Neuronal Death Caused by HMGB1-Evoked via Inflammasomes from Thrombin-Activated Microglia Cells.

Microglial cells are a macrophage-like cell type residing within the CNS. These cells evoke pro-inflammatory responses following thrombin-induced brain damage. Inflammasomes, which are large caspase-1-activating protein complexes, play a critical role in mediating the extracellular release of HMGB1 in activated immune cells. The exact role of inflammasomes in microglia activated by thrombin remains unclear, particularly as it relates to the downstream functions of HMGB1. After receiving microinjections of thrombin, Sprague Dawley rats of 200 to 250 gm were studied in terms of behaviors and immunohistochemical staining. Primary culture of microglia cells and BV-2 cells were used for the assessment of signal pathways. In a water maze test and novel object recognition analysis, microinjections of thrombin impaired rats' short-term and long-term memory, and such detrimental effects were alleviated by injecting anti-HMGB-1 antibodies. After thrombin microinjections, the increased oxidative stress of neurons was aggravated by HMGB1 injections but attenuated by anti-HMGB-1 antibodies. Such responses occurred in parallel with the volume of activated microglia cells, as well as their expressions of HMGB-1, IL-1ß, IL-18, and caspase-I. In primary microglia cells and BV-2 cell lines, thrombin also induced NO release and mRNA expressions of iNOS, IL-1ß, IL-18, and activated caspase-I. HMGB-1 aggravated these responses, which were abolished by anti-HMGB-1 antibodies. In conclusion, thrombin induced microglia activation through triggering inflammasomes to release HMGB1, contributing to neuronal death. Such an action was counteracted by the anti-HMGB-1 antibodies. The refinement of HMGB-1 modulated the neuro-inflammatory response, which was attenuated in thrombin-associated neurodegenerative disorder.

Improved analysis ZIC-HILIC-HCD-Orbitrap method for mapping the glycopeptide by mass spectrometry.

Glycosylation is one of the most common post-translational modifications (PTMs). Protein glycosylation analysis is the bottleneck to deeply understand their functions. At present, the LC-MS analysis of glycosylated post-translational modification is mainly focused on the analysis of glycopeptides. However, the factors affecting the identification of glycopeptides were not fully elucidated. In the paper, we have carefully studied the factors, e.g., HILIC materials, search engines, protein amount, gradient duration, extraction solution, etc. According to the results, HILIC materials were the most important factors affecting the glycopeptides identification, and the amphoteric sulfoalkyl betaine stationary phase enriched glycopeptides 6-fold more compared to the amphiphilic ion-bonded fully porous spherical silica stationary phase. We explored the influence of the extraction solutions on glycan identification. Comparing sodium dodecyl sulfate (SDS) and urea (UA), the results showed that N-glycolylneuraminic acid (NeuGc) type of glycan content was found to be increased 1.4-fold in the SDS compared to UA. Besides, we explored the influence of the search engine on glycopeptide identification. Comparing pGlyco3.0 and MSFragger-Glyco, it was observed that pGlyco3.0 outperformed MSFragger-Glyco in identifying glycopeptides. Then, using our optimized method we found that there was a significant difference in the distribution of monosaccharide types in plasma and brain tissue, e.g., the content of NeuAc in brain was 5-fold higher than that in plasma. To importantly, two glycoproteins (Neurexin-2 and SUN domain-containing protein 2) were also found for the first time by our method. In summary, we have comprehensively studied the factors influencing glycopeptide identification than any previous research, and the optimized method could be widely used for identifying the glycoproteins or glycolpeptides biomarkers for disease detection and therapeutic targets.

Thrombin-Induced Microglia Activation Modulated through Aryl Hydrocarbon Receptors.

Thrombin is a multifunctional serine protein which is closely related to neurodegenerative disorders. The Aryl hydrocarbon receptor (AhR) is well expressed in microglia cells involving inflammatory disorders of the brain. However, it remains unclear as to how modulation of AhR expression by thrombin is related to the development of neurodegeneration disorders. In this study, we investigated the role of AhR in the development of thrombin-induced neurodegenerative processes, especially those concerning microglia. The primary culture of either wild type or AhR deleted microglia, as well as BV-2 cell lines, was used for an in vitro study. Hippocampal slice culture and animals with either wild type or with AhR deleted were used for the ex vivo and in vivo studies. Simulations of ligand protein docking showed a strong integration between the thrombin and AhR. In thrombin-triggered microglia cells, deleting AhR escalated both the NO release and iNOS expression. Such effects were abolished by the administration of the AhR agonist. In thrombin-activated microglia cells, downregulating AhR increased the following: vascular permeability, pro-inflammatory genetic expression, MMP-9 activity, and the ratio of M1/M2 phenotype. In the in vivo study, thrombin induced the activation of microglia and their volume, thereby contributing to the deterioration of neurobehavior. Deleting AhR furthermore aggravated the response in terms of impaired neurobehavior, increasing brain edema, aggregating microglia, and increasing neuronal death. In conclusion, thrombin caused the activation of microglia through increased vessel permeability, expression of inflammatory response, and phenotype of M1 microglia, as well the MMP activity. Deleting AhR augmented the above detrimental effects. These findings indicate that the modulation of AhR is essential for the regulation of thrombin-induced brain damages and that the AhR agonist may harbor the potentially therapeutic effect in thrombin-induced neurodegenerative disorder.

