Effects of sevoflurane exposure during different stages of pregnancy on the brain development of rat offspring.

OBJECTIVE: This study explored the effects of sevoflurane exposure during different stages of pregnancy on the brain development of offspring. METHODS: Thirty-six pregnant SD rats were randomly divided into 4 groups: control, sevoflurane exposure in early (S1) pregnancy, sevoflurane exposure in middle (S2) pregnancy, and sevoflurane exposure in late (S3) pregnancy. After natural birth, the learning and memory capacity of offspring rats was analyzed using the Morris water maze experiment. The hippocampi of offspring rats were collected. The levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus were measured by ELISA. Additionally, the Nissl bodies in the hippocampus were analyzed using Nissl staining. Immunohistochemistry was used to examine the expression of BDNF and CPEB2 in the hippocampus of offspring. Proteins related to the NR4A1/NF-κB pathway were analyzed using western blotting. RESULTS: The memory and learning capacity of offspring rats was significantly reduced in the S1 and S2 groups compared to the control group (p < 0.05), while there was no obvious difference between the control and S3 groups (p > 0.05). The level of IL-1ß was significantly increased (p < 0.05) in the S1 group compared with the control group. Sevoflurane anesthesia received in early and middle pregnancy could significantly affect the formation of Nissl bodies in the hippocampi of offspring rats. In addition, the expression of BDNF and CPEB2 in the hippocampi of offspring rats was greatly decreased in the S1 group compared with the control group (p < 0.05). The expression of NR4A1 in the hippocampi of rat offspring was significantly decreased in the S1 and S2 groups compared with the control group (p < 0.05). The expression of proteins related to the NF-κB pathway was increased in the S1 group compared to the control group (p < 0.05). CONCLUSIONS: The neurotoxic effect of maternal sevoflurane anesthesia on the brain development of offspring is higher when the exposure occurs in early pregnancy than in late pregnancy, and its mechanism might involve the NR4A1/NF-κB pathway to increase the secretion of inflammatory cytokines.

Impact of MDM2 single nucleotide polymorphism on oral squamous cell carcinoma risk.

Mouse Double Minute 2 (MDM2) has emerged as a pivotal cellular antagonist of p53 by destructing the suppressive function of p53 against tumorigenesis. The MDM2 309 T > G polymorphism has been studied for its association with oral squamous cell carcinoma (OSCC) susceptibility, but the evidence was confusing and inconclusive. Here, we performed a meta-analysis to estimate the effects of the 309 T > G polymorphism on the development of OSCC. The relevant studies were searched on both PubMed and Embase. We estimated the risk of OSCC using odds ratio (OR) and 95 % confidence interval (CI). In addition, between-study heterogeneity was measured by the χ (2)-based statistic test; sensitivity analysis, and the funnel plots and Egger's test were also performed in this meta-analysis. Based on five case-control studies with a total of 1,369 OSCC cases and 2,167 control subjects, the meta-analysis result showed neither increased nor decreased risk of OSCC associated with any genetic model of the 309 T > G polymorphism. Similar results were observed in the subgroup of Asians. No significant heterogeneity and publication bias were detected in the meta-analysis. The evidence provided in our study indicated that the 309 T > G polymorphism might have no significant contribution to susceptibility toward OSCC.

Matrix metalloproteinase-1 (MMP1) polymorphism is associated with lowered risk of nasopharyngeal carcinoma in Asian population.

Data on the association between -1607 1G > 2G polymorphism in the promoter region of matrix metalloproteinase-1 (MMP1) and nasopharyngeal carcinoma (NPC) are conflicting. The aim of this study was to confirm whether this polymorphism was a causative factor of NPC. We searched PubMed, Embase, and China National Knowledge Infrastructure (CNKI) for studies on the present topic. A total of four publications (1,044 NPC patients and 1,284 healthy control subjects) were included and meta-analysis was performed to assess the association between -1607 1G > 2G polymorphism and NPC risk. Odds ratio (OR) with 95 % confidence interval (95 % CI) was calculated for 1G1G versus 2G2G, 1G1G + 1G2G versus 2G2G, 1G1G versus 1G2G + 2G2G, 1G versus 2G, and 1G2G versus 2G2G contrast models. Meta-analysis results showed significantly reduced risk of NPC associated with the 1G1G versus 2G2G, 1G versus 2G and 1G2G versus 2G2G contrast models (OR = 0.61, 95 % CI 0.49-0.77; OR = 0.78, 95 % CI 0.65-0.92; OR = 0.86, 95 % CI 0.74-0.99, respectively). When we continued to perform subgroup analysis by ethnicity, the significant association persisted in Asian population and was most pronounced under the 1G2G versus 2G2G model (OR = 0.85, 95 % CI 0.73-0.99). These data suggested that MMP1 -1607 1G > 2G polymorphism was associated with reduced risk of NPC, particularly in the population of Asian descent.

Impact of epidural analgesia on quality of life and pain in advanced cancer patients.

