Temporin-GHaR Peptide Alleviates LPS-Induced Cognitive Impairment and Microglial Activation by Modulating Endoplasmic Reticulum Stress.

Neuroinflammation is considered an important factor that leads to cognitive impairment. Microglia play a crucial role in neuroinflammation, which leads to cognitive impairment. This study aimed at determining whether temporin-GHaR peptide (GHaR) could improve cognitive function and at uncovering the underlying mechanisms. We found that GHaR treatment alleviated LPS-induced cognitive impairment and inhibited activation of microglia in LPS-induced mice. Furthermore, GHaR inhibited activation of endoplasmic reticulum stress (ERS) and the NF-κB signaling pathway in LPS-induced mice. In vitro, GHaR inhibited M1 polarization of BV2 cells and suppressed TNF-α and IL-6 secretion. Additionally, GHaR neuronal cell viability and apoptosis were induced by LPS-activated microglia-conditioned medium. Moreover, in LPS-induced BV2 cells, GHaR inhibited activation of ERS and the NF-κB signaling pathway. In summary, GHaR improved LPS-induced cognitive and attenuated inflammatory responses via microglial activation reversal. In conclusion, the neuroprotective effects of GHaR were mediated via the ERS signaling pathway.

Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Antimicrobial peptide temporin derivatives inhibit biofilm formation and virulence factor expression of Streptococcus mutans.

Introduction: Temporin-GHa obtained from the frog Hylarana guentheri showed bactericidal efficacy against Streptococcus mutans. To enhance its antibacterial activity, the derived peptides GHaR and GHa11R were designed, and their antibacterial performance, antibiofilm efficacy and potential in the inhibition of dental caries were evaluated. Methods: Bacterial survival assay, fluorescent staining assay and transmission electron microscopy observation were applied to explore how the peptides inhibited and killed S. mutans. The antibiofilm efficacy was assayed by examining exopolysaccharide (EPS) and lactic acid production, bacterial adhesion and cell surface hydrophobicity. The gene expression level of virulence factors of S. mutans was detected by qRT-PCR. Finally, the impact of the peptides on the caries induced ability of S. mutans was measured using a rat caries model. Results: It has been shown that the peptides inhibited biofilm rapid accumulation by weakening the initial adhesion of S. mutans and reducing the production of EPS. Meanwhile, they also decreased bacterial acidogenicity and aciduricity, and ultimately prevented caries development in vivo. Conclusion: GHaR and GHa11R might be promising candidates for controlling S. mutans infections.

In vitro bloodbrain barrier permeability study of four main active ingredients from Alpiniae oxyphyllae fructus.

The fruits of Alpinia oxyphylla Miq., a broadly utilized traditional Chinese medicine, have a number of effects on the central nervous system (CNS). The main active constituents of Alpiniae oxyphyllae fructus (AOF) were nootkatone, tectochrysin, chrysin and protocatechuic acid. An immortalized human brain microvascular endothelial cell (hCMEC/D3) and astrocyte (HA1800) coculture model was used to investigate the permeability of the blood-brain barrier (BBB). The validation of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methods for the four compounds was conducted following industry guidelines. Calibration curves were generated with mean coefficients (R2) better than 0.99. The inter-day and intra-day precisions were less than 8.53% and 7.12%, respectively. The accuracies were lower than ± 11.57%, and recoveries were greater than 86.07%. The samples of the transport experiment were examined, and the apparent permeability coefficients (Papp) were calculated. The efflux ratios of the four compounds are all less than 2. The Papp values of protocatechuic acid, chrysin, nootkatone, tectochrysin were at the level of 10-5, 10-6, 10-6, and 10-7 cm/s, respectively. All four compounds crossed the BBB by passive diffusion, with protocatechuic acid having high permeability, and tectochrysin having poor permeability. This research indicated the permeability of protocatechuic acid, chrysin, nootkatone and tectochrysin through the BBB and offered a foundation for related research on AOF in the treatment of CNS illnesses.

A ratiometric fluorescence sensor for tetracycline detection based on two fluorophores derived from Partridge tea.

