PRMT6 deficiency or inhibition alleviates neuropathic pain by decreasing glycolysis and inflammation in microglia.

Microglia induced chronic inflammation is the critical pathology of Neuropathic pain (NP). Metabolic reprogramming of macrophage has been intensively reported in various chronic inflammation diseases. However, the metabolic reprogramming of microglia in chronic pain remains to be elusive. Here, we reported that immuno-metabolic markers (HIF-1α, PKM2, GLUT1 and lactate) were related with increased expression of PRMT6 in the ipsilateral spinal cord dorsal horn of the chronic construction injury (CCI) mice. PRMT6 deficiency or prophylactic and therapeutic intrathecal administration of PRMT6 inhibitor (EPZ020411) ameliorated CCI-induced NP, inflammation and glycolysis in the ipsilateral spinal cord dorsal horn. PRMT6 knockout or knockdown inhibited LPS-induced inflammation, proliferation and glycolysis in microglia cells. While PRMT6 overexpression exacerbated LPS-induced inflammation, proliferation and glycolysis in BV2 cells. Recent research revealed that PRMT6 could interact with and methylate HIF-1α, which increased HIF-1α protein stability. In sum, increased expression of PRMT6 exacerbates NP progress by increasing glycolysis and neuroinflammation through interacting with and stabilizing HIF-1α in a methyltransferase manner, which outlines novel pathological mechanism and drug target for NP.

Toxic dinoflagellate Karenia mikimotoi induces apoptosis in Neuro-2a cells through an oxidative stress-mediated mitochondrial pathway.

The dinoflagellate Karenia mikimotoi is a toxic bloom-forming species that threatens aquaculture and public health worldwide. Previous studies showed that K. mikimotoi induces neurotoxicity; however, the underlying mechanism is poorly understood. In this study, three neural cell lines were used to investigate the potential neurotoxicity of K. mikimotoi. The tested cells were exposed to a ruptured cell solution (RCS) of K. mikimotoi at different concentrations (0.5 × 105, 1.0 × 105, 2.0 × 105, 4.0 × 105, and 6 × 105 cells mL-1) for 24 h, and the RCS decreased cell viabilities and promoted Neuro-2a (N2A) cell apoptosis in a dose-dependent manner. The underlying mechanism was further investigated in N2A cells. At the biochemical level, the RCS stimulated reactive oxygen species (ROS) and malondialdehyde (MDA) formation, decreased SOD activity, and reduced mitochondrial membrane potential (MMP). At the gene level, the moderate RCS treatment (2.0 × 105 cells mL-1) upregulated antioxidant response genes (e.g., nrf-2, HO-1, NQO-1, and cat) to alleviate RCS-induced oxidative stress, while the high RCS treatment (4.0 × 105 cells mL-1) downregulated these genes, thereby aggravating oxidative stress. Meanwhile, apoptosis-related genes (e.g., p53, caspase 3, and bax2) were significantly upregulated and the anti-apoptotic gene bcl2 was suppressed after RCS treatment. Western blotting results for Caspase 3, Bax2 and Bcl2 were consistent with the mRNA trends. These results revealed that K. mikimotoi RCS can induce neural cell apoptosis via the oxidative stress-mediated mitochondrial pathway, providing novel insights into the neurotoxicity of K. mikimotoi.

Sound Field Reconstruction Using Prolate Spheroidal Wave Functions and Sparse Regularization.

Near-field acoustic holography (NAH) based on compressing sensing (CS) theory enables accurate reconstruction of sound fields using a limited number of sampling points. However, the successful implementation of this technique depends on two crucial factors: (1) the appropriate selection or construction of the spatial basis and (2) an effective sparse regularization process. To enhance reconstruction performance for elongated sound sources, this paper proposes a novel sound field reconstruction method that combines prolate spheroidal wave functions (PSWFs) with the orthogonal matching pursuit (OMP) algorithm. In this method, PSWFs serve as a sparse spatial basis for representing the radiated sound field. The sparse coefficients are determined by the OMP algorithm in a linear subspace composed of basic functions that best match the residual error. The OMP algorithm effectively identifies significant components before potentially selecting incorrect ones by setting an appropriate stopping rule. Numerical simulations are conducted using a line-array source model. The results show that the proposed method can accurately reconstruct the sound pressures of the elongated source model using a relatively small number of samplings. In addition, the proposed method exhibits robustness across a wide frequency range, diverse array configurations and various sampling numbers. The experimental results further validate the feasibility and reliability of the proposed method.

