Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Clostridium piliforme.

BACKGROUND: Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme. OBJECTIVES: To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. METHODS: In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system. RESULTS: The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7  ng/µL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes. CONCLUSIONS: The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.

Baicalein attenuates vinorelbine-induced vascular endothelial cell injury and chemotherapeutic phlebitis in rabbits.

Chemotherapy is one of the major strategies for cancer treatment. Several antineoplastic drugs including vinorelbine (VRB) are commonly intravenously infused and liable to cause serious phlebitis. The therapeutic drugs for preventing this complication are limited. In this study, the mechanism of baicalein (BCN) was investigated on VRB-induced phlebitis in vivo and vascular endothelial cell injury in vitro. Treatment with BCN obviously attenuated vascular endothelial cell loss, edema, inflammatory cell infiltration and blood clots, and reduced the serum levels of TNF-α, IL-1ß, IL-6 and ICAM-1 in the rabbit model of phlebitis induced by intravenous injection of VRB compared with vehicle. Further tests in vitro demonstrated that BCN lessened VRB-induced endothelial cell apoptosis, decreased intracellular ROS levels, suppressed phosphorylation of p38 and eventually inhibited activation of NF-κB signaling pathway. And these effects could be reversed by p38 agonist P79350. These results suggested that BCN exerted the protective effects against VRB-induced endothelial disruption in the rabbit model of phlebitis via inhibition of intracellular ROS generation and inactivation of p38/NF-κB pathway, leading to the decreased production of pro-inflammatory cytokines. Thus, BCN could be used as a potential agent for the treatment of phlebitis.

Matrix metalloproteinase activity in early-stage lung cancer.

BACKGROUND: The matrix metalloproteinases (MMPs)-2, -9 and -7 are thought to be associated with tumor invasion, metastasis, and angiogenesis. However, their possible roles in early-stage lung cancer are not clear. We measured the activity of MMP-2, -7 and -9 in early-stage lung cancer tissues. MATERIAL AND METHODS: Normal lung tissues and cancer tissues were collected from 60 consecutive stage-I non-small cell lung cancer (NSCLC) patients. The activities of MMP-2 and MMP-9 were determined by gelatin zymography, and the activity of MMP-7 was determined by casein zymography. Furthermore, the ratio of the active form of MMP-2 in tumor tissue (T) compared with normal tissue (N) was determined, and the survival in the groups with different MMP-2 T:N ratio was compared. RESULTS: The activity of both MMP-2 and MMP-9 was detected in all cancer and normal tissues. Interestingly, MMP-9 activity was significantly reduced, whereas MMP-2 activity was significantly increased, in cancer tissues compared to normal tissues. The survival rate of the MMP-2 T:N ratio > 2.5 group was 57.45%, which was significantly reduced compared with that of the T:N ratio ≤ 2.5 group (86.78%). CONCLUSION: Our findings suggest that MMP-2, but not MMP-9 and MMP-7, may be implicated in early-stage tumor invasion, metastasis, and angiogenesis in NSCLC.

Multifunctional kaolinite-supported nanoscale zero-valent iron used for the adsorption and degradation of crystal violet in aqueous solution.

Kaolinite-supported nanoscale zero-valent iron (K-nZVI) was successfully synthesized as a multifunctional composite and used for the degradation of crystal violet (CV). The presence of kaolinite not only decreased the aggregation of zero-valent iron nanoparticles (nZVI) with maintenance of reactivity, but also facilitated reaction by increasing the local concentration of CV in the vicinity of nZVI as an adsorbent. This was confirmed by scanning electron microscopy (SEM) and batch experiments, which showed that 97.23% of CV was removed using K-nZVI, while only 78.72% and 39.22% of CV were removed using nZVI and kaolinite after 30 min, respectively. Different factors impacting on degradation of CV were investigated as well. On the basis of these results, a removal mechanism was proposed including: (i) prompt adsorption of CV to the K-nZVI phase, and (ii) reduction of CV by Fe(0) on K-nZVI. Furthermore, different adsorption and reduction kinetics were employed to examine the removal process of CV, where a better fit with the pseudo-second-order model for adsorption and pseudo-first-order model for reduction process was observed, and reduction was the rate limiting step. In addition, isotherm and thermodynamic parameters were evaluated for a specific study of the important adsorption step. Finally, the application of K-nZVI to treat wastewater showed the removal efficiency higher than 99.9%.

Confirmation and determination of carboxylic acids in root exudates using LC-ESI-MS.

Reversed-phase liquid chromatography with UV detection is of limited applicability in the separation and identification of carboxylic acids because of the column's poor separation efficiency and the non-selective nature of the UV detector. To address this issue, RP-LC with electrospray ionization mass spectrometry has been explored for the confirmation and determination of carboxylic acids in plant root exudates, with ESI-MS providing structural information, high selectivity, and high sensitivity. The separation of 10 carboxylic acids (pyruvic, lactic, malonic, maleic, fumaric, succinic, malic, tartaric, trans-aconitic, and citric acid) was performed on a C(18) column using an eluent containing 0.1% (v/v) acetic acid within 10 min, where the acidic eluent not only suppressed the ionization of the carboxylic acids to be retained on the column, but was also compatible with ESI-MS detection. In addition, an additional standard was used to overcome the matrix effect. The results showed that peak areas correlated linearly with the concentration of carboxylic acids over the range 0.05-10 mg/L. The detection limits of target acids (signal-to-noise S/N ratio of 3) ranged from 20 to 30 microg/L. Finally, the proposed method was used for the confirmation and determination of low-molecular-weight carboxylic acids in plant root exudates, and provided a simple analytical procedure, including sample processing, fast separation, and high specificity and sensitivity.