Blue fluorescent carbon dots (PCDs) were prepared by hydrothermal method with Partridge tea. The ethanol extract of Partridge tea (PEE) was found to emit red fluorescence. Thus, a novel ratiometric sensor was constructed by simply mixing the two fluorophores derived from Partridge tea. The presence of tetracycline (TET) at lower concentrations enhanced the emission peak at 508 nm of PCDs and had a negligible effect on the emission peak at 680 nm of PEE. TET at higher concentrations led to  quenching  both the fluorescence of PCDs and PEE via inner filter effect and fluorescence resonance energy transfer, separately. Good linearities for the detection of TET were obtained in the ranges 0.67 to 15.00 µM and 33.33 to 266.67 µM, with limit of detection of 0.095 µM. The sensor was successfully applied to detect TET in lake water and milk samples with good recoveries ranging from 93.27 ± 4.04% to 107.30 ± 6.16%. This study provided a simple, selective, sensitive, rapid, and environmentally friendly method of monitoring TET residues in the environment and food.

Preparation of thioglycerol-modified silica through thiol-epoxy click reaction and its application in HILIC for detection of oligosaccharide in beverages.

A novel preparation scheme of thioglycerol-modified silica through thiol-epoxy click reaction was proposed aiming at introducing additional quantities of hydroxyl groups into the structure. When applied as the stationary phase of hydrophilic interaction liquid chromatography (HILIC) for separation of oligosaccharide compounds, the material revealed higher polar separation capability than which synthesized through traditional thiol-ene click reaction. Hydrogen-bond interactions were speculated to be the predominant retention mechanism, while partitioning also participated in the retention of disaccharides and trisaccharides. The column also showed good stability and inter-batch reproducibility. Finally, the column was employed for determination of oligosaccharide compounds in commercial beverages, and good linearities, high accuracy, favorable precision, satisfactory reproducibility and resistance to matrix interference were achieved. In the detection of real samples, the determined content was consistent with the labeled content. This work provided an efficient and practicable method for quantity monitoring of commercial diet drinks in routine laboratory.

Seasonal patterns of rhizosphere microorganisms suggest carbohydrate-degrading and nitrogen-fixing microbes contribute to the attribute of full-year shooting in woody bamboo Cephalostachyum pingbianense.

Compared with the ordinary single-season shooting among woody bamboos in Poaceae, the attribute of full-year shooting in Cephalostachyum pingbianense represents a unique shooting type or mechanism. Nevertheless, except for the overall physiological mechanism, the effect of ecological factors, especially soil microorganisms, on this full-year shooting characteristic remains unclear. In this study, 16S rRNA and ITS rRNA genes were sequenced using the Illumina platform. Our aims were to detect the seasonal changes in rhizospheric microbial communities of C. pingbianense and to discover the correlations of soil microbes with soil properties and bamboo shoot productivity. The results showed that seasonal change had no significant effect on bacterial alpha diversity, but significantly affected bacterial and fungal community structures as well as fungal richness. Among all soil properties examined, soil temperature, soil moisture and organic matter were the predominant factors affecting bacterial community diversity and structure. Soil temperature and soil moisture also significantly influenced fungal community structure, while available phosphorus had the greatest effect on fungal diversity. In each season, bacterial genera Acidothermus, Roseiarcus, and Bradyrhizobium, along with fungal genera Saitozyma, Mortierella, Trichoderma, etc., were dominant in bacterial and fungal communities, respectively. Bacterial community functions in four seasons were dominated by chemoheterotrophy, cellulolysis, and nitrogen fixation. Saprotrophic fungi occupied a high proportion in soil samples of all seasons. In addition, correlation analysis revealed that the bamboo shoot productivity was positively correlated with multiple microbial taxa involved in carbon and nitrogen cycles. It is proposed that highly abundant microbes involved in carbohydrate degradation and nitrogen fixation in the rhizosphere soil may contribute to the attribute of producing bamboo shoots all year round in C. pingbianense. This study is among the few cases revealing the connection between bamboo shooting characteristics and soil microorganisms, and provides new physiological and ecological insights into the forest management of woody bamboos.

Novel ß-cyclodextrin doped carbon dots for host-guest recognition-assisted sensing of isoniazid and cell imaging.

In the present study, novel ß-cyclodextrin doped carbon dots (CCDs) were prepared via a simple one-pot hydrothermal method at a mild temperature (140 °C), using mixtures of ß-cyclodextrin and citric acid as precursors. By characterizing the chemical properties of CCDs prepared at 140 °C and 180 °C, the importance of low-temperature reaction for preservation of the specific structure of ß-CD was elucidated. The CCDs showed excellent optical properties and were stable to changes in pH, ionic strength and light irradiation. Since the fluorescence of the CCDs could be selectively quenched by isoniazid (INZ) through specific host-guest recognition effects, a convenient isoniazid fluorescence sensor was developed. Under the optimal conditions, the sensor exhibited a relatively low detection limit of 0.140 µg mL-1 and a wide detection range from 0.2 µg mL-1 to 50 µg mL-1 for INZ detection. Furthermore, the sensor was employed successfully for the determination of INZ in urine samples with satisfactory recovery (91.1-109.5%), displaying potential in clinical applications. Finally, low cytotoxicity of the prepared CCDs was confirmed using the CCK-8 method, followed by application in HepG2 cell imaging.

