Reductive amination of ω-conotoxin MVIIA: synthesis, determination of modification sites, and self-assembly.

Peptide drugs have disadvantages such as low stability, short half-life and side effects, which limit their widespread use in clinical practice. Therefore, peptide drugs can be modified to improve these disadvantages. Numerous studies have shown that alkyl-modified peptide drugs can self-assemble to prolong the duration of efficacy and/or reduce side effects. However, the commonly used solid-phase synthesis method for alkyl-modified peptides is time-consuming. To overcome this, a simple reductive amination reaction was employed, which can directly graft the alkyl chain to the peptide sequence and effectively avoid stepwise synthesis from C- to N-terminal with amino acids. In this study, ω-conotoxin MVIIA was used as the peptide drug, while myristic aldehyde was used as the alkylating agent. To obtain the maximum productivity of modified peptides, the molar ratio of peptide MVIIA to myristic aldehyde in the reductive amination reaction was optimized. Furthermore, the peptide modification sites in this reaction were confirmed by secondary mass spectrometry analysis. Besides, alkyl-modified peptide MVIIA was able to form micelles by self-assembly and improved stability in serum, which was related to our previous work where myristoylated peptide MVIIA micelles can improve the drug stability. Finally, this study was intended to provide a methodological basis for modifying the alkyl chain of peptide drugs.

Nonivamide inhibits proliferation of human corneal epithelial cells by inducing cell cycle arrest and oxidative stress.

Nonivamide, an agonist of transient receptor potential vanilloid type 1 (TRPV1), is widely used as a riot control agent, police incapacitant spray and pesticide. Although generally considered non-fatal, eye discomfort and even ocular injuries caused by such products are common. Little research has been conducted on the effects of nonivamide on corneal epithelial cells. Cell viability, impedance, flow cytometry, western blotting, and real-time fluorescence analyses were performed to investigate the effects of nonivamide on human corneal epithelial cells (HCE-T cells). We found that nonivamide impaired proliferation at subtoxic doses (100 µM and 200 µM) in HCE-T cells. Next, we described the mechanisms of action of nonivamide. Nonivamide caused cell cycle arrest by increasing p21 and decreasing cyclin D1. TRPV1 was activated by nonivamide, leading to an influx of Ca2+. Enhanced Ca2+ influx partially contributed to oxidative stress. Mitochondrial membrane potential (MMP) also decreased. All combined stress resulted in the inhibition of cell proliferation in HCE-T cells. In summary, nonivamide inhibited the proliferation of HCE-T cells at sub-toxic doses by inducing cell cycle arrest and oxidative stress. Our data demonstrate the corneal toxicity of nonivamide and explain the mechanisms underlying nonivamide-induced corneal injury.

Different Size Formulations of Fluopyram: Preparation, Antifungal Activity, and Accumulation in the Fungal Pathogen Botrytis cinerea.

Nanotechnology is revolutionizing the efficient production and sustainable development of modern agriculture. Understanding the pesticide activity of both nano- and conventional methods is useful for developing new pesticide formulations. In this study, three solid fluopyram formulations with varying particle sizes were developed, and the mechanisms underlying the difference in the antifungal activity among these formulations were investigated. Wet media milling combined with freeze drying was used to prepare fluopyram nanoparticles (FLU-NS) and a micron-sized solid formulation (FLU-MS), and a jet grinding mill was employed to fabricate fluopyram wettable powder (FLU-WP). The mean particle sizes of FLU-NS, FLU-MS, and FLU-WP were 366.8 nm, 2.99 µm, and 10.16 µm, respectively. Notably, FLU-NS displayed a toxicity index against Botrytis cinerea (gray mold) that was approximately double those of FLU-MS and FLU-WP. Similar trends were noticed in the antifungal tests on Alternaria solani. The uptake of FLU-NS by B. cinerea was approximately twice that of FLU-MS and FLU-WP, indicating that fluopyram nanoparticles are more easily taken up by the pathogen (B. cinerea), and display better bioactivity than the larger fluopyram particles. Therefore, the nanosizing of pesticides appears to be a viable strategy to enhance efficiency without increasing the amount of pesticide used.

