基于先进材料的纸基传感器在多毒素联合检测中的应用

农产品、食品和饲料等常因霉菌侵染而被多种真菌毒素污染,研发高通量真菌毒素快速检测产品是热点研究领域之一。目前主流的真菌毒素快检产品中,传统纸基传感器大多以胶体金作为信号标记物用于单一毒素检测,获得信息相对有限。多毒素纸基传感器常常偶联多种真菌毒素抗原,成本更低,耗时更短,然而检测灵敏度因测试样液量变化而受到影响。近年来,在真菌毒素快检领域引入贵金属纳米粒子、量子点、碳纳米材料、上转换粒子、镧系金属纳米粒子等先进材料,可以改善传感器的检测性能。笔者总结了多毒素纸基传感器的检测原理和组成,综述近年来文献报道中基于先进材料的纸基传感器同时检测多种真菌毒素的研究与应用,分析多毒素纸基传感器未来的研究趋势,以期为多种真菌毒素快速高通量检测相关研究提供参考。

国内外真菌毒素检测方法研究现状及进展

真菌毒素是真菌产生的小分子有毒代谢产物,通常以痕量形式存在于基质中,不表现为急性毒性,但具有蓄积性,长期摄入易引发癌症、突变及畸形,从而对人类安全造成威胁。本文着重介绍了黄曲霉毒素、赭曲霉毒素、伏马菌素、玉米赤霉烯酮及其代谢物、单端孢霉烯族类毒素以及麦角碱等毒性相对较强的真菌毒素的来源、结构和毒性。由于许多真菌毒素结构相似且含量较低,需要采用高选择性、准确、快速定性定量方法,因而本文就目前国内外常用的传统及新型的真菌毒素检测方法进行了概述和比较,主要包括:薄层色谱法,液相色谱法,气相色谱法,免疫化学法,核酸适配体法以及液相色谱-质谱联用技术(LC-MS/MS)在多毒素检测中的应用,拟为真菌毒素的检测研究提供参考。

食品中主要真菌毒素检测方法研究进展

文章重点介绍了黄曲霉毒素、脱氧雪腐镰刀菌烯醇、链格孢霉毒素等毒性较强的真菌毒素污染食品状况和毒性,并就当前国内外常用的真菌毒素检测方法进行了概述,分析了各方法的优缺点,最后对今后真菌毒素检测方法的发展方向进行了展望。

“十三五”期间中国真菌毒素监测之发展思考

针对我国真菌毒素污染状况,在“十三五”期间探索了一套适合中国开展食品安全风险监测的方式,即技术上立足于采取多毒素同时检测,涵盖各类重点食品,尽可能全面掌握当前污染现状。实践证明,目前采取的方式取得的效果良好,也为“十四五”开展更全面的监测奠定了扎实的实践基础。

Effect of Echinacea Polysaccharide on Secretion of TNF-α mRNA by IEC-6 Injured by LPS

Objective] This study was conducted to investigate Echinacea polysaccha-ride （EPS） on expression of tumor necrosis factor （TNF） under injury of intestinal epithelial cel s （lEC-6） by lipopolysaccharide （LPS）, so as to discuss the action mechanism of EPS to injured cel s. [Method] Total DNA was extracted with TRlzon reagent, TNF-α mRNA was amplified, and the amplification products were subjected to agarose gel electrophoresis and imaging. [Result] 50 μg/ml EPS could partial y in-hibited the production of TNF-α mRNA by lEC-6 under the stimulation of LPS, while the inhibition of 200 and 500 μg/ml EPS on the level of TNF-α mRNA gradual y in-creased with the concentration increasing; and lEC-6 cel s pretreated with 50, 100, 200 and 500 μg/ml EPS for 24 h and then stimulated by 10 μg/ml LPS for 1 and 4 h were analyzed by RT-PCR method, and it was found that the expression of TNF-α mRNA induced by LPS could be effectively inhibited by EPS, and the inhibition rate at 4 h was higher than that at 1 h. [Conclusion] EPS could play its role of protecting intestinal mucosa by inhibiting the secretion of TNF-α mRNA by cel s un-der the stimulation of LPS, and such inhibition effects of EPS had concentration dependency and time dependency.

