Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

[Progress in the application of evaluation criteria for clinical efficacy of acupuncture in the treatment of knee osteoarthritis].

Knee osteoarthritis is a chronic joint disease characterized by degeneration of knee cartilage and secondary osteoproliferation, pain and dysfunction, with a high morbidity.Clinical evaluation of efficacy was mainly based on scales, including pain scales, knee function scales, quality of life scales. In order to fully present comprehensive evaluation criteria of acupuncture effect of knee osteoarthritis, this paper reviewed the scales, contrasted their characteristics and scope of application, analyzed the existing problems, and offered proposals to develop and choose efficacy evaluation criteria. Expecting to provide refe-rence frame to evaluate the clinical efficacy of acupuncture in treating knee osteoarthritis in the future.

Pedunculoside, a novel triterpene saponin extracted from Ilex rotunda, ameliorates high-fat diet induced hyperlipidemia in rats.

Pedunculoside (PE) is a novel triterpene saponin extracted from the dried barks of Ilex rotunda Thunb. The present study aims to explore lipid-lowering effects of PE on hyperlipidemia rat induced by high-fat diet. The rats were fed with the high-fat diet and subjected to intragastric administration of PE at doses of 30, 15, or 5 mg/kg daily for 7 weeks. The results demonstrated that treatment with PE for 7-week dramatically decreased serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and reduced liver TC in hyperlipidemia rat induced by high-fat diet. Furthermore, the results also showed that PE modulated the expression of enzymes involved in lipid metabolism including peroxisome proliferator-activated receptor α (PPAR-α), sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FAS) and stearoyl CoA desaturase-1 (SCD-1) mRNA in liver. Besides, PE-treated group decreased weights and diameters of epididymal adipose hyperlipidemia rat. Mechanism study demonstrated that PE regulated PPAR-γ, CCAAT/Enhancer-binding Protein α (C/EBPα)、and SREBP-1 expression as well as inhibited phosphorylation of AMPK in MDI (methylisobutylxanthine, dexamethasone, insulin) induced-3T3L1 cells. Molecular Docking confirmed interaction between PE with proteins involving PPAR-γ, C/EBPα and SREBP-1. In summary, these findings may support that PE is a novel lipid-lowering drug candidate.

[Degradation Process of Exogenous Naphthenic Acids and Their Effects on Microbial Community Structure in Soil].

Naphthenic acids (NAs) are a natural component of petroleum, which account for about 2% of severe ecological toxicity in addition to polycyclic aromatic hydrocarbons. With the growth in demand for energy, a large number of NAs have leaked into soil environments through oil industry processes, which have caused enormous potential threats to human health and ecosystems. However, there are few studies about the degradation process of exogenous NAs and their effects on microbial community structures in soil. This research explores the degradation process of NAs and their dynamics in microbial communities in soil by adding a high concentration of 180 mg·kg-1 of NAs to natural, clean soil with the aid of liquid chromatography and high-throughput sequencing technologies. This study found that:① Natural clean soil has a strong capability to degrade high concentration of NAs with about 50% of the NAs degraded within 5 days, which stabilized at 80% after 30 days of the experiment; ② Pollution with NAs obviously alters the microbial community structure as the number of specific OTU increased and were mainly distributed in phylum of unidentified Proteobacteria and Bacteroidetes; ③ Under high concentrations of NAs, the content of Bacteroidetes and Acidobacteria phylum and the γ-Proteobacteria of Proteobacteria phylum all increased swiftly and were speculated to be a potential agents for NA degradation, with the relative abundance ratio of Bacteroidetes and Acidobacteria increasing from 4.2% and 2% to 20.3% and 5.5%, respectively, while a 24.8% decrease was found in Actinobacteria phylum; ④ This study revealed the degradation process of exogenous NAs and their effects on microbial community structure in soil, which provided scientific support for the ecological restoration of petroleum pollution and further study in this area.

ß-Thymosins participate in antiviral immunity of red swamp crayfish (Procambarus clarkii).

ß-Thymosins participate in numerous biological activities, including cell proliferation and differentiation, wound healing, and anti-inflammatory and antimicrobial activities. Many studies have investigated vertebrate ß-thymosins, whereas few reports have focused on invertebrate ß-thymosins. In this study, nine isoforms of ß-thymosins (PcThy-1 to PcThy-8) were identified from the red swamp crayfish Procambarus clarkii. The isoforms contained different numbers of the thymosin ß actin-binding motif. PcThy-1 contained one thymosin ß actin-binding motif, whereas PcThy-8 contained eight motifs. Western blot analysis with anti-PcThy-4 antibody showed that three to six isoforms were present in one tissue, and PcThy-4, PcThy-5, PcThy-6, and PcThy-7 were the main isoforms in several tissues. Time course expression analysis of PcThys at the protein level showed that PcThy-4 was upregulated in hemocytes and gills after white spot syndrome virus (WSSV) challenge. PcThy-4, which contained four thymosin ß actin-binding motifs, was selected for further research. Tissue distribution analysis by quantitative real-time PCR showed that PcThy-4 was present in tissues of the hemocytes, heart, hepatopancreas, gills, stomach, and intestine at the transcriptional level. Transcriptional expression profiles showed that PcThy-4 was upregulated after WSSV challenge. In vivo RNAi and protein injection assay results showed that PcThy-4 inhibited the replication of WSSV in crayfish and enhanced the survival rate after WSSV infection. Furthermore, PcThy-4 promoted hemocyte phagocytosis of WSSV. Overall, results suggested that PcThys protected crayfish from WSSV infection and played an important role in antiviral immune response.

A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response.