Long noncoding RNA uc007nnj.1 mediates neuronal death induced by retinal ischemia/reperfusion in mice via the miR-155-5p/Tle4 axis.

BACKGROUND: Retinal ganglion cells (RGCs) apoptosis is a vital manifestation of retinal ischemia/reperfusion (I/R) injury, yet the underlying mechanisms are not well understood. The contribution of long noncoding RNAs (lncRNAs) to this cellular process is currently being explored. Based on a lncRNA chip assay, we aimed to investigate the role of lncRNA uc007nnj.1 in the pathological process of ischemia-induced RGCs apoptosis. METHODS: Hank's balanced salt solution containing 10 µM antimycin A and 2 µM calcium ionophore for 2 h to construct an ischemic model in RGCs, and elevation of intraocular pressure to 120 mm Hg for 1 h was used to construct a mouse model of retinal I/R injury. RESULTS: In this study, lncRNA uc007nnj.1 was highly upregulated in response to I/R injury in RGCs and mouse retinas. In addition, lncRNA uc007nnj.1 knockdown reduced retinal neuronal cell apoptosis in vitro and in vivo and significantly improved retinal function. DISCUSSION: Mechanistically, the results demonstrated that lncRNA uc007nnj.1 acts as ceRNA competitively binding miR-155-5p, thereby enhancing the expression levels of Tle4, thus aggravating ischemia-related apoptosis in RGCs. CONCLUSIONS: Finally, our study identifies the lncRNA uc007nnj.1/miR-155-5p/Tle4 axis as a potential target for the prevention of I/R-induced retinal neuronal death.

A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a.

Aeromonas hydrophila is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic A. hydrophila is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA- , aerA-hlyA+ , and aerA+hlyA+ ). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.

Alterations of serine racemase expression determine proliferation and differentiation of neuroblastoma cells.

Although the role of serine racemase (SR) in neuropsychiatric disorders has been extensively studied, its role in cell proliferation and differentiation remains unclear. Deletion of Srr, the encoding gene for SR, has been shown to reduce dendritic arborization and dendritic spine density in the brains of adult mice, whereas increased SR levels have been associated with differentiation in cell cultures. Previously, we demonstrated that valproic acid induces differentiation in the N2A neuroblastoma cell line, and that this differentiation is associated with increased SR expression. These observations suggest that SR may have a role in cell proliferation and differentiation. We herein found that both valproic acid and all-trans retinoic acid induced N2A differentiation. In contrast, knockdown of SR reduced levels of differentiation, increased N2A proliferation, promoted cell cycle entry, and modulated expression of cell cycle-related proteins. To further evaluate the effects of SR expression on cell proliferation and differentiation, we used an in vivo model of neuroblastoma in nude mice. N2A cells stably expressing scramble shRNA (Srrwt -N2A) or specific Srr shRNA (Srrkd -N2A) were subcutaneously injected into nude mice. The weights and volumes of Srrwt -N2A-derived tumors were lower than Srrkd -N2A-derived tumors. Furthermore, Srrwt -N2A-derived tumors were significantly mitigated by intraperitoneal injection of valproic acid, whereas Srrkd -N2A-derived tumors were unaffected. Taken together, our findings demonstrate for the first time that alterations in SR expression determine the transition between proliferation and differentiation in neural progenitor cells. Thus, in addition to its well-established roles in neuropsychiatric disorders, our study has highlighted a novel role for SR in cell proliferation and differentiation.

Environmentally relevant concentrations of triclosan exposure promote the horizontal transfer of antibiotic resistance genes mediated by Edwardsiella piscicida.