Arginine replacement of histidine on temporin-GHa enhances the antimicrobial and antibiofilm efficacy against Staphylococcus aureus.

Antimicrobial peptides (AMPs) show broad-spectrum microbicidal activity against bacteria, fungi, and viruses, and have been considered as one of the most promising candidates to overcome bacterial antimicrobial resistance. Structural modification of AMPs is an effective strategy to develop high-efficiency and low-toxicity antibacterial agents. A series of peptides GHaR6R, GHaR7R, GHaR8R, and GHaR9W with arginine replacement of histidine (His) derived from temporin-GHa of Hylarana guentheri were designed and synthesized. These derived peptides exhibit antibacterial activity against Staphylococcus aureus, and GHaR8R exerts bactericidal effect within 15 min at 4 × MIC (25 µm). The derived peptides caused rapid depolarization of bacteria, and the cell membrane damage was monitored using quartz crystal microbalance with dissipation assay, which suggests that they target cell membranes to exert antibacterial effects. The derived peptides can effectively eradicate mature biofilms of S. aureus. Taken together, the derived peptides are promising antibacterial agent candidates against S. aureus.

Integrated untargeted fecal metabolomics and gut microbiota strategy for screening potential biomarkers associated with schizophrenia.

Schizophrenia (SZ) is a serious neurodevelopmental disorder. As the etiology of SZ is complex and the pathogenesis is not thoroughly understood, the diagnosis of different subtypes still depends on the subjective judgment of doctors. Therefore, there is an urgent need to develop early objective laboratory diagnostic biomarkers to screen different subtypes of patients as early as possible, and to implement targeted prevention and precision medicine to reduce the risk of SZ and improve patients' quality of life. In this study, untargeted metabolomics and 16S rDNA sequencing were used to analyze the differences in metabolites and gut microflora among 28 patients with two types of schizophrenia and 11 healthy subjects. The results showed that the metabolome and sequencing data could effectively discriminate among paranoid schizophrenia patients, undifferentiated schizophrenia patients and healthy controls. We obtained 65 metabolites and 76 microorganisms with significant changes, and fecal metabolite composition was significantly correlated with the differential genera (|r|>0.5), indicating that there was a regulatory relationship between the gut microbiota and the host metabolites. The gut microbiome, as an objective and measurable index, showed good diagnostic value for distinguishing schizophrenia patients from healthy people, especially with a combination of several differential microorganisms, which had the best diagnostic effect (AUC>0.9). Our results are conducive to understanding the complicated metabolic changes in SZ patients and providing valuable information for the clinical diagnosis of SZ.

Four temporin-derived peptides exhibit antimicrobial and antibiofilm activities against methicillin-resistant.

Temporin-GHa (GHa) was cloned from , showing a weak antimicrobial activity. In order to improve its bactericidal efficacy, GHaR6R, GHaR7R, GHaR8R and GHaR9W were designed and synthesized. Compared to the parent peptide, the GHa-derived peptides show potent antimicrobial activities against methicillin-resistant (MRSA), which is the main pathogen with high morbidity and mortality that causes various infections in humans. These peptides exert bactericidal actions on MRSA by permeabilizing the cytoplasmic membranes and damaging membrane integrity. All of the four peptides exhibit excellent stability under harsh conditions, including extreme temperature and salts. Furthermore, they inhibit the formation of biofilm and eradicate mature biofilm of MRSA. The GHa-derived peptides decrease bacterial surface hydrophobicity, autoaggregation and polysaccharide intercellular adhesion synthesis in concentration-dependent manner. Real-time quantitative reverse transcription PCR analysis revealed that the peptides downregulate the expression of adhesion genes involved in biofilm formation. Except for GHaR7R, the other three peptides have low hemolytic toxicity against human erythrocytes. In the presence of human erythrocytes, GHaR7R, GHaR8R and GHaR9W interact with MRSA preferentially. GHaR6R, GHaR8R and GHaR9W show less toxicity toward normal cells HL-7702 and hFOB1.19. These results suggest that the GHa-derived peptides may be promising antimicrobial candidates against MRSA infections.

