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.

Temporin-GHb-Derived Peptides Exhibit Potent Antibacterial and Antibiofilm Activities against Staphylococcus aureus In Vitro and Protect Mice from Acute Infectious Pneumonia.

With the continuous development of drug resistance in bacteria to traditional antibiotics, the demand for novel antibacterial agents is urgent. Antimicrobial peptides (AMPs) are promising candidates because of their unique mechanism of action and low tendency to induce drug resistance. Previously, we cloned temporin-GHb (hereafter referred to simply as "GHb") from Hylarana guentheri. In this study, a series of derived peptides were designed, namely, GHbR, GHbK, GHb3K, GHb11K, and GHbK4R. The five derived peptides had stronger antibacterial activities against Staphylococcus aureus than the parent peptide GHb and could effectively inhibit the formation of biofilms and eradicate mature biofilms in vitro. GHbR, GHbK, GHb3K, and GHbK4R exerted bactericidal effects by disrupting membrane integrity. However, GHb11K exhibited bacteriostatic efficacy with toroidal pore formation on the cell membrane. In comparison to GHbK4R, GHb3K showed much lower cytotoxicity against A549 alveolar epithelial cells, with an IC50 > 200 µM, which was much higher than its minimal inhibitory concentration (MIC = 3.1 µM) against S. aureus. The anti-infection potential of GHbK4R and GHb3K was investigated in vivo. Compared with vancomycin, the two peptides displayed significant efficacy in a mouse model of acute pneumonia infected with S. aureus. Both GHbK4R and GHb3K also had no obvious toxicity to normal mice after intraperitoneal administration (15 mg/kg) for 8 days. Our results indicate that GHb3K and GHbK4R might be promising candidates for the treatment of bacterial pneumonia infected with S. aureus.

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.

A Temporin Derived Peptide Showing Antibacterial and Antibiofilm Activities against Staphylococcus aureus.

BACKGROUND: Temporin is one family of the shortest antimicrobial peptides found in Ranidae frogs. Staphylococcus aureus is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections in humans. Temporin-GHa (GHa) was previously obtained from Hylarana guentheri, showing weak antibacterial activity against S. aureus. Most temporin peptides are positively charged by arginine and lysine; however, GHa contains histidine. OBJECTIVE: In order to investigate the impact of positively charged amino acid on its antibacterial and antibiofilm activity, GHa4R was designed and synthesized by replacing histidine with arginine in GHa. METHODS: The antibacterial activity and efficacy against S. aureus were detected by minimum inhibitory concentration, minimum bactericidal concentration, and time-killing kinetics assays. The action mechanism was determined by propidium iodide uptake and scanning electron microscopy assays. The antibiofilm activity was measured by the MTT method. Eradication of biofilm was observed by fluorescence microscope. RESULTS: Compared to GHa, GHa4R had stronger antibacterial activity and bactericidal efficacy against S. aureus. Impressively, GHa4R presented antibacterial activity against methicillin-resistant S. aureus (MRSA). It was barely affected by temperature, pH, and storage period, showing high stability. Furthermore, it increased the permeability of the cell membrane and damaged the membrane integrity, leading to cell death. In addition, GHa4R did not induce antibiotic resistance in S. aureus in 30 days, but the MIC of vancomycin was doubled. It not only inhibited S. aureus biofilm formation but also eradicated 24 h-biofilms. CONCLUSION: The above-mentioned characteristics make GHa4R a promising candidate for the treatment of S. aureus infections.

Preparation of ß-CD-Vitexin Microspheres and their Effects on SW480 Cell Proliferation.