The implication of mesenteric functions and the biological effects of nanomaterials on the mesentery.

A growing number of nanomaterials are being broadly used in food-related fields as well as therapeutics. Oral exposure to these widespread nanomaterials is inevitable, with the intestine being a major target organ. Upon encountering the intestine, these nanoparticles can cross the intestinal barrier, either bypassing cells or via endocytosis pathways to enter the adjacent mesentery. The intricate structure of the mesentery and its entanglement with the abdominal digestive organs determine the final fate of nanomaterials in the human body. Importantly, mesentery-governed dynamic processes determine the distribution and subsequent biological effects of nanomaterials that cross the intestine, thus there is a need to understand how nanomaterials interact with the mesentery. This review presents the recent progress in understanding the mesenteric structure and function and highlights the importance of the mesentery in health and disease, with a focus on providing new insights and research directions around the biological effects of nanomaterials on the mesentery. A thorough comprehension of the interactions between nanomaterials and the mesentery will facilitate the design of safer nanomaterial-containing products and the development of more effective nanomedicines to combat intestinal disorders.

Self-Assembly Nanostructure of Myristoylated ω-Conotoxin MVIIA Increases the Duration of Efficacy and Reduces Side Effects.

Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.

[Application of self-assembly in polypeptide drugs: a review].

Self-assembly refers to the spontaneous process where basic units such as molecules and nanostructured materials form a stable and compact structure. Peptides can self-assemble by non-covalent driving forces to form various morphologies such as nanofibers, nano layered structures, and micelles. Peptide self-assembly technology has become a hot research topic in recent years due to the advantages of definite amino acid sequences, easy synthesis and design of peptides. It has been shown that the self-assembly design of certain peptide drugs or the use of self-assembled peptide materials as carriers for drug delivery can solve the problems such as short half-life, poor water solubility and poor penetration due to physiological barrier. This review summarizes the formation mechanism of self-assembled peptides, self-assembly morphology, influencing factors, self-assembly design methods and major applications in biomedical field, providing a reference for the efficient use of peptides.

Establishment of a High Throughput Screening System for GABAA1 Modulators in Living Cells.

BACKGROUND: The incidence of sleep disorders is more than 27% in the worldwide, and the development of novel sleep drugs that target GABAA receptors is of great interest. Traditional drug screening methods restrict the discovery of lead compounds, the high-throughput screening system is a powerful means for the lead compounds discovery of sleep drug. METHODS: The GABAA1-CHO cell line stably expressing α1ß2γ2L was constituted by cotransfection of α1, ß2 and γ2L subunits into CHO-T-Rex cells. The high-throughput screening method of membrane potential targeting GABAAR was established and optimized. The optimized method was used to screen the compound library, and the compounds with high activity were obtained. The active compounds were confirmed in vitro by electrophysiological detection technique, and the sleep effects of compounds in vivo were detected by pentobarbital sodium sleep model in mice. RESULTS: A stable cell line expressing human GABAA1 receptor in CHO-T-Rex cells was generated and used to establish a functional high-throughput screening assay based on the measurement of membrane potential changes in living cells by fluorometric imaging plate reader (FLIPR). The assay was further used to detect the dose-effect relationships of tool compounds, the EC50 values of agonist GABA (137.42 ± 26.31 nM), positive allosteric modulator diazepam (3.22 ± 0.73 µM), and antagonist gabazine (0.16 ± 0.04 µM), blocking agents bicuculine (0.47 ± 0.06 µM) and PTX (6.39 ± 1.17 µM). In the meanwhile, the compounds were screened from a compound library (10000) by the membrane potential dye assay. Selected 4 active compounds were further identified for their EC50 values in vitro by electrophysiological method, the EC50 values of 4 compounds were further determined as 1.37 ± 0.43 µM, 0.69 ± 0.17 µM, 0.77 ± 0.16 µM, and 1.62 ± 0.29 µM. Furthermore, the pentobarbital sleep rate and the sleep time of mice pretreated with 4 active compounds by oral administration were significantly increased compared with mice pretreated with a negative control in vivo experiment. CONCLUSION: We successfully generated a stable CHO cell line expressing human GABAA1 by induced expression strategy which decreased cytotoxicity. Then, developed an efficient membrane potential detection method for high-throughput screening, the assay based on the stable cell line could distinguish different types of GABAA1 modulators, which would be an effective in vitro system to screen the GABAAR-targeted compounds. Compared with the patch clamp electrophysiological detection method, the membrane potential detection method has higher detection flux for compounds and higher detection sensitivity for active compounds.