Optimized approach for active peptides identification in Cerebrolysin by nanoLC-MS.

Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neurotrophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent). Then the samples were analyzed using nanoLC-MS, followed by the identification of peptides using different sequence analysis software (PEAKS, pNovo and novor). Finally, bioinformatics analysis was performed to predict peptides with potential neuroprotective functions in CBL, such as anti-inflammatory and antioxidant peptides. Results showed that the number of peptides obtained by the MCX method coupled with PEAKS was the highest and the method was the most stable. Bioinformatic analysis of the detected peptides showed that two anti-inflammatory peptides (LLNLQPPPR and LSPSLRLP) and an antioxidant peptide (WPFPR) might be neuroprotective peptides in CBL. In addition, this study found that some peptides in CBL were present in myelin basic protein and tubulin beta chain. The results of this study for the detection of active peptides in CBL laid the foundation for the subsequent study of its active ingredients.

Spider venom-derived peptide JZTX-14 prevents migration and invasion of breast cancer cells via inhibition of sodium channels.

Nav1.5 channel is crucial for the proliferation and migration of breast cancer cells. In this study, we investigated the anticancer effect of JZTX-14, a natural peptide considered an effective antagonist of Nav1.5. First, we successfully isolated and purified the 31 amino acid peptide JZTX-14 containing three pairs of disulfide bonds from spider venom and synthesised JZTX-14 by solid phase synthesis. We then predicted their physiochemical properties and structures in the peptide database. Further, we investigated the effects of natural and synthetic JZTX-14 on the proliferation and migration of MDA-MB-231 breast cancer cells via modulation of sodium current through the Nav1.5 channel. The results showed that both synthetic and natural JZTX-14 inhibited Nav1.5 currents, indicating the successful synthesis of JZTX-14. However, JZTX-14 did not affect MDA-MB-231 cell proliferation but inhibited its migration. Transcriptome analysis revealed that JZTX-14 downregulated S100A4 and FBXO2 and upregulated SERPINB2 in MDA-MB-231 cells. Western blot analysis demonstrated an increased level of the epithelial marker, E-cadherin, and decreased levels of the mesenchymal markers, N-cadherin and vimentin, and matrix metalloproteinase (MMP2), indicating the possible underlying mechanism of the inhibition of MDA-MB-231 cell migration by JZTX-14. This study provides a new target for inhibiting breast cancer metastasis and identifies a potent natural peptide for treating Triple-negative breast cancer.

Roles of Phosphorylation of N-Methyl-D-Aspartate Receptor in Chronic Pain.

Phosphorylation of N-methyl-D-aspartate receptor (NMDAR) is widely regarded as a vital modification of synaptic function. Various protein kinases are responsible for direct phosphorylation of NMDAR, such as cyclic adenosine monophosphate-dependent protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src family protein tyrosine kinases, cyclin-dependent kinase 5, and casein kinase II. The detailed function of these kinases on distinct subunits of NMDAR has been reported previously and contributes to phosphorylation at sites predominately within the C-terminal of NMDAR. Phosphorylation underlies both structural and functional changes observed in chronic pain, and studies have demonstrated that inhibitors of kinases are significantly effective in alleviating pain behavior in different chronic pain models. In addition, the exploration of drugs that aim to disrupt the interaction between kinases and NMDAR is promising in clinical research. Based on research regarding the modulation of NMDAR in chronic pain models, this review provides an overview of the phosphorylation of NMDAR-related mechanisms underlying chronic pain to elucidate molecular and pharmacologic references for chronic pain management.

Biogeography and Ecological Differentiation of Pseudasphondylia gall midges (Diptera: Cecidomyiidae) Distributed in Taiwan and Japan, with Description of a New Species P. kiwiphila sp. nov. and the Southernmost Record of P. elaeocarpi.

