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Pernille Johansen Jannik Vindeløv Nils Arneborg Elke Brockmann 《Systematic and applied microbiology》2014
Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited. 相似文献
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Members of the K(v)7 family generate a subthreshold potassium current, termed M-current, that regulates the excitability of principal central neurons. Mutations in two members of this family, K(v)7.2 (KCNQ2) and K(v)7.3 (KCNQ3) are associated with a neurological disorder known as benign familial neonatal convulsion (BFNC). Despite their importance in normal and pathological brain function, developmental expression and function of these channels remains relatively unexplored. Here, we examined the temporal expression of K(v)7 channel subunits in zebrafish larvae using a real-time quantitative PCR approach. Spatial expression in the larval zebrafish brain was assessed using whole-mount in situ hybridization. The mRNA for three members of the K(v)7 family (KCNQ2, 3 and 5) is reported in zebrafish between two and seven days post-fertilization (dpf). Using electrophysiological techniques, we show that inhibitors of K(v)7 channels (linopirdine and XE991) induce burst discharge activity in immature zebrafish between 3 and 7 dpf. This abnormal electrical activity is blocked by a K(v)7 channel opener (retigabine) and was also shown to evoke convulsive behaviors in freely swimming zebrafish. Using morpholino oligonucleotides directed against KCNQ3, we confirmed a role for KCNQ channels in generation of electrical burst discharges. These results indicate that functional K(v)7 channels are expressed in the larval zebrafish nervous system and could play a direct role in generation of seizure activity. 相似文献
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Omar García-Prez Leticia Melgar-Vilaplana Elizabeth Crdoba-Lanús Ricardo Fernndez-de-Misa 《Current issues in molecular biology》2021,43(3):2167
Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours. 相似文献
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《International journal for parasitology》2021,51(11):893-898
The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice. 相似文献
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Michael J. Rothrock Jr. Kelli L. Hiett John Gamble Andrew C. Caudill Kellie M. Cicconi-Hogan J. Gregory Caporaso 《Journal of visualized experiments : JoVE》2014,(94)
The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. 相似文献
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J.V. Lee S. Lai M. Exner J. Lenz V. Gaia S. Casati P. Hartemann C. Lück B. Pangon M.L. Ricci M. Scaturro S. Fontana M. Sabria I. Sánchez S. Assaf S. Surman‐Lee 《Journal of applied microbiology》2011,110(4):1032-1044
Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l?1) and culture (CFU l?1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l?1 always exceeded CFU l?1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection. 相似文献
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Kai‐Yu Liu Yu‐Qian Xia Jing Zhou Zu‐Wen Chen Dandan Lu Ning‐Zhao Zhang Xu‐Sheng Liu Hui Ai Li‐Lin Zhou 《Archives of insect biochemistry and physiology》2016,92(4):225-241
Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry‐SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl‐HP cells and partially co‐localized with BmATG6‐GFP, but it almost has no significant co‐localization with GFP‐HaATG8. Then, the Western blot analysis demonstrated that GFP‐SeATG5 conjugated with ATG12. Moreover, real‐time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions. 相似文献