Hormone and carbohydrate metabolism associated genes play important roles in rhizome bud full-year germination of Cephalostachyum pingbianense.

Cephalostachyum pingbianense is the only woody bamboo species that can produce bamboo shoots in four seasons under natural conditions. So far, the regulatory mechanism of shoot bud differentiation and development is unknown. In the present study, indole-3-acetic acid (IAA), zeatin riboside (ZR), gibberellin A3 (GA3 ) and abscisic acid (ABA) contents determination, RNA sequencing and differentially expressed gene analysis were performed on dormant rhizome bud (DR), growing rhizome bud (GR), and germinative bud (GB) in each season. The results showed that the contents of IAA and ZR increased while ABA content decreased, and GA3 content was stable during bud transition from dormancy to germination in each season. Moreover, rhizome bud germination was cooperatively regulated by multiple pathways such as carbohydrate metabolism, hormone signal transduction, cell wall biogenesis, temperature response, and water transport. The inferred hub genes among these candidates were identified by protein-protein interaction network analyses, most of which were involved in hormone and carbohydrate metabolism, such as HK and BGLU4 in spring, IDH and GH3 in winter, GPI and talA/talB in summer and autumn. It is speculated that dynamic phytohormone changes and differential expression of these genes promote the release of rhizome bud dormancy and contribute to the phenological characteristics of full-year shooting. Moreover, the rhizome buds of C. pingbianense may not suffer from ecodormancy in winter. These findings would help accumulate knowledge on shooting mechanisms in woody bamboos and provide a physiological insight into germplasm conservation and forest management of C. pingbianense.

Transcriptome-wide analysis reveals core transcriptional regulators associated with culm development and variation in Dendrocalamus sinicus, the strongest woody bamboo in the world.

Transcription factors (TFs) play indispensable roles in plant development and stress responses. As the largest woody bamboo species in the world, Dendrocalamus sinicus is endemic to Yunnan Province, China, and possesses two natural variants characterized by culm shape, namely straight or bent culms. Understanding the transcriptional regulation network of D. sinicus provides a unique opportunity to clarify the growth and development characteristics of woody bamboos. In this study, 10,236 TF transcripts belonging to 57 families were identified from transcriptome data of two variants at different developmental stages, from which we constructed a transcriptional regulatory network and unigene-coding protein-TFs interactive network of culm development for this attractive species. Gene function enrichment analysis revealed that hormone signaling and MAPK signaling pathways were two most enriched pathways in TF-regulated network. Based on PPI analysis, 50 genes interacting with nine TFs were screened as the core regulation components related to culm development. Of them, 18 synergistic genes of seven TFs, including nuclear cap-binding protein subunit 1, transcription factor GTE9-like, and ATP-dependent DNA helicase DDX11 isoform X1, involved in culm-shape variation. Most of these genes would interact with MYB, C3H, and ARF transcription factors. Six members with two each from ARF, C3H, and MYB transcription factor families and six key interacting genes (IAA3, IAA19, leucine-tRNA ligase, nuclear cap-binding protein subunit 1, elongation factor 2, and coiled-coil domain-containing protein 94) cooperate with these transcription factors were differentially expressed at development stage of young culms, and were validated by quantitative PCR. Our results represent a crucial step towards understanding the regulatory mechanisms of TFs involved in culm development and variation of D. sinicus.

Temporin-Like Peptides Show Antimicrobial and Anti-Biofilm Activities against Streptococcus mutans with Reduced Hemolysis.

In our previous study, temporin-GHaR (GHaR) showed potent antimicrobial activity with strong hemolytic toxicity. To overcome its weakness, we designed GHaR6R, GHaR7R, GHaR8R, GHaR9R, and GHaR9W by changing the number of positive charges and the hydrophobic surface of GHaR. With the exception of GHaR7R, the hemolytic toxicity of the derived peptides had been reduced, and the antimicrobial activities remained close to the parent peptide (except for GHaR9R). GHaR6R, GHaR7R, GHaR8R, and GHaR9W exhibited a great bactericidal effect on Streptococcus mutans (S. mutans), which is one of the main pathogens causing dental caries. According to the membrane permeation and scanning electron microscope (SEM) analysis, these derived peptides targeted to the cell membranes of planktonic bacteria, contributing to the disruption of the membrane integrity and leakage of the intracellular contents. Moreover, they inhibited the formation of biofilms and eradicated the mature biofilms of S. mutans. Compared with GHaR7R, the derived peptides showed less cytotoxicity to human oral epithelial cells (HOECs). The derived peptides are expected to be the molecular templates for designing antibacterial agents to prevent dental caries.