Association of promoter polymorphism of the CD14 C (-159) T endotoxin receptor gene with chronic hepatitis B

AIM: To investigate whether single-nucleotide polymor- phisms in the promoter regions of endotoxin-responsive genes CD14 C (-159) T is associated with chronic hepatitis B. METHODS: We obtained genomic DNA from 80 patients with established diagnosis of chronic hepatitis B and 126 healthy subjects served as a control population. The CD 14 C (-159) T polymorphism was investigated using an allele specific PCR method. RESULTS: Twenty seven percent of chronic hepatitis B patients and 75% of controls were heterozygous for CT genotype. The difference between the chronic hepatitis B and control groups was statistically significant [P < 0.0001; Odds ratio (OR) = 2.887; 95% CI: 1.609-5.178]. Twenty four point six percent of chronic hepatitis B and patients 12.3% of the control group were heterozygous for TT genotype. The difference between groups was not statistically significant (P = 0.256; OR = 0.658; 95% CI: 0.319-1.358). Forty eight point four percent of chronic hepatitis B patients and 12.7% of control were homozy- gote for CC genotype (P < 0.004; OR = 0.416; 95% CI: 0.229-0.755). The frequency of allele C was 61.9% and allele T was 38.1% in hepatitis B patients group. The frequency of allele C was 55.2% and allele T was 44.8% for the control group (P = 0.179; OR = 1.319; 95% CI: 0.881-1.977). CONCLUSION: The TT heterozygous genotype was not a risk factor for chronic hepatitis B. CC homozygote genotype is protective for hepatitis B. Lack of heterozy- gosis of genotype CT is a risk factor for chronic hepatitis B. Alleles C or T were not risk factors for chronic hepatitis B. These findings show the role of a single-nucleotide polymorphism at CD14/-159 on the development ofchronic hepatitis B. Endotoxin susceptibility may play a role in the pathogenesis of chronic hepatitis B.

Effect of JIANPI HUOXUE decoction on inflammatory cytokine secretion pathway in rat liver with lipopolysaccharide challenge

AIM： To evaluate the effect of Chinese traditional medicinal prescription, JIANPI HUOXUE decoction （JHD） on cytokine secretion pathway in rat liver induced by lipopolysaccharide （LPS）. METHODS： Twenty-four male SD rats were divided into normal group （n = 4）, model group （n = 10） and JHD group （n = 10） randomly. Rats in model group and JHD group were administrated with normal saline or JHD via gastrogavage respectively twice a day for 3 d. One hour after the last administration, rats were injected with LPS via tail vein, 50 μg/kg. Simultaneously, rats in normal group were injected with equivalent normal saline. After LPS stimulation for 1.5 h, serum and liver tissue were collected. Pathological change of liver tissues was observed through hematoxylineosin （H.E.） staining. Tumor necrosis factor alpha （TNF-α） in serum were assayed by enzyme linked immunosorbent assay （ELISA）. The protein expression of TNF-α, phosphorylated inhibit-κB （p-κB） and CD68 in liver were assayed by Western blot. The distribution of CD68 protein in liver was observed through immunohistochemical staining. The mRNA expression of TNF-α, interleukin-6 （IL-6）, CD14, toll-like receptor 2 （TLR2） and TLR4 in liver were assayed by real-time RT-PCR.RESULTS： Predominant microvesicular change, hepatocyte tumefaction and cytoplasm dilution were observed in liver tissues after LPS administration as well as obvious CD68 positive staining in hepatic sinusoidal. After LPS stimulation, serum TNF-α （31.35 ± 6.06 vs 12225.40 ± 9007.03, P 〈 0.05）, protein expression of CD68 （1.13 ± 0.49 vs 3.36 ±1.69, P 〈 0.05）, p-IκB （0.01 ±0.01 vs 2.07 ＋ 0.83, P 〈 0.01） and TNF-α （0.27 ± 0.13 vs 1.29 ± 0.37, P 〈 0.01） in liver and mRNA expression of TNF-α （1.96 ± 2.23 vs 21.45 ±6.00, P 〈 0.01）, IL-6 （4.80 ± 6.42 vs 193.50 ± 36.36, P 〈 0.01） and TLR2 （1.44 ± 0.62 vs 4.16 ± 0.08, P 〈 0.01） in liver were also increased significantly. These pathological changes were all improved in .1HD group. On the other hand, TLR4 mRNA （1.22 ± 0.30 vs 0.50 ± 0.15, P 〈 0.05） was down-regulated and CD14 mRNA increased but not significantly after LPS stimulation. CONCLUSION： JHD can inhibit cytokine secretion pathway induced by LPS in rat liver, which is probably associated with its regulation on CD68, p-IκB and endotoxin receptor TLR2.