Anti-lipopolysaccharide factors (ALFs) are a group of critical effector molecules with a broad spectrum of antimicrobial activities in crustaceans. Four groups of ALFs (A, B, C, and D) have been identified in peneaid shrimp. In the study, we identified a new group of ALFs (designated as MjALF-E) from Marsupenaeus japonicus. This new group (group E) included MjALF-E1 and E2. MjALF-E1 was highly expressed in hemocytes, heart, and intestine, whereas E2 was highly expressed in gills, stomach, and intestine. Expressions of both MjALF-E1 and E2 were upregulated by bacterial challenge. Synthesized LPS-binding domain peptides of MjALF-E1 and E2 strongly bind to bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). The recombinant rMjALF-E2 showed relatively weak binding activity to LPS and PGN. Both synthesized peptides and rMjALF-E2 exhibited antimicrobial activity against Gram-negative bacteria, whereas rMjALF-E2 could promote the clearance of bacteria in vivo. After knockdown of MjALF-E2 and infection with Vibrio anguillarum, shrimp showed high and rapid mortality compared with GFPi shrimp. These results suggest that MjALF-Es serves a protective function against bacterial infection in shrimp.

L-Type lectin from the kuruma shrimp Marsupenaeus japonicus promotes hemocyte phagocytosis.

L-Type lectins (LTLs) contain a luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These type I membrane proteins are involved in the early secretory pathway of animals, and have functions in glycoprotein sorting, trafficking and targeting. Recent studies suggest that LTLs may be involved in immune responses in vertebrates, but no functional studies have been reported. This study reports an LTL, designated as MjLTL1, from the kuruma shrimp Marsupenaeus japonicus. MjLTL consists of a signal peptide, leguminous lectin domain, and transmembrane region. It was upregulated following challenge of shrimp with Vibrio anguillarum. MjLTL1 could agglutinate several bacteria with the presence of calcium, and bind to several Gram-positive and Gram-negative bacteria through lipopolysaccharide and peptidoglycan binding. MjLTL1 could enhance the clearance of V. anguillarum in vivo. MjLTL1 silencing by RNA interference could impair bacterial clearance ability. Further study suggested that MjLTL1 promoted hemocyte phagocytosis. To analyze the possible mechanism, a disintegrin and metalloprotease-like protein (MjADAM) mediating the proteolytic release of extracellular domains from the membrane-bound precursors was also studied in the shrimp. MjADAM exhibited similar tissue location and expression profiles to MjLTL1. After knockdown of MjADAM, the hemocyte phagocytosis rate also declined significantly. ADAM was reported to have an ectodomain shedding function to LTL and release the ectodomain of the lectin from cell membrane. Therefore, our results suggest that the extracellular domain of MjLTL1 might be released from the cell surface as a soluble protein by MjADAM, and function as an opsonin involved in the antibacterial immune responses in shrimp.

Antibacterial activity of serine protease inhibitor 1 from kuruma shrimp Marsupenaeus japonicus.

Serine protease inhibitors (Serpins) are a large family of protease inhibitors involved in many critical biological processes such as blood coagulation, fibrinolysis, programmed cell death, development, and innate immunity. We identified MjSerp1, a serpin in the kuruma shrimp Marsupenaeus japonicus. The MjSerp1 cDNA has a 1239 bp open reading frame (ORF) that encodes a 412-amino acid protein with a 23 aa signal peptide and a classic serpin domain. MjSerp1 has a calculated molecular mass of 46.3 kDa and a predicted isoelectric point of 5.51. MjSerp1 is mainly expressed in the hepatopancreas and the intestines, and is moderately expressed in hemocytes. Expression pattern analysis indicated that MjSerp1 is upregulated in the hepatopancreas after Vibrio anguillarum challenge. rMjSerp1 inhibits three Gram-positive bacteria and two Gram-negative bacteria, but does not inhibit phenoloxidase activity. The microorganism binding assay showed that rMjSerp1 closely binds to both Gram-positive and Gram-negative bacteria. MjSerp1 also exhibits inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, indicating that MjSerp1 acts as a microbial serine protease inhibitor. rMjSerp1 injection into shrimp enhances V. anguillarum clearance, but MjSerp1 knockdown through RNA interference impairs Vibrio clearance in vivo. These results indicate that MjSerp1 functions as a direct effector in the bacterial clearance of M. japonicus. All together, our findings provide novel evidences for the serine protease inhibitor in shrimp immunity.

Two novel C-type lectins with a low-density lipoprotein receptor class A domain have antiviral function in the shrimp Marsupenaeus japonicus.

C-type lectins (CTLs) are pattern-recognition receptors (PRRs) that play important roles in immune response. In this study, two new CTLs containing a low-density lipoprotein receptor class A domain (LDLR) and a carbohydrate recognition domain (CRD) were identified in Marsupenaeus japonicus and designated as LdlrLec1 and LdlrLec2. The two CTLs expressed in all the tested tissues of shrimp, however, LdlrLec1 was mainly expressed in hemocytes, heart, gill and intestines, whereas LdlrLec2 was expressed in hepatopancreas and heart. The expression patterns of both LdlrLec1 and LdlrLec2 mRNA were obviously upregulated upon white spot syndrome virus (WSSV) challenge. Injection of recombinant LdlrLec1 or LdlrLec2 into shrimp inhibited WSSV replication, whereas knocking down the expression of LdlrLec1 and LdlrLec2 by RNA interference increased WSSV replication in vivo. The infection rates of WSSV incubated with LdlrLecs were reduced significantly compared with the control group. The LdlrLec proteins could interact with VP28, a major envelope protein of WSSV, which is necessary for the attachment and penetration of WSSV into shrimp cells. These results indicate that LdlrLec1 and LdlrLec2 may function in antiviral response by binding to WSSV and inhibiting their pervasion and replication in shrimp.