Synthesis of sheet-like polypyrrole nanowires for the microextraction of trace residues of pyrethroid pesticides in human plasma and molecular dynamics-aided study of adsorption mechanism.

The synthesized sheet-like polypyrrole (ppy) nanowires were used as solid phase extraction materials, followed by gas chromatography-mass spectrometry (GC-MS) for the detection of traces residues of pyrethroid pesticides in human plasma. A multiresidue method was developed and verified for the determination of trace pyrethroid residues (transfluthrin, allethrin, resmethrin, fenpropathrin, etofenprox, fenvalerate) in human plasma. In this study, using the cationic surfactant cetyltrimethylammonium bromide (CTAB) as a soft template, ppy nanowires with regular morphology were prepared by oxidative polymerization. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and other techniques were employed for characterization. Molecular dynamics analyses were used to simulate the adsorption mechanism of each pyrethroid and ppy nanowires. Based on density analysis, molecular recognition analysis, and binding energy, the van der Waals force was considered as an important driving force for the adsorption of pyrethroids and ppy nanowires. The limits of detection (LOD) of six pyrethroids were 0.008-0051 ng mL-1, and the limits of quantification (LOQ) were 0.028-0.162 ng mL-1. The relative standard deviations of ppy nanowires were 2.12-5.09%, and the recoveries of six pyrethroids ranged from 76.9 to 110.4%. The enrichment factors were within the range of 47.09-51.30. The experimental results showed that the method could be an efficient detection method for trace residue analysis of pyrethroid pesticides in complex biological samples. It would be advantageous for clinical monitoring and toxicological studies of pyrethroids.

Amino Acid Profiling Study of Psidium guajava L. Leaves as an Effective Treatment for Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) has become a major disease threatening human health worldwide. At present, the treatment of T2DM cannot cure diabetes and is prone to many side effects. Psidium guajava L. leaves have been reported to possess hypoglycemic activity, and they have been widely used in diabetes treatment in the folk. However, the antidiabetic mechanism has not been clearly explained. Also, the change in amino acid profile can reflect a metabolic disorder and provide insights into system-wide changes in response to physiological challenges or disease processes. The study found that P. guajava L. leaves can decrease fasting blood glucose and lipid levels in type 2 diabetic rats induced by streptozotocin. Through the analysis of amino acid profiling following 20 days of gavage administration, the concentration data were modeled by principal component analysis and orthogonal partial least squares discriminant analysis to find the different metabolites and related metabolic pathways (including cysteine and methionine metabolism, valine, leucine, and isoleucine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis) for the explanation of the hypoglycemic mechanism of P. guajava L., which provides an experimental and theoretical basis for diabetes prediction and for the development of new drugs for the treatment of diabetes.

Photodegradation kinetics, transformation, and toxicity prediction of ketoprofen, carprofen, and diclofenac acid in aqueous solutions.

Photodegradation of 3 commonly used nonsteroidal anti-inflammatory drugs, ketoprofen, carprofen, and diclofenac acid, was conducted under ultraviolet (UV) irradiation. The kinetic results showed that the 3 pharmaceuticals obeyed the first-order reaction with decreasing rate constants of 1.54 × 10-4 , 5.91 × 10-5 , and 7.78 × 10-6 s-1 for carprofen, ketoprofen, and diclofenac acid, respectively. Moreover, the main transformation products were identified by ion-pair liquid-liquid extraction combined with injection port derivatization-gas chromatography-mass spectrometry and high-performance liquid chromatography-quadrupole-time of flight mass spectrometric analysis. There were 8, 3, and 6 transformation products identified for ketoprofen, carprofen, and diclofenac acid, respectively. Decarboxylation, dechlorination, oxidation, demethylation, esterification, and cyclization were proposed to be associated with the transformation of the 3 pharmaceuticals. Toxicity prediction of the transformation products was conducted on the EPI Suite software based on ECOSAR model, and the results indicate that some of the transformation products were more toxic than the parent compounds. The present study provides the foundation to understand the transformation behavior of the studied pharmaceuticals under UV irradiation. Environ Toxicol Chem 2017;36:3232-3239. © 2017 SETAC.