Aquaculture pathogen and antibiotic resistance genes (ARGs) co-occur in the aquatic environment. Accumulated evidence suggests that aquaculture pathogens can facilitate the horizontal transfer of plasmid-mediated ARGs. However, the role of Edwardsiella piscicida (E. piscicida) in ARG dissemination is still not fully understood. In addition, the potential impact of triclosan (TCS) on the spread of ARGs mediated by E. piscicida is still unknown, so a mating model system was established to investigate the transfer process of ARGs. The results showed that E. piscicida disseminated ARGs on RP4 by horizontal gene transfer (HGT). Furthermore, TCS exposure promoted this process. The conjugative transfer frequencies were enhanced approximately 1.2-1.4-fold by TCS at concentrations from 2 to 20 µg/L, when compared with the control. TCS promoted the HGT of ARGs by stimulating reactive oxygen species (ROS) production, increasing cell membrane permeability, and altering expressions of conjugative transfer-associated genes. Together, the results suggested that aquaculture pathogens spread ARGs and that the emerging contaminant TCS enhanced the transfer of ARGs between bacteria.

Mbd2 deficiency alleviates retinal cell apoptosisvia the miR-345-5p/Atf1 axis in high glucoseinjury and streptozotocin-induced diabetic mice.

DNA methylation is considered to play an important role in the development of diabetic retinopathy. Here, our goal was to investigate the precise role of methyl-CpG binding domain protein 2 (Mbd2) in the apoptosis of retinal ganglion cells (RGCs) in the early diabetic retina. Mbd2 was significantly upregulated after high glucose (HG) treatment and played a proapoptotic role in RGCs during HG-induced apoptosis. Combining ChIP and gene microarray datasets, the results showed that Mbd2 possessed potential binding sites for miR-345-5p, thereby elevating the expression levels of miR-345-5p via the enhancement of promoter demethylation. Activating transcription factor 1 (Atf1) played an anti-apoptotic role during the process of apoptosis in RGCs and acted as the target gene for miR-345-5p. Furthermore, the number of surviving RGCs in the diabetic retina was increased in Mbd2-knockout mice when compared with wild-type mice and the visual function became better accordingly. Collectively, our data demonstrated that the HG-induced overexpression of Mbd2 in the retina was partly responsible for the apoptosis of retinal neuronal cells through the miR-345-5p/Atf1 axis. Therefore, the targeting of Mbd2 might represent a novel therapeutic strategy for the treatment of neurodegeneration in the early diabetic retina.

Effects of embryonic exposure to bixafen on zebrafish (Danio rerio) retinal development.

Bixafen, a pyrazole-carboxamide fungicide, is a potent toxicant that may elicit multiple adverse effects in non-target organisms. However, knowledge of the mechanisms involved in developmental defects caused by bixafen in aquatic organisms remains limited. In this study, the effects of bixafen on retinal development were evaluated in embryo-larval zebrafish. We exposed zebrafish embryos to 0, 0.1, and 0.3 µM bixafen. Exposure of zebrafish embryos to bixafen caused severe retinal defects, including extreme microphthalmia and a significantly increased cell density of the ganglion cell layer (GCL). Compared with the controls, the expression levels of rod and cone photoreceptor marker genes (rho, opn1sw2, opn1mw1, opn1lw1, and opn1sw1) in the outer nuclear layer (ONL) were significantly downregulated after bixafen exposure. Furthermore, bixafen caused significantly increased expression levels in the GCL marker ath5 and decreased expression levels in the inner nuclear layer (INL) markers prox1a, vsx1, and sox2. Accordingly, we observed a significantly increased rate of cell apoptosis in the retina after bixafen exposure. Taken together, our data demonstrate that bixafen exhibits retinal developmental toxicity to zebrafish embryos/larvae, and thus, it may pose a significant environmental threat to aquatic organisms.

The distal arthrogryposis-linked p.R63C variant promotes the stability and nuclear accumulation of TNNT3.

BACKGROUND: Distal arthrogryposis (DA) is comprised of a group of rare developmental disorders in muscle, characterized by multiple congenital contractures of the distal limbs. Fast skeletal muscle troponin-T (TNNT3) protein is abundantly expressed in skeletal muscle and plays an important role in DA. Missense variants in TNNT3 are associated with DA, but few studies have fully clarified its pathogenic role. METHODS: Sanger sequencing was performed in three generation of a Chinese family with DA. To determine how the p.R63C variant contributed to DA, we identified a variant in TNNT3 (NM_006757.4): c.187C>T (p.R63C). And then we investigated the effects of the arginine to cysteine substitution on the distribution pattern and the half-life of TNNT3 protein. RESULTS: The protein levels of TNNT3 in affected family members were 0.8-fold higher than that without the disorder. TNNT3 protein could be degraded by the ubiquitin-proteasome complex, and the p.R63C variant did not change TNNT3 nuclear localization, but significantly prolonged its half-life from 2.5 to 7 h, to promote its accumulation in the nucleus. CONCLUSION: The p.R63C variant increased the stability of TNNT3 and promoted nuclear accumulation, which suggested its role in DA.