Subacute sarin exposure disrupted the homeostasis of purine and pyrimidine metabolism in guinea pig striatum studied by integrated metabolomic, lipidomic and proteomic analysis.

Sarin was used as a chemical weapon due to its high neurotoxicity and mortality. Subacute sarin induced cognitive and behavioral disorder. However, the underlying mechanism is still unclear. Here we offered a multi-omic approach for the analysis of altered metabolites, lipids, and proteins to explore the neurotoxicity of subacute sarin. Guinea pigs were administered between the shoulder blades 16.8 µg/kg of sarin in a volume of 1.0 ml/kg body weight by subcutaneous injection once daily for 14 days. At the end of the final injection, guinea pigs were sacrificed, and striatum were dissected for analysis. A total of 138 different metabolites were identified in the metabolome analysis. Lipids and lipid-like molecules is the largest group (38.41%). For lipidomic analysis, a total of 216 lipids were identified. In proteomic study, over 4300 proteins were identified and quantified. By integrating these enriched components, we demonstrated that the joint pathways disturbed by subacute sarin mainly involving lipid, purine and pyrimidine metabolism in guinea pig striatum. Overall, this study highlights the powerfulness of omics platforms to deepen the understanding of nerve agents caused neurotoxicity.

Editing flagellin derivatives for exploration of potent radioprotective agents.

Exploration of medical radiation countermeasures (MRCs) has great implications in protection of mammals from radiation damages. While flagellin has been recently reported to show radioprotective effects, the relationships between flagellin structure and radioprotective activity are rarely explored. Herein, we deliberately edited the amino acid sequence of flagellin in its binding domain with toll-like receptor 5 (TLR5) for exploration of potent flagellin derivatives (Fds). An in vitro screening paradigm was developed to examine the radioprotective effects of six engineered Fds. Notably, mutation of 103 threonine on flagellin into asparagine resulted in a potent MRC candidate (Fd-T103N) displaying 1.28-fold increment of interactions with TLR5. Fd-T103N was able to further activate NF-κB pathway, induce immune protective cytokine (e.g. G-CSF) release, and significantly ameliorate γ-irradiation induced cell death. The protection effects of Fd-T103N were further validated in mice exposed to 10 Gy γ-irradiations. Compared to parent flagellin, Fd-T103N treatment showed higher G-CSF release in mouse blood, lower intestine damages, and 13% increments of mouse survival rates. In short, the established predictive paradigm could greatly reduce the labor-, time- and animal-costs in exploration of MRC candidates. Fd-T103N is a promising candidate of investigational new drug for radioprotection.

Repeated low-dose exposures to sarin disrupted the homeostasis of phospholipid and sphingolipid metabolism in guinea pig hippocampus.