Patients with advanced cancer often experience chronic postoperative pain and poor quality of life. The objective of this study was to determine if epidural self-controlled analgesia reduced the incidence of chronic pain and improved the quality of life when compared with intravenous self-controlled analgesia. A total of 50 patients diagnosed with advanced cancer who received analgesia treatment were randomly divided into two groups, epidural self-controlled analgesia group (EA group, n = 26) and intravenous self-controlled analgesia group (IA group, n = 24). Visual analog scale (VAS) and Karnofsky score were used to assess the pain and the quality of life, respectively. A multifunction monitor was used to continuously record the physical signs of patients after treatment. The physical signs, such as heart failure, respiration, pulse, blood pressure, and oxygen saturation, in the two groups were better after analgesia treatment. Meanwhile, the respiration and oxygen saturation in the EA group were significantly improved compared with that of the IA group (p < .05). The VAS in the EA group was significantly lower than that in the IA group (p < .05), and the Karnofsky score in the EA group was significantly higher than that in the IA group (p < .05). Moreover, patients treated with EA felt more satisfied and experienced fewer complications than those with IA (p < .05). The epidural self-controlled analgesia may greatly improve the quality of life and relieve the pain in patients with advanced cancer.

Supression of chronic central pain by superoxide dismutase in rats with spinal cord injury: Inhibition of the NMDA receptor implicated.

Superoxide dismutase (SOD) is used to manage chronic pain, including neuropathic and inflammatory pain. However, data regarding the clinical effectiveness are conflicting and the neurophysiological mechanism of SOD has yet to be elucidated. The aim of the present study was to investigate whether SOD relieved chronic central pain (CCP) following spinal cord injury (SCI) and the possible underlying mechanisms. A CCP model was established using the Allen method and the CCP of the rats was measured using the paw withdrawal threshold. SOD was administered intraperitoneally following the establishment of CCP as a result of SCI. The results demonstrated that SOD relieved CCP in rats following SCI. In addition, the expression of spinal phosphorylated N-methyl-D-aspartate(NMDA) receptor subunit 1 (pNR-1) was inhibited in the CCP rats that had been treated with SOD. These observations indicated that SOD reduced mechanical allodynia and attenuated the enhancement of spinal pNR1 expression in rats with CCP. In addition, the results indicated that superoxide, produced via xanthine oxidase, and the participation of superoxide and nitric oxide (NO) as a precursor of peroxynitrite in NMDA, were involved in the mediation of central sensitization. Therefore, the observations support the hypothesis that SOD may have a potential therapeutic role for the treatment of CCP following SCI via the manipulation of superoxide and NO.

Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

E-cadherin expression and prognosis of oral cancer: a meta-analysis.

This study aims to evaluate the association of E-cadherin expression with the prognosis of oral squamous cell carcinoma (OSCC). Literature retrieval, selection and assessment, data extraction, and meta-analyses were performed according to the Revman 5.0 guidelines. In the meta-analysis, we utilized either fixed effects or random effects model to pool the HR according to the test of heterogeneity. A total of nine eligible studies included 973 OSCC patients were analyzed. Of the patients, 76.3 % had low expression of E-cadherin according to the cutoff value defined by the authors. The pooled hazard ratio (HR) of low expression of E-cadherin for overall survival (OS) was 0.65 (95 % CI 0.52 to 0.80, P<0.001); in Asian population, the HR for overall survival of the patients with reduced expression of E-cadherin was 0.84 (95 % CI 0.75 to 0.95, P=0.006), and in non-Asian population, the HR for overall survival of the patients with reduced expression of E-cadherin was 0.54 (95 % CI 0.41 to 0.69, P<0.001). Patients with reduced expression of E-cadherin appear to have a poorer OS compared with those with normal or higher expression of E-cadherin.

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology.

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

High-yield soluble expression and simple purification of the antimicrobial peptide OG2 using the intein system in Escherichia coli.

OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2.

Effects of thioredoxin: SUMO and intein on soluble fusion expression of an antimicrobial peptide OG2 in Escherichia coli.

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.

Effects of amino acid deletion and substitution on the chemical properties, biological activities of the frog peptide palustrin-OG1.

Palustrin-OG1 (OG1) is a host defense peptide isolated from the frog Odorrana grahami. In this study, we analyzed the chemical properties, antimicrobial activities and cytotoxicities of OG1 and its derivatives to identify the most promising peptide as an antimicrobial agent. By increasing the net positive charge, amphipathicity and decreasing the mean hydrophobicity of OG1, the derivative named as OG2 exerted higher antimicrobial activity against bacteria but lower cytotoxicity against both porcine erythrocytes and peripheral blood mononuclear cells than did OG1 (P<0.01). After substitution of Cys residues of OG2 by Ala or Trp residues, two derivatives named as OG2A and OG2W were less effective against bacteria and induced greater hemolysis than did OG2, indicating the importance of Cys residues. The substitution of the C-terminal Thr of OG2 resulted OG2N, which decreased the cytotoxicity and improved killing kinetics against gram-positive bacteria by the rapid damage of cell wall and membrane.