Response of Escherichia coli to hydrogen nanobubbles: an in vitro evaluation using synchrotron infrared spectroscopy.

Hydrogen (H2)-rich water, an apparent source of molecular H2, is an emerging functional drink with many purported benefits for human health (Yang et al., 2020; Ostojic, 2021). The preventive and therapeutic effects of H2 on various pathological processes have been intensively investigated in numerous clinical trials; it is commonly believed that the beneficial effects are mainly attributed to its selective antioxidant and anti-inflammatory properties (Lee et al., 2015; Ohta, 2015; LeBaron et al., 2019; Qiu et al., 2020). In recent years, a handful of rodent studies revealed that exogenous H2 can affect the gut microbiota (Sha et al., 2018; Valdes et al., 2018). For example, H2 was reported to induce a higher abundance of butyrate-producing bacteria in a rat model of Parkinson's disease (Bordoni et al., 2019). Recent first-in-human trials have explored the effects of the long-term consumption of H2-rich water on antioxidant activity and the gut flora (Sha et al., 2018; Suzuki et al., 2018). Although these promising results suggest that the intestinal microbiota may be another plausible target for molecular H2, more studies are highly warranted to explain the mechanism(s) of H2 action on bacterial growth and functions.

Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay.

Vibrio vulnificus is an important zoonotic and aquatic pathogen and can cause vibriosis in humans and aquatic animals (especially farmed fish and shrimp species). Rapid and sensitive detection methods for V. vulnificus are still required to diagnose human vibriosis early and reduce aquaculture losses. Herein, we developed a rapid and sensitive diagnostic method comprising a recombinase-aided amplification (RAA) assay and the CRISPR/Cas12a system (named RAA-CRISPR/Cas12a) to detect V. vulnificus. The RAA-CRISPR/Cas12a method allows rapid and sensitive detection of V. vulnificus in 40 min without a sophisticated instrument, and the limit of detection is two copies of V. vulnificus genomic DNA per reaction. Meanwhile, the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood, stool, and shrimp samples. Therefore, our proposed rapid and sensitive V. vulnificus detection method, RAA-CRISPR/Cas12a, has great potential for early diagnosis of human vibriosis and on-site V. vulnificus detection in aquaculture and food safety control.

lncRNA Ttc3-209 Promotes the Apoptosis of Retinal Ganglion Cells in Retinal Ischemia Reperfusion Injury by Targeting the miR-484/Wnt8a Axis.

Purpose: Apoptosis of the retinal ganglion cells (RGCs) can cause irreversible damage to visual function after retinal ischemia reperfusion injury (RIR). Using a lncRNA chip assay, we selected lncRNA Ttc-209 and characterized its role in RGCs during ischemia reperfusion (I/R)-induced apoptosis. Methods: We created an ischemic model of RGCs by applying Hank's balanced salt solution containing 10 µM antimycin A and 2 µM calcium ionophore for 2 hours. RIR was induced in mice by elevating the intraocular pressure to 120 mm Hg for 1 hour by cannulation of the cornea; this was followed by reperfusion. Real-time quantitative PCR was used to detect the expression levels of long noncoding RNA (lncRNA), microRNA (miRNA), and target gene mRNA. Western blotting, flow cytometry, immunofluorescent staining, and TUNEL assays were performed to detect cell apoptosis. Dual-luciferase reporter assays and FISH were used to identify endogenous competitive RNA (ceRNA) mechanisms that link lncRNAs, miRNAs, and target genes. We also used scotopic electroretinography examinations to evaluate visual function in treated mice. Results: lncRNA Ttc3-209 was significantly upregulated after I/R injury and played a proapoptotic role in RGCs during I/R-induced apoptosis. Mechanistically, lncRNA Ttc3-209 is a ceRNA that competitively binds to miR-484 and upregulates the translation of its target (Wnt8a mRNA), thus promoting apoptosis in RGCs. Conclusions: Reducing the expression of lncRNA Ttc3-209 had a protective effect against apoptosis in RGCs. This may provide a new therapeutic option for the prevention of RGC apoptosis in response to RIR injury.

