ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Efficient production of 1,2,4-butanetriol from corn cob hydrolysate by metabolically engineered Escherichia coli.

Corn cob is a major waste mass-produced in corn agriculture. Corn cob hydrolysate containing xylose, arabinose, and glucose is the hydrolysis product of corn cob. Herein, a recombinant Escherichia coli strain BT-10 was constructed to transform corn cob hydrolysate into 1,2,4-butanetriol, a platform substance with diversified applications. To eliminate catabolite repression and enhance NADPH supply for alcohol dehydrogenase YqhD catalyzed 1,2,4-butanetriol generation, ptsG encoding glucose transporter EIICBGlc and pgi encoding phosphoglucose isomerase were deleted. With four heterologous enzymes including xylose dehydrogenase, xylonolactonase, xylonate dehydratase, α-ketoacid decarboxylase and endogenous YqhD, E. coli BT-10 can produce 36.63 g/L 1,2,4-butanetriol with a productivity of 1.14 g/[L·h] using xylose as substrate. When corn cob hydrolysate was used as the substrate, 43.4 g/L 1,2,4-butanetriol was generated with a productivity of 1.09 g/[L·h] and a yield of 0.9 mol/mol. With its desirable characteristics, E. coli BT-10 is a promising strain for commercial 1,2,4-butanetriol production.

Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca.

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively.

Dexmedetomidine Protects Cortical Neurons from Propofol-Induced Apoptosis via Activation of Akt-IKK-NF-κB Signaling Pathway by α2A-adrenoceptor.

CONTEXT: Propofol can induce neuroapoptosis. It has been reported that dexmedetomidine (DEX) has a protective effect on propofol-induced neuroapoptosis, but the specific mechanism needs to be further explored to provide a theoretical basis for their combined use. OBJECTIVE: We aimed to explore the neuroprotective effect of DEX on primary cortical neurons treated by propofol and to elucidate the underlying mechanistic pathways. METHODS: Cortical neurons were isolated from fetal rats and treated with propofol. MTT assays were performed to detect cell viability, α-tubulin immunofluorescent assays were conducted to observe cell abnormalities, and c-caspase3 immunofluorescent assays and flow cytometry were performed to examine cell apoptosis. Further, neurons were cotreated with propofol and DEX to study DEX's neuroprotective effects on propofol-caused neuronal injuries. Finally, the α2A-adrenoceptor was knocked out and/or the Akt activator (SC-79) was added to cells co-treated with propofol and DEX. The expression levels of Akt-IKK-NF-κB pathway-related proteins were detected by western blot. RESULTS: Propofol decreased cell viability in a dose-dependent manner, triggered apoptosis, caused morphological abnormalities and down-regulated the phosphorylation levels of Akt, IKK, NF-κB and IκB in cortical neurons. DEX ameliorated the decrease of cell viability, alleviated neuronal apoptosis and promoted the downregulated expression levels of p-Akt, IKK, NF-κB, and IκB proteins which had been induced by propofol treatment. Western blot findings following the transfection of α2A-siRNA and the addition of SC-79 suggested that DEX's neuroprotective functions arose from the stimulation of α2A-adrenoceptors to activate the Akt-IKK-NF-κB signal pathway. CONCLUSION: DEX protected neurons against propofol-induced apoptosis via activation of the Akt-IKK-NF-κB signal pathway through α2A-adrenoceptors.

Microglial priming induced by loss of Mef2C contributes to postoperative cognitive dysfunction in aged mice.