OBJECTIVE: In order to overcome the insolution and low bioavailability of the vitexin in vivo, ß-cyclodextrin-vitexin (ß-CD-vitexin) microspheres were prepared, and their effects on the proliferation of SW480 cells were observed. METHODS: Scanning electron microscopy, ultraviolet spectrum, Fourier transform infrared spectroscopy, and release rate analysis identified the formation of ß-CD-vitexin microspheres. MTT assay detected the effect of ß-CD-vitexin microspheres on tumor cell proliferation at 6, 12, 24, and 48 h. Fluorescence microscopy and flow cytometry were used to observe the effect of ß-CD-vitexin microspheres on the apoptosis of SW480 cells. The mRNA expression of the p53 gene was measured by qPCR. RESULTS: ß-CD-vitexin microspheres were successfully prepared. SW480 cell proliferation was inhibited by 0.1, 0.2, and 0.4 mg/mL of ß-CD-vitexin microspheres in a dose- and time-dependent manner, and the mechanism of proliferation inhibition was related to cell apoptosis caused by the upregulated expression of p53 gene. CONCLUSION: The preparation of ß-CD-vitexin sustained release microspheres is feasible, and ß-CDvitexin microspheres have potential anti-colorectal cancer value.

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.

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.

A review of cardiovascular benefits of SGLT2 inhibitors.

Sodium-glucose cotransporter 2 inhibitor (SGLT2I) is a new type of hypoglycemic drug that targets the kidney. As research continues to advance on this topic, it has been found that SGLT2I has multiple protective effects, such as hypoglycemic, cardio-renal protective, antihypertensive, and lipid-lowering effects. This review discusses the current concepts and possible mechanisms of SGLT2I in the treatment of heart failure, myocardial infarction, hypertension, cardiomyopathy and arrhythmia to provide a reference for clinicians to use drugs more reasonably and scientifically.

Distribution and model prediction of antibiotic resistance genes in Weishan Lake based on the indication of Chironomidae larvae.

The widespread contamination of antibiotic resistance genes (ARGs) in freshwater environment are becoming a serious challenge to human health and ecological safety. Rapid and efficient monitoring of ARGs pollution is of great significance to ARGs control. Water, bottom mud, and fish have all been used to indicate ARG contamination in aquatic environments. However, it is unclear whether macrobenthic invertebrates in the food chain of aquatic environments can be indicators of ARG contamination. In this study, we demonstrated that ARGs including tetA gene, sul2 gene, and km gene were distributed in Chironomidae larvae in Weishan Lake. The ARG distribution was related to animal species, body parts, sampling sites, time, urban environment, animal farming, south-to-north water diversion, food chain, antibiotics, and water storage. Mathematical model predictions of ARG contamination in Weishan Lake were constructed based on the structural equation model (SEM) and the distribution of ARG sul2 in Chironomidae larvae. Influencing factors such as water storage, metal elements, antibiotic, and temperature were found to be closely related to the prediction of ARG contamination. This study provided a new indicator for ARG contamination in freshwater environments and a method to predict ARGs contamination.

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.

Structure and Activity of a Selective Antibiofilm Peptide SK-24 Derived from the NMR Structure of Human Cathelicidin LL-37.

The deployment of the innate immune system in humans is essential to protect us from infection. Human cathelicidin LL-37 is a linear host defense peptide with both antimicrobial and immune modulatory properties. Despite years of studies of numerous peptides, SK-24, corresponding to the long hydrophobic domain (residues 9-32) in the anionic lipid-bound NMR structure of LL-37, has not been investigated. This study reports the structure and activity of SK-24. Interestingly, SK-24 is entirely helical (~100%) in phosphate buffer (PBS), more than LL-37 (84%), GI-20 (75%), and GF-17 (33%), while RI-10 and 17BIPHE2 are essentially randomly coiled (helix%: 7-10%). These results imply an important role for the additional N-terminal amino acids (likely E16) of SK-24 in stabilizing the helical conformation in PBS. It is proposed herein that SK-24 contains the minimal sequence for effective oligomerization of LL-37. Superior to LL-37 and RI-10, SK-24 shows an antimicrobial activity spectrum comparable to the major antimicrobial peptides GF-17 and GI-20 by targeting bacterial membranes and forming a helical conformation. Like the engineered peptide 17BIPHE2, SK-24 has a stronger antibiofilm activity than LL-37, GI-20, and GF-17. Nevertheless, SK-24 is least hemolytic at 200 µM compared with LL-37 and its other peptides investigated herein. Combined, these results enabled us to appreciate the elegance of the long amphipathic helix SK-24 nature deploys within LL-37 for human antimicrobial defense. SK-24 may be a useful template of therapeutic potential.

