, to interact with DPPC membranes by molecular dynamics (MD) simulations and Langmuir monolayers. The experimental results indicate that the presence of menthone and pulegone in the subphase modify the interfacial characteristics of DPPC isotherms. The changes were reflected as expansion of the isotherms and disappearance or bringing forward of DPPC phase transition. MD simulation corroborate these results and indicate that both ketones are located at the region of the carbonyl group, at the interface with the acyl chains. Ketone intercalation between lipid molecules would induce an increasing intermolecular interaction, diminishing the film elasticity and causing an ordering effect. Our results suggest that the insecticidal activity of both ketones could involve their interaction with lipid molecules causing disturbance of the cell membrane as postulated for several larvicide compounds, or at least modulating the receptor surrounding.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Stability of cytoplasmic nanoviscosity during cell cycle of HeLa cells synchronized with Aphidicolin.

Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.

Biophysical Elucidation of Amyloid Fibrillation Inhibition and Prevention of Secondary Nucleation by Cholic Acid: An Unexplored Function of Cholic Acid.

Protein misfolding and its deviant self-assembly to converge into amyloid fibrils is associated with the perturbation of cellular functions and thus with debilitating neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, etc. A great deal of research has already been carried out to discover a potential amyloid inhibitor that can slow down, prevent, or remodel toxic amyloids. In the present study with the help of a combination of biophysical, imaging, and computational techniques, we investigated the mechanism of interaction of cholic acid (CA), a primary bile acid, with human insulin and Aß-42 and found CA to be effective in inhibiting amyloid formation. From ThT data, we inferred that CA encumbers amyloid fibrillation up to 90% chiefly by targeting elongation of fibrils with an insignificant effect on lag time, while in the case of Aß-42, CA stabilizes the peptide in its native state preventing its fibrillation. Strikingly upon adding initially at the secondary nucleation stage, CA also detained the progression/growth of insulin fibrils. CA is unable to prevent the conformational changes completely during fibrillation but tends to resist and maintain an α helical structure up to a significant extent at a primary nucleation stage while reducing the ß sheet rich content at the secondary nucleation stage. Moreover, CA treated samples exhibited reduced cytotoxicity and different morphology. Furthermore, the results obtained after molecular docking indicated that CA is interacting with insulin via hydrogen bonds. For future research, this study can be considered as preliminary research for the development of CA, a metabolite of our body, as a potential therapeutic agent against Alzheimer's disease without even stimulating the immunological responses.

Inhibition and assessment of the biophysical gating properties of GluA2 and GluA2/A3 AMPA receptors using curcumin derivatives.

The development of efficacious and safe drugs for the treatment of neurological diseases related to glutamate toxicity has been a focus in neuropharmacological research. Specifically, discovering antagonists to modulate the activity and kinetics of AMPA receptors, which are the fastest ligand-gated ion channels involved in excitatory neurotransmission in response to glutamate. Thus, the current study investigated novel curcumin derivatives on the biophysical properties of AMPA receptors, specifically on the homomeric GluA2 and the heteromeric GluA2/A3 subunits and assessed for inhibitory actions. The biophysical parameter (i.e., desensitization, deactivation, and peak currents) were measured by using whole-cell patch clamp electrophysiology with and without the administration of the derivatives onto HEK293 cells. CR-NN, CR-NNPh, CR-MeNH, and CR-NO of the tested derivatives showed inhibition on all AMPA receptors up to 6 folds. Moreover, the inhibitory derivatives also increased desensitization and deactivation, which further intensifies the compounds' neuroprotective effects. However, CR-PhCl, CR-PhF, and CR-PhBr did not show any significant changes on the peak current, deactivation or desensitization rates. By comparison to other discovered and widely used antagonist, the prepared curcumin derivatives are not selective to a specific AMPA subunit, instead implement its effect in the same way between all types of AMPA receptors. Additionally, the obtained results provide derivatives that not only noncompetitively inhibit AMPARs but also decrease its biophysical kinetics, specifically desensitization and deactivation rates. Hence, to potentially serve as a new AMPAR inhibitor with therapeutic potential, the current study provides compounds that are non-selective and non-competitive antagonist, which also effect the desensitization and deactivation rates of the receptor.

