Mitochondrial Membranes of Human SH-SY5Y Neuroblastoma Cells Express Serotonin 5-HT7 Receptor

Mitochondria in neurons contribute to energy supply, the regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. In recent years, several studies have highlighted that the neurotransmitter serotonin (5-HT) plays an important role in mitochondrial biogenesis in cortical neurons, and regulates mitochondrial activity and cellular function in cardiomyocytes. 5-HT exerts its diverse actions by binding to cell surface receptors that are classified into seven distinct families (5-HT1 to 5-HT7). Recently, it was shown that 5-HT3 and 5-HT4 receptors are located on the mitochondrial membrane and participate in the regulation of mitochondrial function. Furthermore, it was observed that activation of brain 5-HT7 receptors rescued mitochondrial dysfunction in female mice from two models of Rett syndrome, a rare neurodevelopmental disorder characterized by severe behavioral and physiological symptoms. Our Western blot analyses performed on cell-lysate and purified mitochondria isolated from neuronal cell line SH-SY5Y showed that 5-HT7 receptors are also expressed into mitochondria. Maximal binding capacity (Bmax) obtained by Scatchard analysis on purified mitochondrial membranes was 0.081 pmol/mg of 5-HT7 receptor protein. Lastly, we evaluated the effect of selective 5-HT7 receptor agonist LP-211 and antagonist (inverse agonist) SB-269970 on mitochondrial respiratory chain (MRC) cytochrome c oxidase activity on mitochondria from SH-SY5Y cells. Our findings provide the first evidence that 5-HT7 receptor is also expressed in mitochondria.

Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions

Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.

Adiposome Targeting and Enzymatic Activity of Lipid Droplet-Specific Proteins

SUMMARYNew strategies to decode the specific protein targeting mechanism on lipid droplet (LD) are urgently needed. Using adiposome, the LD binding of perilipin 2 (PLIN2), perilipin 3 (PLIN3), and adipose triglyceride lipase (ATGL) were studied. Scatchard analysis found that the binding of PLIN2 to the adiposome surface was saturable, pointing to a specific membrane binding partner. Phosphatidylinositol (PI) was found to inhibit PLIN2 binding while it did not impede PLIN3. Structural analysis combined with mutagenesis revealed that the 73rd glutamic acid of PLIN2 is significant for the effect of PI on the protein binding. The presence of PI significantly stimulated the activity of ATGL in vitro. The phosphorylation site mutants of ATGL were found reducing the lipase activity in the adiposome system. Our study demonstrates the utility of adiposome as a powerful, manipulatable model system for the characterization of LD binding and enzymatic activity of LD proteins in vitro.

Exploring Matrix Effects on Binding Properties and Characterization of Cotinine Molecularly Imprinted Polymer on Paper-Based Scaffold

Commercially available sorbent materials for solid-phase extraction are widely used in analytical laboratories. However, non-selective binding is a major obstacle for sample analysis. To overcome this problem, molecularly imprinted polymers (MIPs) were used as selective adsorbent materials prior to determining target analysts. In this study, the use of non-covalent molecularly imprinted polymers (MIPs) for cotinine adsorption on a paper-based scaffold was studied. Fiberglass paper was used as a paper scaffold for cotinine-selective MIP adsorption with the use of 0.5% agarose gel. The effects of salt, pH, sample matrix, and solvent on the cotinine adsorption and extraction process were investigated. Under optimal conditions, the adsorption isotherm of synthesized MIPs increased to 125.41 µg/g, whereas the maximum adsorption isotherm of non-imprinted polymers (NIPs) was stable at 42.86 µg/g. The ability of the MIP paper scaffold to absorb cotinine in water medium was approximately 1.8–2.8-fold higher than that of the NIP scaffold. From Scatchard analysis, two dissociation constants of MIPs were calculated to be 2.56 and 27.03 µM. Nicotine, myosmine, and N-nitrosonornicotine were used for selectivity testing, and the calculated selectivity factor of cotinine to nicotine, myosmine, and N-nitrosonornicotine was 1.56, 2.69, and 2.05, respectively. Overall, the MIP paper scaffold is promising for simple onsite sampling of cotinine and can be used to assess tobacco smoke exposure.

Preparation of Ampicillin Surface Molecularly Imprinted Polymers for Its Selective Recognition of Ampicillin in Eggs Samples

Surface-imprinted polymers (MIPs) microspheres with the ability to specifically recognize water-soluble molecules were prepared using self-made monodisperse porous poly(chloromethylstyrene-co-divinylbenzene) beads as the solid-phase matrix and ampicillin (AMP) as the template molecule. MIPs were synthesized using different template molecule: monomer: crosslinker ratios and the optimum preparation ratio were obtained by measuring adsorption. The maximum equilibrium amount of adsorption by the MIPs reached 115.62 mg/g. Scatchard analysis indicated that the MIPs contained two types of recognition sites: specific and nonspecific. Based on the adsorption kinetics, adsorption equilibrium was reached after 30 minutes. Penicillin G, amoxicillin, and sulbactam acid were used as competitive molecules to research the selective adsorption capacity of the MIPs. The imprinted material was found to have good selectivity with selectivity coefficients for penicillin G, amoxicillin, and sulbactam acid of 5.74, 6.83, and 7.25, respectively. The MIPs were used as solid-phase extraction filler, resulting in successful enrichment and separation of ampicillin residue from egg samples. Standard addition recovery experiments revealed that recovery was good with recoveries from the spiked samples ranging from 91.5 to 94.9% and relative standard deviations from 3.6 to 4.2%. The solid-phase extraction MIPs microcolumn was reused 10 times, where it maintained a recovery rate of over 80%. This work presents a sensitive, fast, and convenient method for the determination of trace ampicillin in food samples.