Endotoxin receptor CD14 gene variants and histological features in chronic HCV infection

AIM:To analyze the correlation between CD14 rs2569190/C-159T single nucleotide polymorphism (SNP) and disease progression in chronic hepatitis C.METHODS: Liver biopsy specimens from a total of 137 and 349 patients with chronic hepatitis C were separately evaluated with respect to necroinflammatory activity (grading) and architectural changes (staging). In one group, further histological lesions characteristic for hepatitis C, hepatitis C virus subtypes, and biochemical parameters of liver disease were also investigated. Samples of genomic DNA were genotyped for the respective SNP by 5'-nuclease assays using fluorescent dye-labeled allele-specif ic probes.RESULTS: Genotype distribution did not deviate from the Hardy-Weinberg equilibrium. In the first group, patients homozygous for the variant allele T were found to be younger than C allele carriers (39.6±12.5 vs 45.7±11.5, P=0.008). Among the histological lesions studied, portal lymphoid aggregates were more frequently observed among TT homozygotes than among C carriers (21/37 vs 32/100, P=0.008). The presence of portal lymphoid aggregates was closely correlated with hepatic inflammation (P=0.003) and with bile duct damage (P<0.001). The degree of fibrosis, in contrast, was not found to be related to the CD14 gene C-159T polymorphism.CONCLUSION: The data suggest a possible relationship between CD14 C-159T polymorphism and the formation of portal lymphoid aggregates, but not liver fibrosis progression in chronic hepatitis C.

Antifungal Activity of Lactic Acid Bacteria, Isolated from Bulgarian Wheat and Rye Flour

The economic losses and the health hazards of the mycotoxins produced by spoilage fungi are the main concerns of the food industry. The spoilage of bakery products by fungi is more common in countries with a high humidity and temperature. About 5-10% of food production is spoiled by the growth of yeast and fungi in food materials. Similarly, in Western Europe, the growth of the spoilage fungi of bread is estimated to reach more than 200 million Euros per year. The history conditions of the food can be a major factor in determining any fungal spoilage--for example, stored and processed foods are more sensitive to spoilage when compared with fresh and prepared foods. Lactic acid bacteria isolated from Bulgarian wheat and rye flour were used in the present study to check their antifungal properties against pathogenic yeast and fungi imperfecta using standard disc diffusion method in vitro. A broad spectrum of antifungal activity of the six newly identified as L. plantarum strains e Tsl, Ts2, Ts3,Ts4 and Ts5, and L. helveticus Ts6 was estimated. Our in vitro studies were performed with wheat and rye sourdough, in order to simulate a real product and to assess the bio-protective potential of the tested lactobacilli. The used test-cultures are representatives of carcinogenic, toxigenic, deteriorative and allergenic fungi from the genera .4spergillus and Penicillium. The all tested strains completely suppress the growth of against C. glabrata 72. Strains L. plantarum Tsl and Ts3 completely suppress the growth against S. cerevisae. While, in the sample with L. plantarum strains e Tsl, Ts2, Ts3,Ts4 and Ts5, and L. helveticus Ts6, a retarded and weak growth of A. niger and P. claviforme was observed. However, the spore germination and the colony growth started only on the fifth day of the mould lactobacilli co-cultivation, which also should be considered as a good result. In this study six isolates Tsl,Ts2, Ts3, Ts4, Ts5 and Ts6, from the traditional Bulgarian wheat and rye flour have been identified as L. plantarum and L. helveticus and characterized as cultures with promising antifungal activity. Obtained results from the combined molecular identification （16S rRNA gene sequencing） approach contribute to give new data on the microbial biodiversity of this not well-studied niche. The antifungal activity of our new isolates, identified as L. plantarum and L. helveticus, seems to be a promising advantage of these six strains, suggesting their potential applications in different food technologies. However, more experiments have to be conducted to clarify the nature and the mechanisms of the reported antifungal activity and they are still in progress. The combination of dairy origin and strong inhibitory activity of the lactobacillus strains is a prerequisite for their possible application as starters and/or bioprotective antifungal adjuncts.

Intestinal microflora in rats with ischemia/reperfusion liver injury

Objectives: To investigate the intestinal microflora status related to ischemia/reperfusion (I/R) liver injury and explore the possible mechanism. Methods: Specific pathogen free grade Sprague-Dawley rats were randomized into three groups: Control group (n=8), sham group (n=6) and I/R group (n=10). Rats in the control group did not receive any treatment, rats in the I/R group were subjected to 20 min of liver ischemia, and rats in the sham group were only subjected to sham operation. Twenty-two hours later, the rats were sacrificed and liver enzymes and malondialdehyde (MDA), superoxide dismutase (SOD), serum endotoxin,intestinal bacterial counts, intestinal mucosal histology, bacterial translocation to mesenteric lymph nodes, liver, spleen, and kidney were studied. Results: Ischemia/reperfusion increased liver enzymes, MDA, decreased SOD, and was associated with plasma endotoxin elevation in the I/R group campared to those in the sham group. Intestinal Bifidobacteria and Lactobacilli decreased and intestinal Enterobacterium and Enterococcus, bacterial translocation to kidney increased in the I/R group compared to the sham group. Intestinal microvilli were lost, disrupted and the interspace between cells became wider in the I/R group.Conclusion: I/R liver injury may lead to disturbance of intestinal microflora and impairment of intestinal mucosal barrier function,which contributes to endotoxemia and bacterial translocation to kidney.