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

Norepinephrine Inhibits Alzheimer's Amyloid-ß Peptide Aggregation and Destabilizes Amyloid-ß Protofibrils: A Molecular Dynamics Simulation Study.

The abnormal self-assembly of amyloid-ß (Aß) peptides into toxic fibrillar aggregates is associated with the pathogenesis of Alzheimer's disease (AD). The inhibition of ß-sheet-rich oligomer formation is considered as the primary therapeutic strategy for AD. Previous experimental studies reported that norepinephrine (NE), one of the neurotransmitters, is able to inhibit Aß aggregation and disaggregate the preformed fibrils. Moreover, exercise can markedly increase the level of NE. However, the underlying inhibitory and disruptive mechanisms remain elusive. In this work, we performed extensive replica-exchange molecular dynamic (REMD) simulations to investigate the conformational ensemble of Aß1-42 dimer with and without NE molecules. Our results show that without NE molecules, Aß1-42 dimer transiently adopts a ß-hairpin-containing structure, and the ß-strand regions of this ß-hairpin (residues 15QKLVFFA21 and 33GLMVGGVV40) strongly resemble those of the Aß fibril structure (residues 15QKLVFFA21 and 30AIIGLMVG37) reported in an electron paramagnetic resonance spectroscopy study. NE molecules greatly reduce the interpeptide ß-sheet content and suppress the formation of the above-mentioned ß-hairpin, leading to a more disordered coil-rich Aß dimer. Five dominant binding sites are identified, and the central hydrophobic core 16KLVFFA21 site and C-terminal 31IIGLMV36 hydrophobic site are the two most favorable ones. Our data reveal that hydrophobic, aromatic stacking, hydrogen-bonding and cation-π interactions synergistically contribute to the binding of NE molecules to Aß peptides. MD simulations of Aß1-42 protofibril show that NE molecules destabilize Aß protofibril by forming H-bonds with residues D1, A2, D23, and A42. This work reveals the molecular mechanism by which NE molecules inhibit Aß1-42 aggregation and disaggregate Aß protofibrils, providing valuable information for developing new drug candidates and exercise therapy against AD.

Biophysical analysis of cancer stem cell-potent copper(ii) coordination complexes.

Copper(ii) coordination complexes, 1 and 2, containing nonsteroidal anti-inflammatory drugs (NSAIDs) potently kill breast cancer stem cells (CSCs) and bulk breast cancer cells. Although detailed biological studies have been conducted to shed light on their mechanism of cytotoxicity, little is known about their molecular level mechanism of action. This biophysical study, aided by the preparation of a fluorophore-containing analogue, 3, reveals that the complexes operate by undergoing reduction to a copper(i) form and releasing the associated NSAIDs.

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi.

BACKGROUND: VhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose. METHODS: VhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water. RESULTS: The role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H2O and D2O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H2O as solvent by D2O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp136 with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp136 was replaced by Arg, or 5-fold and 3-fold for Trp136 replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel. CONCLUSIONS: Hydrogen-bonds contribute to sugar permeation.

Enhanced expression of the Epithelial Sodium Channel in neutrophils from hypertensive patients.

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.

Lipid-Based Inhibitors Act Directly on GlyT2.

The endogenous lipids N-arachidonylglycine and oleoyl-l-carnitine are potential therapeutic leads in the treatment of chronic pain through their inhibition of the glycine transporter GlyT2. However, their mechanism of action is unknown. It has been hypothesized that these "bioactive" lipids either inhibit GlyT2 indirectly, by significantly perturbing the biophysical properties of the membrane, or directly, by binding directly to the transporter (either from a membrane-exposed or solvent-exposed binding site). Here, we used molecular dynamics simulations to study the effects of the lipids anandamide, N-arachidonylglycine, and oleoyl-l-carnitine on (a) the biophysical properties of the bilayer and (b) direct binding interactions with GlyT2. During the simulations, the biophysical properties of the bilayer itself, for example, the area per lipid, bilayer thickness, and order parameters, were not significantly altered by the presence or type of bioactive lipid, regardless of the presence of GlyT2. Our work, together with previous computational and experimental data, suggests that these acyl-inhibitors of GlyT2 inhibit the transporter by directly binding to it. However, these bioactive lipids bound to various parts of GlyT2 and did not prefer a single binding site during 4.5 µs of simulation. We postulate that the binding site is located at the solvent-exposed regions of GlyT2. Understanding the mechanism of action of these and related bioactive lipids is essential in effectively developing high-affinity GlyT2 inhibitors for the treatment of pain.