Protective effect of hawthorn vitexin on the ethanol-injured DNA of BRL-3A hepatocytes.

ABSTRACT: Vitexin is a natural active ingredient in hawthorn leaves, which has a wide range of anti-tumor effects. This study was conducted to assess the protective effect of hawthorn vitexin on the ethanol-injured DNA of hepatocytes in vitro and to explore its mechanism. The effect of different concentrations of hawthorn vitexin on ethanol-injured hepatocytes was detected via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method to study the protective effect of hawthorn vitexin on ethanol-injured DNA damage in hepatocytes. Single-cell gel electrophoresis was used to observe the effect of hawthorn vitexin on ethanol-induced DNA damage in hepatocytes, and the Olive tail moment was measured. Cell physiological and biochemical indexes, such as superoxide dismutase activity, malonaldehyde content, and glutathione peroxidase activity, were detected with kits. The mRNA expression of the superoxide dismutase gene was measured via real-time quantitative polymerase chain reaction. It was showed that 0.2, 0.4, and 0.8 mg mL-1 hawthorn vitexin could significantly repair hepatocyte growth and ethanol-induced DNA damage. This effect was closely related to the improvement in superoxide dismutase, malonaldehyde, and glutathione peroxidase. Hawthorn vitexin could be used to repair ethanol-injured hepatocytes through antioxidation effects, and showed potential for the treatment of liver injury.

Fucoxanthin regulates Nrf2 signaling to decrease oxidative stress and improves renal fibrosis depending on Sirt1 in HG-induced GMCs and STZ-induced diabetic rats.

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial cellular defense factor to cope with oxidative stress. Silent information regulator T1 (Sirt1) is a deacetylase with antioxidative stress activity. Fucoxanthin is a marine-derived carotenoid. This study was conducted to investigate whether fucoxanthin could alleviate oxidative stress by activating Sirt1/Nrf2 signaling to alleviate DN. In streptozotocin-induced diabetic rats, fucoxanthin treatment effectively improved renal function, alleviated glomerulosclerosis. Fucoxanthin reversed the decreased protein levels of Sirt1 and Nrf2 in the kidney of diabetic rats and glomerular mesangial cells cultured in high glucose. Conversely, EX527, a Sirt1 inhibitor, counteracted the effect of fucoxanthin on the expression of Nrf2. Furthermore, in vivo and vitro results showed that fucoxanthin treatment reversed the low expression and activity of superoxide dismutase and heme oxygenase 1, depending on Sirt1 activation. Our results suggest that fucoxanthin improves diabetic kidney function and renal fibrosis by activating Sirt1/Nrf2 signaling to reduce oxidative stress.

Short and Robust Anti-Infective Lipopeptides Engineered Based on the Minimal Antimicrobial Peptide KR12 of Human LL-37.

This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of in vitro filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity in vitro against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. In vivo, C10-KR8d is able to reduce bacterial burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.

Altered Dynamic Amplitude of Low-Frequency Fluctuations in Patients With Migraine Without Aura.

Migraine is a chronic and idiopathic disorder leading to cognitive and affective problems. However, the neural basis of migraine without aura is still unclear. In this study, dynamic amplitude of low-frequency fluctuations (dALFF) analyses were performed in 21 patients with migraine without aura and 21 gender- and age-matched healthy controls to identify the voxel-level abnormal functional dynamics. Significantly decreased dALFF in the bilateral anterior insula, bilateral lateral orbitofrontal cortex, bilateral medial prefrontal cortex, bilateral anterior cingulate cortex, and left middle frontal cortex were found in patients with migraine without aura. The dALFF values in the anterior cingulate cortex were negatively correlated with pain intensity, i.e., visual analog scale. Finally, support vector machine was used to classify patients with migraine without aura from healthy controls and achieved an accuracy of 83.33%, sensitivity of 90.48%, and specificity of 76.19%. Our findings provide the evidence that migraine influences the brain functional activity dynamics and reveal the neural basis for migraine, which could facilitate understanding the neuropathology of migraine and future treatment.