Inhibitory effect of emodin and Astragalus polysaccharide on the replication of HBV

AIM: To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice.METHODS: Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrifi ced and serum as well as liver tissues were collected for ELISA and histological examination.RESULTS: After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a signif icant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A.CONCLUSION: Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.

MyD88/PI3-K/NF-κB pathway mediates the antagonism of lipoxin A_4 on LPS-induced synthesis of interleukins in endothelial cells

In order to investigate whether lipoxin A4 （LXA4） has an antagonistic effect on lipopolysaccharide （LPS）-induced synthesis of interleukin （IL）-β3, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells （PMVEC）, and to explore the molecular mechanisms of signal pathway in LXA4 actions, cultured PMVEC were treated with LPS, with or without preincubation with LXA4. Proteins of IL-β3, IL-6 and IL-8 in supernatant were analyzed by enzyme-linked immunosorbent assay （ELISA）. Expressions of mRNA of IL-β3, IL-6 and IL-8 were determined by RT-PCR. Expressions of phosphorylation of phosphoinositide 3-kinase （PI3-K） and myeloid differentiation factor 88 （MyD88） were analyzed by Western blot. Activities of DNA-binding of nuclear factor-kappa B （NF-κB） and activator protein-1 （AP-1） were measured by electrophoretic mobility shift assay （EMSA）. The results showed that LPS induced production of IL-β3, IL-6 and IL-8 in rat PMVEC via MyD88/PI3-K/NF-κB and AP-1 pathway-dependent signal transduction. LPS-stimulated expression of PI3-K, activities of NF-κB and AP-1, secretion of protein and expression of mRNA of IL-β3, IL-6 and IL-8 but not MyD88 expression in PMVEC were inhibited by LXA4 in a dose-dependent manner. In conclusion, LXA4 inhibits synthesis of IL-β3, IL-6 and IL-8 by down-regulation of PI3-K/NF-κB and AP-1 signal pathway in PMVEC.

EFFECT OF HEMORRHAGIC SHOCK ON ENDOTOXININDUCED TNF PRODUCTION AND ITS MOLECULAR MECHANISM IN RATS

The present study was designed to investigate the production of tumor necrosis factor a (TNFα) in-duced by low-dose (1μg/kg) lipopolysaccharide (LPS) and its cellular source after hemorrhagic shock(HS) in rats, and to further analyze the mechanism for increased sensitivity to LPS through looking at ex-pression of lipopolysaccharide -binding protein (LBP ) mRN A in t he liver, lungs and kidneys. lt wa s foundin uiuo that plasma TNFa levels in the HS+LPS group were 20-fold higher than that in the HS group (P<0. 01 ), and 2. 7-fold higher than that in the LPS group (P<0. 05). lt was shown in ndro that the ca-pacity of peripheral white blood cells to produce TNFa in response to LPS stimulation was significantly de-creased by 126 % (P<0. 01 ) and 57% (P<0. 05) compared with pre-shock levels and the sham group re-spectively at the end of resuscitation following shock, and was still markedly decreased 3 hours after resus-citation, while the capacity of Kupffer Cells was significantly increased by 110% compared with the shamgroup (P<0. 01) after shock and resuscitation. Results from RT-PCR showed that expression of LBPmRNA in the liver, lungs and kidneys was increased after shock and resuscitation. It is suggested thathemorrhagic shock could significantly enhance endotoxin-induced TNFa production, which might be due toup-regulation of LBP expression in tissues after shock, and tissue macrophages might be the main source ofcytokine production.

Expression of Overlapping PCR-generated Shiga Toxin B Gene Fragment in E. Coli and Its Ascitic Polyclonal Antibody

The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.