Sp4 controls constitutive expression of neuronal serine racemase and NF-E2-related factor-2 mediates its induction by valproic acid.

Serine racemase (SR) synthesizes l-type serine to its enantimor, d-serine which participates in physiological processes and in pathological conditions. In the central nervous system, SR is highly expressed in neurons and astrocytes but expressed at relatively lower amount in microglia. However, the mechanism by which SR is highly expessed in neurons is hitherto unknown. We report that the SR mRNA and protein levels in Neuro-2a were increased by valproic acid (VPA), a neuron differentiation stimulator as well as a histone deacetylase inhibitor. SR proximal promoter contained nine putative Sp-binding elements and in the exon 1, three putative anti-oxidant elements (AREs) were conservative among human, rat, and mouse genome. The promoter constructs including 5'-, 3'-fragment, and full length fragment from mouse were individually cloned into a luciferase reporter. Using dual-luciferase assay, the promoter harboring 3'-fragment contained much lower activity than the construct containing 5'-fragment which was though resistant to VPA induction, relative to 3'-fragment. Overexpression of Sp4 or Nrf2 increased whereas knockdown of either decreased Srr mRNA and SR protein. Using site-directed mutagenesis, mutation of Sp-binding elements or AREs in the constructs significantly decreased luciferase activity of the corresponding promoter construct. With chromatin immunoprecipitation, Sp4 was demonstrated to interact directly with the Sp-binding elements whereas Nrf2 bound AREs in Srr mRNA promoter. Altogether, our study highlights that Sp4 controls constitutive expression of SR in neuron and VPA mediates SR expression in N2A cells which is associated with its effect on neuron differentiation, that is, the effect is mediated via Nrf2.

The deubiquitinase USP6 affects memory and synaptic plasticity through modulating NMDA receptor stability.

Ubiquitin-specific protease (USP) 6 is a hominoid deubiquitinating enzyme previously implicated in intellectual disability and autism spectrum disorder. Although these findings link USP6 to higher brain function, potential roles for USP6 in cognition have not been investigated. Here, we report that USP6 is highly expressed in induced human neurons and that neuron-specific expression of USP6 enhances learning and memory in a transgenic mouse model. Similarly, USP6 expression regulates N-methyl-D-aspartate-type glutamate receptor (NMDAR)-dependent long-term potentiation and long-term depression in USP6 transgenic mouse hippocampi. Proteomic characterization of transgenic USP6 mouse cortex reveals attenuated NMDAR ubiquitination, with concomitant elevation in NMDAR expression, stability, and cell surface distribution with USP6 overexpression. USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells. Together, these results indicate that USP6 enhances NMDAR stability to promote synaptic function and cognition.

[Mutation analysis and prenatal diagnosis for 50 pedigrees affected with Duchenne/Becker muscular dystrophy].

OBJECTIVE: To establish individualized prenatal diagnosis program for families affected with Duchenne/Becker muscular dystrophy (DMD/BMD) and different clinical background using a variety of methods. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was performed on 50 patients suspected for DMD/BMD. For single exon deletions of the DMD gene, PCR was used for validating the results. For those without any deletion or duplication, Sanger sequencing was used to screen for DMD gene mutations in the children and their mothers. Prenatal genetic testing was provided to female carriers using chorionic villus, amniocentesis or cord blood samples. To ensure the accuracy of diagnosis, all prenatal specimens were also subjected to linkage analysis. RESULTS: Among the 50 patients with DMD/BMD, 23 harbored large deletions, 11 only had single exon deletions, 10 harbored duplications, and 5 had small scare mutations. No mutation was detected in one family. For 37 women undergoing prenatal diagnosis, 10 fetuses were identified as affected males, 6 were female carriers, while 21 were not found to carry any mutation. Testing of creatine kinase was consistent with the results of prenatal diagnosis. For a patient harboring exon 51 deletion, the same mutation was found in a fetus but not in their mother. The proband and fetus had inherited the same haplotype, which suggested that the mother probably has germline mosaicism for the mutation. CONCLUSION: Application of individualized methods for analyzing pregnant women with different clinical background can minimize the risk for giving birth to further children affected with DMD/BMD.