Repeated low-level exposure to sarin results to hippocampus dysfunction. Metabonomics involves a holistic analysis of a set of metabolites in an organism in the search for a relationship between these metabolites and physiological or pathological changes. The objective of the present study was to evaluate the effects of repeated exposure to low-level sarin on the metabonomics in hippocampus of a guinea pig model. Guinea pigs were divided randomly into control and sarin treated groups (n = 14). Guinea pigs in the control group received saline; while the sarin-treated group received 0.4×LD50 (16.8 µg/kg) sarin. Daily injections (a total of 14 days) were administered sc between the shoulder blades in a volume of 1.0 ml/kg body weight. At the end of the final injection, 6 animals in each group were chosen for Morris water maze test. The rest guinea pigs (n = 8 for each group) were sacrificed by decapitation, and hippocampus were dissected for analysis. Compared with the control-group, the escape latency in sarin-group was significantly (p < 0.05) longer while the crossing times were significantly decreased in the Morris water task (p < 0.05). Sarin inhibited activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in hippocampus. The AChE activity of hippocampus from sarin-treated groups is equivalent to 59.9 ± 6.4 %, and the NTE activity of hippocampus from sarin-groups is equivalent to 78.1 ± 8.3 % of that from control-group. Metabolites were identified and validated. A total of 14 variables were selected as potential biomarkers. Phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), Lysophosphatidylethanolamine (LysoPE or LPE)] and sphingolipids (SPs) [sphinganine (SA), phytosphingosine (PSO) and sphinganine-1-phosphate (SA1P)] were clearly modified. In conclusion, repeated low-dose exposures to sarin disrupted the homeostasis of phospholipid and sphingolipid metabolism in guinea pig hippocampus and may lead to a neuronal-specific function disorders. Identified metabolites such as SA1P need to be studied more deeply on their biological function that against sarin lesions. In future research, we should pay more attention to characterize the physiological roles of lipid metabolism enzymes as well as their involvement in pathologies induced by repeated low-level sarin exposure.

Toll-like receptor 5 agonist CBLB502 induces radioprotective effects in vitro.

CBLB502 derived from Salmonella flagellin is a novel agonist of Toll-like receptor 5 (TLR5). It has been shown that CBLB502 can exert high radioprotective efficacy on mice and primates from both GI and hematopoietic syndromes during whole-body irradiation with low toxicity and immunogenicity. However, no effective system has been used to investigate the protective effect of CBLB502 against irradiation and the related mechanism in vitro. In this study, we investigated the radioprotective properties of CBLB502 in HEK293-N-T cells constitutively expressing human TLR5 and NF-κB-dependent luciferase. HEK293-N-T cells were treated with different doses of CBLB502 prior to 60Co-γ ray irradiation. After irradiation, cell viability was real-time measured for 4 days by using the real-time cell analysis system. We found that CBLB502 was capable of efficiently maintaining the survival rate of irradiated HEK293-N-T cells. Then apoptotic cell death and cell cycle were detected by flow cytometry. The results showed that CBLB502 pre-treatment could reduce the apoptosis and promote the recovery of irradiated HEK293-N-T cells from G2-phase arrest in a dose-dependent manner. Our data indicated that CBLB502 has a direct radioprotective effect in vitro via anti-apoptosis and promotes cell cycle recovery. The method developed here could be an effective in vitro system to screen other TLR5-target radioprotectants like CBLB502.

Influence of Paeonia lactiflora roots extract on cAMP-phosphodiesterase activity and related anti-inflammatory action.

BACKGROUND: Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in TCM. Research has shown baishao to have positive pharmacological actions, including, particularly, anti-inflammatory properties. In this paper we studied the influence of baishao extract on cAMP-phosphodiesterase (PDE) activity and related anti-inflammatory action to identify new pharmacologic action for its clinically widespread use. METHODS: PDE activity was calculated by cAMP change examined with HPLC, respiratory burst of neutrophils was detected with method of cytochrome C reduction, elastase release was indicated with the substrate reduction, rat arthritis model was caused by complete Freund's adjuvant, mouse capillary permeability model was made by acetic acid, and chemical constituents of baishao extract was identified by HPLC, mass spectroscopy and NMR spectrum. RESULTS: Baishao extract had significant inhibition on cAMP-PDE activity (p<0.01), had dose dependent restraint on neutrophils respiratory burst (p<0.001), had inhibition at low concentration and promotion at high concentration on elastase release (p<0.05), and had obvious restraint on local inflammation of animal model (p<0.01). Analysis of HPLC, mass spectroscopy and NMR spectrum showed baishao extract mainly had five components (identified as gallic acid, paeoniflorin sulfonate, albiflorin, paeoniflorin and benzoic acid), among which gallic acid had the largest inhibition on cAMP-PDE activity. CONCLUSION: The anti-inflammatory effects of baishao may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inhibition on cAMP-PDE.