Pseudasphondylia species (Diptera: Cecidomyiidae) are known to induce fruit galls on Actinidia rufa (Siebold & Zucc.) Planch. ex Miq. and finger-like leaf galls on Elaeocarpus sylvestris (Lour.) Poir. in Taiwan, but their taxonomic positions remain undetermined. Based on gall morphology and host plants, they were supposed to be the same or allied species of known Japanese congeners, i.e., P. matatabi Yuasa & Kumazawa inducing flower-bud galls on Actinidia polygama (Sieb. et Zucc.) Maxim and P. elaeocarpi Tokuda & Yukawa inducing finger-like leaf galls on E. sylvestris. Species identifications of these Taiwanese species provide us an opportunity to study biogeographical aspects and transition of ecological features in these Pseudasphondylia species distributed in East Asian Arc. Morphological comparisons and species delimitation by molecular analysis indicated that the cecidomyiid on the fruit of A. rufa is distinct from P. matatabi and thus it is described as a species new to science, P. kiwiphila sp. nov. Lin, Tokuda, & Yang. The leaf galler on E. sylvestris was identical to P. elaeocarpi, whose southernmost distribution range extended to Taiwan, a new record of its distribution. COI-based phylogenetic tree (Bayesian inference and IQ tree) of Pseudasphondylia suggested that leaf galling habitat and univoltine life history are ancestral, whereas fruit or flower-bud galling and multivoltine life history are derived. In addition, the monophyletic Actinidia-associated species lineage is sistered to the clade including the remaining Japanese fruit and flower-bud gallers, suggesting that Pseudasphondylia has colonized on the host genus Actinidia once and later speciated on different plant species of the host genus. As a biogeographical aspect of P. elaeocarpi, 2.7% of the COI distance between Japanese and Taiwanese individuals indicates that they have diverged around 1.2 mya, which corresponds to the last but second separation of Taiwan and Japan in the Pleistocene. As for Actinidia-associated Pseudasphondylia species, the two valid species are allopatric and have distinct areas of origin, suggesting they may have speciated allopatrically. Nevertheless, there is still the possibility of ecological speciation due to the following reasons: (1) Host species (and varieties) and unidentified congener of Actinidia-associated Pseudasphondylia are occurring China, revealing potential occurrence of these gall midges. (2) The divergence time (2.2-2.9 mya) of the two known species corresponds to the late Pliocene to Pleistocene, when China, Taiwan, and Japan were part of the East Asian continent. During this period, their host species were sympatric in southeast China. (3) The host of two named Actinidia-associated Pseudasphondylia species each belong to different plant groups with distinct fruit features. These presume that the speciation might have been caused via sympatric host shift.

Original Hosts, Clinical Features, Transmission Routes, and Vaccine Development for Coronavirus Disease (COVID-19).

The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to public concern worldwide. Although a variety of hypotheses about the hosts of SARS-CoV-2 have been proposed, an exact conclusion has not yet been reached. Initial clinical manifestations associated with COVID-19 are similar to those of other acute respiratory infections, leading to misdiagnoses and resulting in the outbreak at the early stage. SARS-CoV-2 is predominantly spread by droplet transmission and close contact; the possibilities of fecal-oral, vertical, and aerosol transmission have not yet been fully confirmed or rejected. Besides, COVID-19 cases have been reported within communities, households, and nosocomial settings through contact with confirmed COVID-19 patients or asymptomatic individuals. Environmental contamination is also a major driver for the COVID-19 pandemic. Considering the absence of specific treatment for COVID-19, it is urgent to decrease the risk of transmission and take preventive measures to control the spread of the virus. In this review, we summarize the latest available data on the potential hosts, entry receptors, clinical features, and risk factors of COVID-19 and transmission routes of SARS-CoV-2, and we present the data about development of vaccines.

Evaluation of current medical approaches for COVID-19: a systematic review and meta-analysis.

BACKGROUND: Because of the lack of vaccination, it is urgent to find effective antiviral agents for COVID-19 treatment. METHOD: Online databases were searched for articles published before or on 22 June 2020. Studies reporting the effectiveness and safety of antiviral agents for COVID-19 were analysed. RESULTS: A total of 42 studies were included in this analysis. Hydroxychloroquine (HCQ) was not associated with the incidence of death (risk ratio (RR)=1.08; 95% CI 0.81 to 1.44) and severe cases (RR=1.05; 95% CI 0.61 to 1.81). Patients treated with HCQ obtained few benefits with respect to the clearance of viral RNA and were more likely to have adverse reactions. HCQ treatment could shorten the body temperature recovery time (weighted mean difference = -1.04; 95% CI -1.64 to -0.45). Lopinavir/ritonavir (LPV/r) (RR=0.90; 95% CI 0.76 to 1.07) and Arbidol (RR=1.09; 95% CI 0.92 to 1.29) were not associated with the negative conversion rate. Integrative Chinese-Western medicine alleviated clinical symptoms and decreased the incidence of severe cases (RR=0.38; 95% CI 0.25 to 0.59). Remdesivir treatment reduced the 14-day mortality rate of patients with severe COVID-19 (RR=0.64; 95% CI 0.44 to 0.94). Convalescent plasma (CP) tended to increase the negative conversion rate (RR=2.47; 95% CI 1.70 to 3.57). CONCLUSION: HCQ, LPV/r and Arbidol bring little benefit in COVID-19 treatment. Integrative Chinese-Western medicine improved the clinical symptoms of patients with COVID-19. Remdesivir and CP might be the potential treatments for patients with severe COVID-19. However, large-scale clinical randomised trials are needed to validate our conclusions.