Exploring the effect of vitamin E in cancer chemotherapy-A biochemical and biophysical insight.

Many oncologists contend that patient undergoing chemotherapy must avoid antioxidant supplementation as it may interfere with the activity of the drug. In the present investigation, we have explored the influence of vitamin E, a well-known antioxidant on Camptothecin (CPT), a potent anti-cancer drug induced cell apoptosis and death of cervical cancer cells. HeLa cells were treated with different concentrations of CPT in presence and absence of 100 µm vitamin E. Treated cells were subjected to cytotoxicity studies, catalase assay, DNA fragmentation assay, clonogenic assay and flow cytometry based apoptosis detection. Also, Raman spectroscopy a label free technique which provides global information, in conjunction with multivariate tools like PCA, PCLDA and FDA, was investigated to explore vitamin E supplementation induced alterations. Our data based on biochemical and biophysical experimental analysis reveals that CPT causes DNA damage along with protein and lipid alteration culminating in cell death. Importantly, Raman spectroscopic analysis could uniquely differentiate the cluster of control and vitamin E control from CPT and CPT + Vit E treated cells. We conclusively prove that presence of vitamin E at 100 µM concentration shows promising antioxidant activity and displays no modulatory role on CPT induced effect, thereby causing no possible hindrance with the efficacy of the drug. Vitamin E may prove beneficial to alleviate chemotherapy associated side effects in patients during clinical settings which may open the doors further for subsequent exploration in in vivo preclinical studies.

Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons.

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.

Neochamaejasmin A inhibits KV1.4 channel activity via direct binding to the pore.

Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb and widespread in Mongolia and the northern parts of China. Previous studies have revealed that Neochamaejasmin A (NCA), one of the main active ingredients in the plant roots, has many bioactivities such as inhibiting the P-gp-mediated efflux. But whether NCA affects ion channels is unknown. Here the whole cell patch clamp technique was used to investigate whether NCA affects ion channels, especially how it inhibits KV1.4. Mutagenesis and structure-based molecular simulation were used for analysis of inhibition mechanism and identification of binding site. Among all the channels assayed, KV1.4 stood out as the one on which NCA showed strongest inhibition activity with IC50 of 7.55 µM. Compared with NCA's isomerides, neochamaejasmin B (NCB) and chamaechromone (CMC), NCA also exhibited superior inhibition ability on KV1.4. Three mutations, V549A, A553V and V560A, occurred inside the pore, were found to significantly alleviate the NCA blocking effects, suggesting that they are the important binding sites of NCA. Structure-based modelling showed that the phenolic hydroxyl group of NCA can form hydrogen bonds with main chains of Val549 and Ala553 in IS6 and IVS6 segment respectively, which support our in vitro results. In conclusion, data suggest that NCA might inhibit KV1.4 channels via direct binding to the pore domain.

Increased functional coupling of 5-HT1A autoreceptors to GIRK channels in Tph2-/- mice.

Firing activity of serotonergic neurons is under regulatory control by somatodendritic 5-HT1A autoreceptors (5-HT1AARs). Enhanced 5-HT1AAR functioning may cause decreased serotonergic signaling in brain and has thereby been implicated in the etiology of mood and anxiety disorders. Tryptophan hydroxylase-2 knockout (Tph2-/-) mice exhibit sensitization of 5-HT1A agonist-induced inhibition of serotonergic neuron firing and thus represents a unique animal model of enhanced 5-HT1AAR functioning. To elucidate the mechanisms underlying 5-HT1AAR supersensitivity in Tph2-/- mice, we characterized the activation of G protein-coupled inwardly-rectifying potassium (GIRK) conductance by the 5-HT1A receptor agonist 5-carboxamidotryptamine using whole-cell recordings from serotonergic neurons in dorsal raphe nucleus. Tph2-/- mice exhibited a mean twofold leftward shift of the agonist concentration-response curve (p < 0.001) whereas the maximal response, proportional to the 5-HT1AAR number, was not different (p = 0.42) compared to Tph2+/- and Tph2+/+ littermates. No differences were found in the basal inwardly-rectifying potassium conductance, determined in the absence of agonist, (p = 0.80) nor in total GIRK conductance activated by intracellular application of GTP-γ-S (p = 0.69). These findings indicate increased functional coupling of 5-HT1AARs to GIRK channels in Tph2-/- mice without a concomitant increase in 5-HT1AARs and/or GIRK channel density. In addition, no changes were found in α1-adrenergic facilitation of firing (p = 0.72) indicating lack of adaptive changes Tph2-/- mice. 5-HT1AAR supersensitivity may represents a previously unrecognized cause of serotonergic system hypofunction and associated disorders and provides a possible explanation for conflicting results on the correlation between 5-HT1AAR density and depression in clinical imaging studies.