Smart Bandage Based on Molecularly Imprinted Polymers (MIPs) for Diclofenac Controlled Release

The aim of the present study was the development of a “smart bandage” for the topical administration of diclofenac, in the treatment of localized painful and inflammatory conditions, incorporating Molecularly Imprinted Polymers (MIPs) for the controlled release of this anti-inflammatory drug. For this purpose, MIP spherical particles were synthesized by precipitation polymerization, loaded with the therapeutic agent and incorporated into the bandage surface. Batch adsorption binding studies were performed to investigate the adsorption isotherms and kinetics and the selective recognition abilities of the synthesized MIP. In vitro diffusion studies were also carried out using Franz cells and the obtained results were reported as percentage of the diffused dose, cumulative amount of diffused drug, steady-state drug flux and permeability coefficient. Moreover, the biocompatibility of the developed device was evaluated using the EPISKIN™ model. The Scatchard analysis indicated that the prepared MIP is characterized by the presence of specific binding sites for diclofenac, which are not present in the corresponding non-imprinted polymer, and the obtained results confirmed both the ability of the prepared bandage to prolong the drug release and the absence of skin irritation reactions. Therefore, these results support the potential application of the developed “smart bandage” as topical device for diclofenac sustained release.

Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1–3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1–3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1–3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. We also showed that recombinant proteins Ly2.1–3 (rLy2.1–3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1–3 could specifically bind to LPS. Finally, we found that Ly2.1–3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1–3 can function as AMPs.

Identification of resolvin D2 receptor mediating resolution of infections and organ protection

Endogenous mechanisms that orchestrate resolution of acute inflammation are essential in host defense and the return to homeostasis. Resolvin (Rv)D2 is a potent immunoresolvent biosynthesized during active resolution that stereoselectively stimulates resolution of acute inflammation. Here, using an unbiased G protein–coupled receptor-β-arrestin–based screening and functional sensing systems, we identified a receptor for RvD2, namely GPR18, that is expressed on human leukocytes, including polymorphonuclear neutrophils (PMN), monocytes, and macrophages (MΦ). In human MΦ, RvD2-stimulated intracellular cyclic AMP was dependent on GPR18. RvD2-stimulated phagocytosis of Escherichia coli and apoptotic PMN (efferocytosis) were enhanced with GPR18 overexpression and significantly reduced by shRNA knockdown. Specific binding of RvD2 to recombinant GPR18 was confirmed using a synthetic 3H-labeled-RvD2. Scatchard analysis gave a Kd of ∼10 nM consistent with RvD2 bioactive concentration range. In both E. coli and Staphylococcus aureus infections, RvD2 limited PMN infiltration, enhanced phagocyte clearance of bacteria, and accelerated resolution. These actions were lost in GPR18-deficient mice. During PMN-mediated second organ injury, RvD2’s protective actions were also significantly diminished in GPR18-deficient mice. Together, these results provide evidence for a novel RvD2–GPR18 resolution axis that stimulates human and mouse phagocyte functions to control bacterial infections and promote organ protection.

Selective Solid-Phase Extraction of Zinc(II) from Environmental Water Samples Using Ion Imprinted Activated Carbon

A simple ion imprinted amino-functionalized sorbent was synthesized by coupling activated carbon withiminodiacetic acid, a functional compound for metal chelating, through cyanoric chloride spacer. The resulting sorbent has been characterized using FTIR spectroscopy, elemental analysis, and thermogravimetricanalysis and evaluated for the preconcentration and determination of trace Zn(II) in environmental watersamples. The optimum pH value for sorption of the metal ion was 6–7.5. The sorption capacity of the functionalized sorbent was 66.6 mg/g. The chelating sorbent can be reused for 10 cycles of sorption-desorption without any significant change in sorptioncapacity. A recovery of 100% was obtained for the metal ion with 0.5 M nitric acid as the eluent. Compared with nonimprinted polymer particles, the preparedZn-imprinted sorbent showed high adsorption capacity, significant selectivity, and good site accessibility for Zn(II). Scatchard analysis revealed that the homogeneous binding sites were formed in the polymer. The equilibrium sorption data of Zn(II) by modified resin were analyzed by Langmuir, Freundlich, Temkin, and Redlich-Peterson models. Based on equilibriumadsorption data, the Langmuir, Freundlich, and Temkin constants were determined as 0.139, 12.82, and 2.34, respectively, at 25°C.

Preparation of Henriol C Molecularly Imprinted Polymer Microspheres

Molecular imprinted polymer micrcosheres (MIPMs) were prepared through precipitation polymerization by henriol C using as the template molecule. The morphologies of synthesized MIPMs were characterized by scanning electronmicroscope (SEM). Systematic investigations of the influences of key synthetic conditions, including functional monomers, cross-linkers and porogens, on the morphologies, yields and the recognition properties of the MIPs were conducted. The results indicated that the morphologies of MIPs with DVB as cross-linker was perfect, but their binding affinity is lower than that of MIPs with TRIM or EDMA as cross-linkers. And particle size of MIPs with TRIM as cross-linkers is small but with high binding affinity. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The application of MIPs with high affinity and excellent stereo-selectivity toward henriol C in solid-phase extraction (SPE) column might offer a novel method for the enrichment and determination of sesquiterpenoids in the traditional herbal medicine.