Postoperative cognitive dysfunction (POCD) is a common postoperative central nervous system (CNS) complication with a higher occurrence among aged individuals than among young individuals. The aim of this study was to explore the mechanisms by which POCD preferentially affects older individuals. We found here that exploratory laparotomy induced cognitive function decline in aged mice but not in young mice and that this decline was accompanied by inflammatory activation of microglia in the hippocampus. Furthermore, microglial depletion by feeding of a standard diet containing a colony stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) markedly protected aged mice from POCD. Notably, the expression of myocyte-specific enhancer 2C (Mef2C), an immune checkpoint that limits overactivation of microglia, was downregulated in aged microglia. Knocking down Mef2C induced a microglial priming phenotype in young mice, resulting in postoperative increases in the hippocampal levels of the inflammatory factors IL1-ß, IL-6 and TNF-α that could impair cognition; these findings were consistent with the observations in aged mice. In vitro, BV2 cells lacking Mef2C released higher levels of inflammatory cytokines upon stimulation with lipopolysaccharide (LPS, a bacterial toxin) than Mef2C-sufficient cells. Moreover, upregulation of Mef2C in aged mice restrained postoperative microglial activation, attenuating the neuroinflammatory response and cognitive impairment. These results reveal that during aging, loss of Mef2C leads to microglial priming, amplifying postsurgical neuroinflammation and contributing to the vulnerability of elderly patients to POCD. Thus, targeting the immune checkpoint Mef2C in microglia may be a potential strategy for the prevention and treatment of POCD in aged individuals.

Dexmedetomidine modulates neuroinflammation and improves outcome via alpha2-adrenergic receptor signaling after rat spinal cord injury.

BACKGROUND: Spinal cord injury induces inflammatory responses that include the release of cytokines and the recruitment and activation of macrophages and microglia. Neuroinflammation at the lesion site contributes to secondary tissue injury and permanent locomotor dysfunction. Dexmedetomidine (DEX), a highly selective α2-adrenergic receptor agonist, is anti-inflammatory and neuroprotective in both preclinical and clinical trials. We investigated the effect of DEX on the microglial response, and histological and neurological outcomes in a rat model of cervical spinal cord injury. METHODS: Anaesthetised rats underwent unilateral (right) C5 spinal cord contusion (75 kdyne) using an impactor device. The locomotor function, injury size, and inflammatory responses were assessed. The effect of DEX was also studied in a microglial cell culture model. RESULTS: DEX significantly improved the ipsilateral upper-limb motor dysfunction (grooming and paw placement; P<0.0001 and P=0.0012), decreased the injury size (P<0.05), spared white matter (P<0.05), and reduced the number of activated macrophages (P<0.05) at the injury site 4 weeks post-SCI. In DEX-treated rats after injury, tissue RNA expression indicated a significant downregulation of pro-inflammatory markers (e.g. interleukin [IL]-1ß, tumour necrosis factor-α, interleukin (IL)-6, and CD11b) and an upregulation of anti-inflammatory and pro-resolving M2 responses (e.g. IL-4, arginase-1, and CD206) (P<0.05). In lipopolysaccharide-stimulated cultured microglia, DEX produced a similar inflammation-modulatory effect as was seen in spinal cord injury. The benefits of DEX on these outcomes were mostly reversed by an α2-adrenergic receptor antagonist. CONCLUSIONS: DEX significantly improves neurological outcomes and decreases tissue damage after spinal cord injury, which is associated with modulation of neuroinflammation and is partially mediated via α2-adrenergic receptor signaling.

Propofol induces apoptosis and increases gemcitabine sensitivity in pancreatic cancer cells in vitro by inhibition of nuclear factor-κB activity.

AIM: To investigate the effect of propofol on human pancreatic cells and the molecular mechanism of propofol action. METHODS: We used the human pancreatic cancer cell line MIAPaCa-2 for in vitro studies measuring growth inhibition and degree of apoptotic cell death induced by propofol alone, gemcitabine alone, or propofol followed by gemcitabine. All experiments were conducted in triplicate and carried out on three or more separate occasions. Data were means of the three or more independent experiments ± SE. Statistically significant differences were determined by two-tailed unpaired Student's t test and defined as P < 0.05. RESULTS: Pretreatment of cells with propofol for 24 h followed by gemcitabine resulted in 24%-75% growth inhibition compared with 6%-18% when gemcitabine was used alone. Overall growth inhibition was directly correlated with apoptotic cell death. We also showed that propofol potentiated gemcitabine-induced killing by downregulation of nuclear factor-κB (NF-κB). In contrast, NF-κB was upregulated when pancreatic cancer cells were exposed to gemcitabine alone, suggesting a potential mechanism of acquired chemoresistance. CONCLUSION: Inactivation of the NF-κB signaling pathway by propofol might abrogate gemcitabine-induced activation of NF-κB, resulting in chemosensitization of pancreatic tumors to gemcitabine.