[Analysis of clinical phenotypes and GJB2 gene mutations in families affected with hearing loss from southern Zhejiang].

OBJECTIVE: To analyze the clinical features and pathological mutations in 44 families affected with hearing loss from southern Zhejiang, and to provide genetic counseling and prenatal diagnosis for 6 of the families. METHODS: Microarray was employed to detect c.35delG, c.176del16, c.235delC and c.299-300delAT mutations of the GJB2 gene among 228 patients. For those carrying a single heterozygous mutation, the whole coding region of the GJB2 gene was analyzed by Sanger sequencing. For prenatal diagnosis, maternal DNA contamination was excluded by application of STR analysis. RESULTS: The microarray assay has detected 49 patients with GJB2 mutations, which included 24 homozygous c.235delC mutations, 5 compound heterozygous c.235delC/c.176del16 mutations, 2 compound heterozygous c.235delC/c.299-300delAT mutations. Respectively, 16, 1 and 1 patients have carried single heterozygous c.235delC, c.176del16, and c.299-300delAT mutation. For the 16 patients, 7, 1, 1, 2, and 3 were detected by Sanger sequencing with a second heterozygous mutation of c.109G>A (2 of which were in conjunction with heterozygous c.176del16 and c.299-300delAT mutations), c.230G>A, c.427C>T, c.508-511 dupAACG, 79G>A+341A>G, respectively. Prenatal diagnosis revealed a compound heterozygous mutation in a fetus, heterozygous mutations in 4 fetuses, and no mutation of the GJB2 gene in 1 fetus. CONCLUSION: The proportion of carriers for GJB2 gene mutations in patients with hearing loss from southern Zhejiang has reached 21.5%. The c.235delC, c.176del16, and compound c.299-300delAT and c.109G>A mutations can cause moderate to severe hearing loss. In most affected families, Heterozygous mutations may be identified by sequencing the whole coding region of the GJB2 gene. Genetic analysis and prenatal diagnosis can prevent birth of further affected children.

Reduced serine racemase expression in aging rat cerebellum is associated with oxidative DNA stress and hypermethylation in the promoter.

Regulation of serine racemase (SR) occurs at transcriptional and translational levels; post-translational modification, cytosolic distribution as well as allosteric effect regulate SR activity. In this study, we report a new route of SR regulation, i.e. oxidative stress and hypermethylation of the srr (gene of SR) promoter correlate with its reduced transcription in aging rat cerebella. We first showed that the mRNA and protein level of srr were decreased in the homogenates of rat cerebellum at age 12 months compared with the counterparts from age 20 days. The reduction of SR protein level in aging cerebella was evidenced by decreased immunostaining observed in the cell body of granule cells or Purkinje cells. Staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress to DNA, was much stronger in granule cell or Purkinje cell nuclei from rat cerebella at 12 months compared with staining at 20 days. We further detected srr promoter hypermethylation at 12 months compared with that at 20 days by use of bisulfite sequencing PCR, coinciding with elevated protein levels of DNA methyltransferase 1 (DNMT1) in homogenates of aging cerebella. In vitro, we demonstrated that chronic treatment with the oxidant, menadione (VK3), reduced srr mRNA levels, which was reversed by the DNA demethylating agent 5-Aza-dC-2'-deoxycytidine (5-Aza-dC) in primary cerebellar granule cell cultures. Together, the in vivo and ex vivo results suggest that oxidative DNA stress and srr promoter hypermethylation are associated with reduced srr gene transcription and corresponding reduced protein expression in aging cerebella.

Identification and functional characterization of the novel Edwardsiella tarda effector EseJ.

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaN mutant, we identified a new effector by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the ΔeseJ mutant in the blue gourami fish model: the 50% lethal dose (LD50) of the ΔeseJ mutant is 2.34 times greater than that of the wild type, and the ΔeseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

Edwardsiella tarda-Induced cytotoxicity depends on its type III secretion system and flagellin.

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection. Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS of E. tarda facilitates its survival and replication in murine bone marrow-derived macrophages, and E. tarda infection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1ß in a T3SS-dependent manner. Deletion of the flagellin gene fliC of E. tarda results in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in the fliC mutant strain reduces its virulence. We propose that the host controls E. tarda infection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity.