Integration of cloud-based molecular networking and docking for enhanced umami peptide screening from Pixian douban.

This study presents an innovative cloud-based approach, using Pixian Douban, a well-known Chinese fermented seasoning, as a case study, to improve the identification of umami peptides and explore their interactions with the T1R1/T1R3 receptor. A feature-based molecular networking method was utilized to rapidly identify a total of eighteen peptides, including seven previously unrecorded ones. Notably, the umami threshold of QIVK in an aqueous solution was determined to be 0.3215 mmol/L, surpassing the majority of peptides reported in the past three years. Molecular docking analysis further revealed the strong binding of QIVK to T1R3 receptor residues through hydrogen bonds, as well as interactions via salt bridges and electrostatic attractions. As a result, this research significantly contributes to the efficient screening of umami peptides and the elucidation of the molecular basis of umami sensory perception in complex food systems.

PAG neuronal NMDARs activation mediated morphine-induced hyperalgesia by HMGB1-TLR4 dependent microglial inflammation.

Morphine is one of the most effective and widely used analgesic drugs. However, chronic morphine use caused opioid-induced hyperalgesia (OIH). The development of OIH limits the use of morphine. The mechanisms of OIH are not fully understood. Toll-like receptor4 (TLR4) and glutamate receptors in the periaqueductal gray (PAG) are critical in OIH, however, the association between TLR4 and N-methyl-D-aspartate Receptors (NMDARs) activation in PAG remains unclear. Microglia activation, increased TLR4/p65 nuclear factor-kappa B (p65 NF-κB) and proinflammatory cytokines in microglia, and phosphorylation of NMDAR1 subunit (NR1) and NMDAR2B subunit (NR2B) in neurons were observed in PAG of OIH mice. Up-regulations of TLR4/p65 NF-κB and proinflammatory cytokines (IL-1ß, IL-6, TNF-α) in BV2 cells were prevented by inhibiting and knocking down TLR4. By inhibiting myeloid differentiation factor 2 (MD2) and knocking down the High-mobility group box 1 (HMGB1), we found that morphine activated TLR4 by HMGB1 but not MD2. We co-cultured Neuro-2a (N2A) with BV2 microglial cell line and found that instead of directly phosphorylating NMDAR subunits, morphine increased the phosphorylation of NR1 and NR2B by inducing TLR4-mediated microglia inflammation. Knocking TLR4 out of PAG by Lentivirus-GFP-TLR4 shRNA reversed these changes and relieved OIH. Our findings suggested that the secretion of HMGB1 induced by morphine-activated TLR4 in microglia, and the proinflammatory factors released by activated microglia phosphorylated NR1 and NR2B of adjacent neurons, induced increased neuronal excitability. In conclusion, TLR4/NMDARs in PAG were involved in the development and maintenance of OIH and supported novel strategies for OIH treatment.

Chronic exposure of alcohol triggers microglia-mediated synaptic elimination inducing cognitive impairment.

BACKGROUND AND AIMS: Long-term alcohol intake leads to cognitive impairment and dementia. The impairment of the cerebral cortex and limbic structures in alcoholics is associated with the loss of synapses instead of neurons. Synapse loss is considered to be an early and key feature of many neurodegenerative diseases, in which microglia-mediated synapse elimination is vital. However, the underlying mechanisms of synapse loss and cognitive impairment caused by long-term alcohol intake are still largely unknown. METHODS: We investigated the relationship of synapse impairment, the microglial innate immune receptor-TREM2, and microglia-mediated synaptic elimination in long-term alcohol exposure. RESULTS: We found that long-term alcohol exposure increased expression of TREM2, decreased expression of synaptic proteins and glutamate receptor subunits, reduced dendrite spine density, and impaired long-term potentiation (LTP) in the hippocampus. Minocycline reduced the amount of the postsynaptic marker PSD95 in microglia, attenuated dendrite spine density loss, and slow down the forgetting process of already-formed memory. Furthermore, we found that TREM2 participated in microglia-mediated synapse elimination in chronic alcohol exposure in vivo. Significantly fewer PSD95 were detectable in microglial phagolysosomes in TREM2 knockdown mice. Besides, TREM2 gene silencing ameliorated synapse loss, LTP impairment, and forgetting of remote memories. CONCLUSIONS: Our data suggests that TREM2 is associated with synaptic plasticity impairment and memory deficits, indicating microglia-mediated synaptic pruning might be the underlying mechanism involved in synapse loss and memory impairment induced by long-term alcohol intake. These findings provide new evidence for the receptor's participation in neurodegeneration diseases.

Morphine-induced microglial immunosuppression via activation of insufficient mitophagy regulated by NLRX1.

BACKGROUND: Chronic morphine exposure induces immunosuppression in the peripheral and central nervous system, resulting in susceptibility of patients to invading pathogens. Mitophagy is a crucial regulator of inflammation, and dysregulated mitophagy may cause immunosuppression, but whether mitophagy is linked with morphine-induced immunosuppression in the brain remains unknown. NLRX1 is the only mitochondrially localized NOD family receptor protein which serves as a critical regulator in immunity and mitophagy activation, but it remains an enigma how NLRX1 functions in the crosstalk between microglial inflammatory defense and mitophagy in the presence of morphine. METHODS: Primary microglia and astrocytes, BV2 and MA cell lines were utilized. Mice were stimulated with repeated morphine treatment to mimic chronic morphine exposure, and activation of mitophagy, lysosomal functions, and inflammation were assayed in specific brain regions and immune organs with or without NLRX1-silencing. RESULTS: Morphine induced microglial mitophagy in a LC3 (microtubule-associated proteins light chain 3)-dependent manner, which was mediated by NLRX1. Contrastingly, morphine impaired lysosomal functions, including generation, acidification and mitophagosome-lysosome fusion, thus leading to insufficient mitophagy activation in microglia. NLRX1-silencing inhibited mitophagy activity and rescued lysosomal functions including generation and acidification in microglia. The NLRX1-mediated incomplete mitophagy in microglial cells contributed to immunosuppression and vulnerability towards pathogenic challenge after morphine treatment. In vivo, NLRX1-mediated microglial mitophagy activation by morphine was mainly located in the murine brain cortex, striatum, and cerebellum, where NLRX1 functioned as a negative immune regulator and facilitated septic shock. Collectively, microglial immune responses to septic shock were amenable to NLRX1 silencing in the brain with morphine treatment. CONCLUSION: Morphine activated insufficient mitophagy in microglia which was regulated by NLRX1, ultimately leading to host immunosuppression and susceptible conditions in the brain.

MPO/HOCl Facilitates Apoptosis and Ferroptosis in the SOD1G93A Motor Neuron of Amyotrophic Lateral Sclerosis.

BACKGROUND: Oxidative stress and reactive oxygen species (ROS) are important in the pathogenesis of amyotrophic lateral sclerosis (ALS). Hypochlorous acid (HOCl) is a powerful oxidant of the reactive oxygen species (ROS) family. HOCl's role in the progress of ALS remains unclear due to the lack of an effective HOCl detection method. Cumulative evidence supports oxidative damage incurred by mutant hSOD1 contributing to motor neuron death; however, whether HOCl as well as its catalytic enzyme myeloperoxidase (MPO) function in the cell death of SOD1G93A ALS remains elusive. METHODS: The hSOD1WT and hSOD1G93A NSC-34 cell and SOD1G93A ALS mouse models were employed. With a novel fluorescent HOCl probe, HKOCl-3, we detected the expressions of HOCl and its catalytic enzyme, MPO, in the above models in vitro and in vivo. The regulation of MPO/HOCl by hSOD1G93A mutation and cell deaths by MPO/HOCl were also assayed, including apoptosis, ferroptosis, and autophagy. RESULTS: Our results showed that hSOD1G93A mutation promoted the activation of the MPO/HOCl pathway in SOD1G93A ALS cell models. The activation of MPO/HOCl pathways facilitated apoptosis and ferroptosis through increasing the Bax/Bcl-2 ratio and expression of caspase-3 or inhibiting the expressions of GPX4 and NQO1 and thus leading to irreversible lipid peroxidation. Overexpressed FSP1, a glutathione-independent suppressor, could ameliorate ferroptosis. In vivo, we demonstrated that the activation of the MPO/HOCl pathway occurred differently in motor neurons of the motor cortices, brain stems, and spinal cords in male and female SOD1G93A transgenic mice. In addition, inhibiting MPO improved the motor performance of SOD1G93A transgenic mice, as demonstrated by the rotarod test. CONCLUSIONS: We concluded that aggregation of mutant hSOD1 proteins contributed to activation of the MPO/HOCl pathway, triggering apoptosis and ferroptosis in motor neuronal deaths and exerting impaired motor performance.

The roles of PAI-1 gene polymorphisms in atherosclerotic diseases: A systematic review and meta-analysis involving 149,908 subjects.

BACKGROUND: The roles of plasminogen activator inhibitor-1 (PAI-1) gene polymorphisms in atherosclerotic diseases were intensively analyzed, but the results of these studies were inconsistent. Therefore, we performed this study to better assess the relationship between PAI-1 genetic variations and atherosclerosis. METHODS: Eligible studies were searched in PubMed, Medline, Embase and Web of Science. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess relationship between PAI-1 polymorphisms and atherosclerotic diseases. RESULTS: Ninety-nine studies involving 62,739 cases and 87,169 controls were finally included. Significant associations with the risk of atherosclerosis were detected for the rs2227631 polymorphism in the dominant model (95% CI 0.84-1.00), for the rs1799889 polymorphism in the dominant (95% CI 1.01-1.18), recessive (95% CI 0.90-0.98) and allele (95% CI 1.01-1.12) models. Further subgroup analyses based on type of disease and ethnicity of participants suggested that the rs2227631 polymorphism was significantly associated with the risk of coronary artery disease in the dominant (95% CI 0.71-0.94) and allele (95% CI 0.80-0.94) models, whereas the rs1799889 polymorphism was significantly associated with the risk of myocardial infarction (dominant model: 95% CI 1.09-1.57; recessive model: 95% CI 0.71-0.96; allele model: 95% CI 1.05-1.28) and cerebral infarction (dominant model: 95% CI 1.68-3.51; additive model: 95% CI 0.39-0.77; allele model: 95% CI 1.23-2.00). Moreover, the rs1799889 polymorphism was also significantly correlated with the risk of atherosclerosis in both Asians (dominant model: 95% CI 1.10-1.83; allele model: 95% CI 1.03-1.41) and Caucasians (recessive model: 95% CI 0.87-0.97; allele model: 95% CI 1.01-1.12). CONCLUSION: In conclusion, our findings indicate that PAI-1 rs2227631 and rs1799889 polymorphisms may serve as genetic biomarkers of atherosclerotic diseases.