Clinicopathological significance of S100A4 expression in human hepatocellular carcinoma.

OBJECTIVE: Prognostic markers for hepatocellular carcinoma (HCC) could help in the clinical management and understanding of its poor prognosis. S100 calcium binding protein A4 (S100A4) is directly involved in tumour metastasis. This study evaluated S100A4 gene expression in human HCC, to identify its role in tumour progression. METHODS: In this retrospective study, S100A4 protein levels in HCC samples from Chinese patients were evaluated using immunohistochemistry and compared with paired normal tumour-adjacent tissue samples (controls). All patients were evaluated for HCC recurrence. RESULTS: S100A4 levels were significantly increased in HCC samples compared with controls (n = 72, each sample type). Moderately or poorly differentiated tumours had significantly higher levels of S100A4 protein than well-differentiated tumours, and those with tumour vascular invasion showed significantly higher S100A4 levels than those without invasion. Risk of recurrence increased in patients positive for S100A4, and S100A4 positivity correlated with a shorter overall survival time. CONCLUSION: This pilot study suggests S100A4 as a likely tumour marker for HCC that correlates with tumour differentiation, invasion, recurrence and overall survival. S100A4 could be a useful marker of tumour aggressiveness and prognosis.

Clusterin confers resistance to TNF-alpha-induced apoptosis in breast cancer cells through NF-kappaB activation and Bcl-2 overexpression.

Secretory clusterin (sClu) is an anti-apoptotic protein that plays a role in protecting cells from Tumour-necrosis factor (TNF)-alpha-induced apoptosis. The aim of the present study was to investigate the molecular mechanisms underlying the effect of sClu on TNF-alpha-induced apoptosis in breast cancer cells. The wild-type p53 expressing MCF-7 cell line was engineered to overexpress sClu (MCF-7/sClu), whereas the MDA-MB-231 cell line with mutant p53 was transfected with a sClu silencing siRNA (MDA-MB-231/sClu siRNA). The effects of clusterin overexpression and downregulation on apoptosis and sensitivity to TNF-alpha were examined in vitro. Our results showed that TNF-alpha treatment increased Bcl-2 mRNA and protein levels in breast cancer cells, suggesting that Bcl-2 is directly regulated by nuclear factor-kappaB (NF-kappaB) in response to TNF-alpha. The induction of Bcl-2 was mediated by the p65 subunit of NF-kappaB. siRNA-mediated silencing of Bcl-2 led to a significant increase in TNF-alpha-induced apoptosis. Silencing of sClu in MDA-MB-231/sClu siRNA cells abrogated TNF-alpha-mediated NF-kappaB activation and Bcl-2 overexpression, and rendered the MDA-MB-231/sClu siRNA cells significantly more sensitive to TNF-alpha-mediated apoptosis than the parental cells. Furthermore, overexpression of sClu in MCF-7/sClu cells promoted TNF-alpha-mediated NF-kappaB activity and Bcl-2 overexpression, and rendered the MCF-7/Clu cells significantly more resistant to TNF-alpha-mediated apoptosis. Inhibition of NF-kappaB activity or p65 and Bcl-2 expression reversed these effects. The present results suggested that sClu confers breast cancer cells resistance to TNF-alpha-induced apoptosis through NF-kappaB activation and Bcl-2 overexpression.