Modalities and Mechanisms of Treatment for Coronavirus Disease 2019.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly throughout the world. Although COVID-19 has a relatively low case severity rate compared to SARS and Middle East Respiratory syndrome it is a major public concern because of its rapid spread and devastating impact on the global economy. Scientists and clinicians are urgently trying to identify drugs to combat the virus with hundreds of clinical trials underway. Current treatments could be divided into two major part: anti-viral agents and host system modulatory agents. On one hand, anti-viral agents focus on virus infection process. Umifenovir blocks virus recognizing host and entry. Remdesivir inhibits virus replication. Chloroquine and hydroxychloroquine involve preventing the whole infection process, including virus transcription and release. On the other hand, host system modulatory agents are associated with regulating the imbalanced inflammatory reaction and biased immune system. Corticosteroid is believed to be commonly used for repressing hyper-inflammation, which is one of the major pathologic mechanisms of COVID-19. Convalescent plasma and neutralizing antibodies provide essential elements for host immune system and create passive immunization. Thrombotic events are at high incidence in COVID-19 patients, thus anti-platelet and anti-coagulation are crucial, as well. Here, we summarized these current or reproposed agents to better understand the mechanisms of agents and give an update of present research situation.

Taxonomy and biology of a new ambrosia gall midge Daphnephila urnicola sp. nov. (Diptera: Cecidomyiidae) inducing urn-shaped leaf galls on two species of Machilus (Lauraceae) in Taiwan.

Recent field surveys show that galls induced by Daphnephila spp. (Cecidomyiidae) on Machilus spp. (Lauraceae) are common in Taiwan, yet only five species, four leaf-gall inducers and one stem-gall inducer on M. thunbergii, have been named in the past. Here we describe a new species, Daphnephila urnicola sp. nov. Chiang, Yang & Tokuda, inducing urn-shaped galls on leaves of both M. zuihoensis and M. mushaensis. Comparisons of D. urnicola populations on M. zuihoensis and on M. mushaensis, indicate that they belong to one species, a result supported by gall midge morphology, life-history traits, gall shape and structure, the developmental process of gall tissues, fungal associations, and DNA-sequencing data. Size and structure of the gall operculum was found to differ between M. zuihoensis and M. mushahaensis.

Is a Gall an Extended Phenotype of the Inducing Insect? A Comparative Study of Selected Morphological and Physiological Traits of Leaf and Stem Galls on Machilus thunbergii (Lauraceae) Induced by Five Species of Daphnephila (Diptera: Cecidomyiidae) in Northeastern Taiwan.

Mature galls induced by Daphnephila truncicola, D. taiwanensis, D. sueyenae, D. stenocalia, and D. ornithocephala on Machilus thunbergii in northern Taiwan were examined to verify the dictum that the morphology of galls is an expression of the extended phenotype of the respective gall-inducing insect. Based on their length-width ratio, the materials were grouped into either fleshy (those induced by D. taiwanensis and D. sueyenae) or slim galls (those induced by D. truncicola, D. stenocalia, and D. ornithocephala). Stem galls induced by D. truncicola showed an energy level of 0.0178 kJ/g. Among leaf galls, the greatest energy level was in the one induced by D. stenocalia (0.0193 kJ/g), followed by D. sueyenae (0.0192 kJ/g), D. taiwanensis (0.0189 kJ/g), and D. ornithocephala (0.0160 kJ/g). The numbers of reserve and nutritive cell layers in galls were greater in the stem galls induced by D. truncicola, similar to those in the fleshy leaf galls, than in the slim leaf galls. Based on the fungal taxa isolated from the larval chambers and considering the similarities and divergences among gall characteristics, the galls induced by D. truncicola and D. taiwanensis clustered into one, whereas those of D. sueyenae aligned with the 'D. stenocalia-D. ornithocephala' cluster. The present study verified that shapes, structure, nutritive tissues, energy levels, and multiple coexisting fungal taxa within galls reinforce that they are extended phenotypes of the respective gall-inducing Daphnephila species and they represent adaptive evolution of Daphnephila on M. thunbergii.