[Antifungal effects of three medicinal crops on Phytophthora nicotianae].

Tobacco black shank is one of the most harmful soil-borne diseases infected by Phytophthora parasitica. In order to probe the control method to this disease, in this study, the mycelial growth rate method was employed to investigate the antifungal effects of extracts from stem-leaf and root, root exudates, and their combination of Scrophularia ningpoensis, Chuanmingshen violaceum and Pinellia ternata The results showed that: ①Stem-leaf and root extracts of S. ningpoensis, C. violaceum and P. ternata exhibited different antifungal activities, and the inhibition increased with the increase of extract concentration. The antifungal effect of S. ningpoensis extracts at 0.5 g•mL⁻¹ was the strongest than other medicinal plants, the inhibition rate of steam-leaf and root extracts reached 74.88%, 69.27%, respectively. The inhibitory effect of C. violaceum and P. ternata was relatively lower, however, there is a significant gain effect after combination of steam-leaf and root extracts of C. violaceum. ②The root exudates of S. ningpoensis, C. violaceum and P. ternata showed fungistasis to Phytophthora nicotianae, and fungistasis was enhanced with the increase of root exudate concentration. The antifungal effect in the order of C. violaceum > S. ningpoensis > P. ternata. ③The antifungal activity of combination of extract and root exudate from S. ningpoensis was similar with the effect of C. violaceum, they were both stronger than P. ternata, and the antifungal activity for three combination were located between the antifungal activity of their extracts and root exudates. S. ningpoensis and C. violaceum can be potentially applied to prevent and control the tobacco black shank.

Serum metabolic changes in rats after intragastric administration of dextromethorphan.

Dextromethorphan is recognized as a substance of abuse around the world. An estimated 3.1 million people between the ages of 12 and 25 years (5.3%) misused over-the-counter cough and cold medications in 2006. In this study, we developed a serum metabolomic method by gas chromatography-mass spectrometry (GC-MS) to evaluate the effect of abuse of dextromethorphan on rats. The dextromethorphan-treated rats were given 12, 24 and 48 mg/kg (low, medium, high) of dextromethorphan by intragastric administration each day for 3 days. Partial least squares-discriminate analysis revealed that intragastric administration of dextromethorphan induced metabolic perturbations. Compared with the control (healthy) group, the levels of propanoic acid, urea, heptafluorobutanoic acid, 2-hexyldecanoic acid and butanedioic acid of the low group decreased; levels of propanoic acid and heptafluorobutanoic acid of the medium group decreased, while that of benzoic acid increased; and levels of 2-hexyldecanoic acid, glycerol and butanedioic acid of the high group increased. These biomarkers are involved in the citric acid cycle, urea cycle, glycerolipid metabolism and tricarboxylic acid cycle. The results indicate that the metabolomic method by GC-MS may be useful to elucidate abuse of dextromethorphan. According to the pathological changes in the liver at different dosages, dextromethorphan is not hepatotoxic after intragastric administration of 12, 24 and 48 mg/kg for 3 days.

[Influence of tobacco-Chuanminshen violaceum rotation on microbe community in soil].

Soil microbes are the important indicator of soil quality. For exploring Chuanminshen violaceum planting to microbial effects in tobacco soil, this paper adopted Illumina MiSeq high-throughput sequencing to research the change of bacteria and fungi at the phylum and genus in the soil. The results showed that the Ch. violaceum planting increased the biodiversity of bacteria and fungi. The influence on fungi was greater than that on bacteria. It greatly increased the sequence of fungi, it obtained 32 978 16S rDNA and 32 229 18S rDNA sequence number. There was no change of the top three phylums in bacteria, but the content changed, Proteobacteria and Acidobacteria reduced by 1.73% and 1.4% respectively, and Actinobacteria increased by 0.65%. The advantage phylum Ascomycete in tobacco reduced by 27.99% to be second advantage phylum after Ch. violaceum planting, and the second advantage phylum Basidiomycete increased by 23.69% to become the first dominant fungi. At the genus, Ch. violaceum planting changed the order of dominant genus and the abundance was also changed. Some changed largely such as uncultured Acidobacteriaceae Subgroup-1, Gemmatimonas, Subgroup-2,uncultured Nitrosomonadaceae for bacteria, norank Sordariales, norank Agaricomycetes, Phialophora for fungi. Especially the rotation increased antagonistic microbes and physiological microbes and decreased pathogenic microbes. So the Ch. violaceum planting can improve the microbe community in tobacco soil.