In vitro inhibition of pigmentation and fiber development in colored cotton

Colored cotton has naturally pigmented fibers.The mechanism of pigmentation in cotton fiber is not well documented.This experiment was conducted to study the effects of respiratory chain inhibitors,i.e.,rotenone and thiourea,on pigmentation and fiber development in colored cotton.After 1 d post-anthesis,ovaries were harvested and developing ovules were cultured on the liquid medium containing different concentrations of rotenone and thiourea for 30 d.The results demonstrate that both respiratory inhibitors reduced fiber length and ovule development under ovule culture conditions,and the inhibition efficiency of rotenone was much higher than that of thiourea.Rotenone and thiourea also showed significant effects on fiber pigment (color) development in colored cotton.In green cotton fiber,rotenone advanced fiber pigment development by 7 d at 200 μmol/L,while thiourea inhibited fiber pigmentation at all treatment levels (400,600,800,1000,and 2000 μmol/L).Both respiratory inhibitors,however,had no significant effects on pigmentation of brown cotton fibers.The activities of cytochrome c oxidase (COX) and polyphenol oxidase (PPO) decreased significantly with increasing levels of both respiratory inhibitors.It is suggested that both respiratory inhibitors have important roles in deciphering the mechanism of pigmentation and fiber development in colored cotton.

In vivo expression of lipopolysaccharide binding protein and its gene induced by endotoxin

Objective: To investigate the expression of lipopolysaccharide binding protein (LBP) and its gene in rats with endotoxemia and explore the role of LBP in the response of host to endotoxin. Methods: Thirty Wistar rats were divided randomly into five groups: the normal group and the endotoxemia groups (1, 3, 6, 12 hours after LPS injection, respectively). The level of plasma endotoxin was determined by the Limulus Amebocyte Lysate assay. The expression of LBP mRNA in hepatic tissue was examined by reverse transcription polymerase chain reaction (RT PCR). Plasma levels of LBP, tumor necrosis factor (TNF) α and interleukin (IL) 6 were measured by enzyme linked immunosorbent assay (ELISA). Morphologic changes of hepatic tissue were observed under transmission electron microscope. Results: The level of plasma endotoxin peaked at 1 h after LPS injection, then declined, but was still higher than that of the normal group at 12 h; intrahepatic expression of LBP mRNA and plasma LBP increased with time after LPS stimulation; TNF α and IL 6 in plasma increased with upregulation of LBP expression; there were significant differences between the normal group and endotoxemia groups (P< 0.05 ). Activation of Kupffer cells and injury of hepatocytes could be seen in rats with endotoxemia. Conclusions: LPS can upregulate the intrahepatic expression of LBP mRNA and increase the plasma LBP level. Under certain conditions, LBP may enhance the sensitivity of host to the toxic effects of LPS.

Lipopolysaccharide/Toll-like receptor 4 signaling pathway involved Qingdu decoction treating severe liver injury merging with endotoxemia

OBJECTIVE: To investigate the protective effects and underlying mechanism of Qingdu decoction(QDD) on experimental rats with severe liver injury induced by thioacetamide(TAA).METHODS: A total of 40 Wistar rats were randomly divided into normal group(n = 10) and experimental group(n = 30). Rats were administrated the same content of saline in normal group. The rats inthe experimental group were pretreated with TAA at dose of 12 mg/kg lasting 8 weeks, and from 9th week to 12 th week, with TAA at concentration of 36mg/kg. During the 9th week to 12 th week period,the rats were randomly divided into three subgroups(n = 10 each) simultaneously based on the treatment categories: model group, lactulose(LA,3.5 m L/kg) group and QDD(5.95 g/kg) group, orally once per day respectively. At the 12 th week, the content of serum alanine transaminase(ALT), aspartate transaminase(AST), total bilirubin(TBIL), endotoxin(ET) and tumor necrosis factor α(TNF-α) was detected by automatic biochemical analyzer. The plasma prothrombin time(PT), prothrombin time-international normalized ratio(PTR) and prothrombin time activity(PTA) were measured by automatic coagulation analyzer. The level of lipopolysaccharide(LPS)-binding protein(LBP), cluster differentiation 14(CD14) and Toll-like receptor 4(TLR4) expressions was measured by both western blot(WB) and real-time polymerase chain reaction(real-time PCR).RESULTS: Compared with the model group, hepatic morphology in the QDD group was improved under light microscope and transmission electron microscope; at the same time, the contents of serum ALT, AST, TBIL, ET and TNF-α, and level of LBP, CD14 and TLR4 expressions in liver tissues were significantly decreased compared with the model group(P < 0.05), while PTA in the QDD group was enhanced(P < 0.05).CONCLUSION: QDD has the functional effect on improving the injured liver through inhibiting the LPS/TLR4 signaling pathway thus decreasing the level of the inflammatory medicators.