Nongenomic Glucocorticoid Suppression of a Postsynaptic Potassium Current via Emergent Autocrine Endocannabinoid Signaling in Hypothalamic Neuroendocrine Cells following Chronic Dehydration.

Glucocorticoids rapidly stimulate endocannabinoid synthesis and modulation of synaptic transmission in hypothalamic neuroendocrine cells via a nongenomic signaling mechanism. The endocannabinoid actions are synapse-constrained by astrocyte restriction of extracellular spatial domains. Exogenous cannabinoids have been shown to modulate postsynaptic potassium currents, including the A-type potassium current (IA), in different cell types. The activity of magnocellular neuroendocrine cells is shaped by a prominent IA. We tested for a rapid glucocorticoid modulation of the postsynaptic IK and IA in magnocellular neuroendocrine cells of the hypothalamic paraventricular nucleus (PVN) using whole-cell recordings in rat brain slices. Application of the synthetic glucocorticoid dexamethasone (Dex) had no rapid effect on the IK or IA amplitude, voltage dependence, or kinetics in magnocellular neurons in slices from untreated rats. In magnocellular neurons from salt-loaded rats, however, Dex application caused a rapid suppression of the IA and a depolarizing shift in IA voltage dependence. Exogenously applied endocannabinoids mimicked the rapid Dex modulation of the IA, and CB1 receptor antagonists and agonists blocked and occluded the Dex-induced changes in the IA, respectively, suggesting an endocannabinoid dependence of the rapid glucocorticoid effect. Preincubation of control slices in a gliotoxin resulted in the partial recapitulation of the glucocorticoid-induced rapid suppression of the IA. These findings demonstrate a glucocorticoid suppression of the postsynaptic IA in PVN magnocellular neurons via an autocrine endocannabinoid-dependent mechanism following chronic dehydration, and suggest a possible role for astrocytes in the control of the autocrine endocannabinoid actions.

Solution conformations of Zika NS2B-NS3pro and its inhibition by natural products from edible plants.

The recent Zika viral (ZIKV) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and Guillain-Barre syndrome but unfortunately no vaccine or medication is effectively available yet. Zika NS2B-NS3pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. Thus NS2B-NS3pro represents an attractive target for anti-Zika drug discovery/design. Here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked Zika NS2B-NS3pro complexes and found that the unlinked complex manifested well-dispersed NMR spectra. Subsequently with selective isotope-labeling using NMR spectroscopy, we demonstrated that C-terminal residues (R73-K100) of NS2B is highly disordered without any stable tertiary and secondary structures in the Zika NS2B-NS3pro complex in the free state. Upon binding to the well-characterized serine protease inhibitor, bovine pancreatic trypsin inhibitor (BPTI), only the extreme C-terminal residues (L86-K100) remain disordered. Additionally, we have identified five flavonoids and one natural phenol rich in edible plants including fruits and vegetables, which inhibit Zika NS2B-NS3pro in a non-competitive mode, with Ki ranging from 770 nM for Myricetin to 34.02 µM for Apigenin. Molecular docking showed that they all bind to a pocket on the back of the active site and their structure-activity relationship was elucidated. Our study provides valuable insights into the solution conformation of Zika NS2B-NS3pro and further deciphers its susceptibility towards allosteric inhibition by natural products. As these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in terms of both inhibitory mode and chemical scaffold, our finding might open a new avenue for development of better allosteric inhibitors to fight ZIKV infection.