Flavonol glycosides and monoterpenoids from Potentilla anserina.

Potentilin A (1), a rare diflavonol ester of mu-truxinic acid and a new normonoterpenoid, 2,6-dimethyl-2,3-dihydro-4-oxo-4H-pyran-2-acetic acid (2), was isolated from Potentilla anserina, together with 19 known flavonol glycosides (3-21) and 2 known monterpenoids (22,23). Their structures were elucidated by means of UV, IR, HR-ESI-MS, 1D and 2D NMR spectroscopic data.

Cytotoxic triterpenoid saponins from the roots of Ardisia crenata.

Ardisiacrenoside I (1), a new triterpenoid pentasaccharide with an unusual glycosyl glycerol side chain, was isolated from Ardisia crenata together with five closely related triterpenoid saponins. Their structures were elucidated by a combination of mass spectrometry, IR, 1D, and 2D NMR spectroscopy. Their cytotoxic activities were evaluated against several different human tumor cell lines by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method.

New hepatoprotective coumarinolignoids from Mallotus apelta.

Three new coumarinolignoids, malloapelins A-C (1-3, resp.), together with three known coumarinolignoids, cleomiscosin A (4), cleomiscosin B (5), and 5'-demethylaquillochin (6), were isolated from the roots of Mallotus apelta MUELL.-ARG. Compounds 1-6 are three pairs of regioisomeric coumarinolignoids. Their structures were elucidated on the basis of spectral evidence. Compounds 3 showed promising hepatoprotective activity against D-galactosamine-induced toxicity in WB-F344 rat hepatic epithelial stem-like cells.

Rare biscoumarins and a chlorogenic acid derivative from Erycibe obtusifolia.

Three coumarins, 7,7'-dihydroxy-6,6'-dimethoxy-3,3'-biscoumarin (1), 7,7'-dihydroxy-6,6'-dimethoxy-8,8'-biscoumarin (2) and 7-O-[4'-O-(3'',4''-dihydroxycinnamyl)-beta-d-glucopyranosyl]-6-methoxycoumarin (3), and a chlorogenic acid derivative, methyl-3-O-(4''-hydroxy-3'',5''-dimethoxybenzoyl)-chlorogenate (4) were isolated from the roots of Erycibe obtusifolia along with four known coumarins, scopoletin (5), scopolin (6), cleomiscosin A (7) and cleomiscosin B (8). Their structures were elucidated by spectroscopic methods. Among them, compounds (1) and (2) are rare carbon-carbon linked symmetrical biscoumarins.

[Determination of primary structure of a novel peptide from mistletoe and its antitumor activity].

AIM: To study the antitumor peptide components in the stems and leaves of mistletoe (Viscum coloratum (Kom.) Nakai), the primary structure of the novel peptide was elucidated. METHODS: Cation exchange, gel filtration and HPLC were employed for isolation and purification. Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry was used to determine the mass. The complete amino acid sequence of the novel peptide was obtained by Edman degradation combined with enzyme digestion. The antitumor activity of the peptide in vitro was studied with MTT method. RESULTS: The primary stucture of the peptide named as viscotoxin B2 is KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK. The IC50 value of viscotoxin B2 on the Rat Osteoblast-like Sarcoma 17/2.8 cells in vitro is 1.6 mg x L(-1). CONCLUSION: Viscotoxin B2 in V. coloratum, which has high similarity with viscotoxins from V. album, showed antitumor activity.