Pyrroloquinoline quinone ameliorates renal fibrosis in diabetic nephropathy by inhibiting the pyroptosis pathway in C57BL/6 mice and human kidney 2 cells.

Diabetic nephropathy (DN), which is characterized by renal fibrosis, is a major complication of diabetes, a disease that afflicted more than 460 million people worldwide in 2019. Pyroptosis is an essential signaling pathway in DN-related injuries, such as renal fibrosis. Pyrroloquinoline quinone (PQQ) is a naturally occurring bioactive compound that protects human kidney 2 (HK-2) cells from oxidative stress-induced damage caused by high glucose concentrations. However, the nature and underlying mechanism of the effect of PQQ on DN-related renal fibrosis remains unclear. In this study, we evaluated whether PQQ has potential protective effects against renal fibrosis due to DN by establishing type 1 diabetes in mice via streptozotocin treatment and then inhibiting their pyroptosis signaling pathway. We found that compared to control mice, the area of renal fibrosis and injury were significantly increased in diabetic mice, and this was accompanied by increased levels of expression of collagen Ⅰ and transforming growth factor-ß1; increased concentrations of the inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α; and activation of the pyroptosis pathway components nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, IL-1ß, and IL-18. All of these changes were reversed by PQQ treatment. Analogously, we treated cultured HK-2 cells with a high concentration of glucose (35 mmol/L), which caused these cells to exhibit significantly increased concentrations of reactive oxygen species (ROS), phosphorylated (p)-nuclear factor kappa B (NF-κB), p-IkappaB, NLRP3, caspase-1, IL-1ß, and IL-18, and the loss of mitochondrial transmembrane potential. However, PQQ treatment significantly blunted these effects. In conclusion, in this study we demonstrated that PQQ attenuates renal fibrosis by alleviating mitochondrial dysfunction, reducing ROS production, and inhibiting the activation of the NF-κB/pyroptosis pathway under conditions of DN and hyperglycemia.

Application of a novel method PCR-ligase detection reaction for tracking predator-prey trophic links in insect-resistant GM rice ecosystem.

Insect-resistant genetically modified (IRGM) rice is on the verge of commercial release in China, however, its potential non-target effect on non-target insect natural enemies remains controversial. Tracking trophic interactions between predators and preys in IRGM rice ecosystem can provide new insights into better understanding of the ecological risks of IRGM rice. In the present study, a novel method based on ligase detection reaction (LDR), PCR-LDR was introduced to track 15 prey species in the gut of a predaceous spider Pirata subpiraticus, a dominant natural enemy in rice field. Our results indicated that PCR-LDR could provide high specificity and sensitivity in tracking prey-predator interactions in rice ecosystems. PCR-LDR could detect as little as 1,000 th of DNA mixture. Reliable detection of DNA samples of prey species using PCR-LDR could be significantly affected by digestion time and prey species. In the analysis of 200 field-collected P. subpiraticus and 105 field-collected Tetragnatha maxillosa individuals using PCR-LDR, prey remains were identified in 78.3 and 74.3% of the individuals, respectively, from which significant predation differences between the two spider species were observed. Predation behavior of the spider species was not significantly different between Bt and non-Bt control rice lines. These results indicated that PCR-LDR can be used as an important tool for ecological studies, especially on the interactions between predators and preys in IRGM rice or other similar ecosystems.

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

[Development, optimization and application of the expression analysis platform based on multiplex quantitative RT-PCR using fluorescent universal primers].

A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. This technology employs a chimeric-primer-induced-universal-primer amplification method that ensures target genes amplified in a constant ratio. This technique was cost-effective, moderate-throughput, and reliable in quantification of gene expression. It is complementary to cDNA chip, which has low quantitative accuracy , and Real-time quantitative PCR with low throughput, through improving the entire process of expression profiling analysis. Eleven genes within a QTL segment regulating mouse puberty onset on chromosome X were investigated to construct and optimize the method. The sensitivity of detection (102 copies) was determined, the concentration ratio of universal primer and chimeric forward primers (1:1) was optimized, and the accuracy and repeatability were validated. The method of Touchdown PCR with addition of universal primers significantly improved amplification of genes expressed in low abundance. After testing the expression profile of 11 genes in hypothalamus and testis in two mouse strains C3H/HeJ and C57BL/6J at the age of 15 d, one gene named PHF6 was found differentially expressed for further function analysis.

Use of Luminex xMAP bead-based suspension array for detecting microRNA in NSCLC tissues and its clinical application.

BACKGROUND: We measured the expression of microRNA (miRNA) in non-small cell lung cancer (NSCLC) tissues using the Luminex xMAP bead-based suspension array. We discuss the feasibility of employing this method to detect miRNA in NSCLC and explore its value as a high-throughput miRNA array. METHODS: We performed the methodological analysis of xMAP with oligoribonucleic acid references. We detected the expression of miR-21, miR, miR-31, miR-222, miR-145 and miR 40 NSCLC cancer tissues and adjacent normal tissues by xMAP bead-based suspension array. We selected miR-191 and miR-103 as the house-keeping genes (internal control). We also analyzed the relationship between xMAP and RT-PCR. RESULTS: The methodological analysis parameters of xMAP are quite good. The expression of miR-21, miR, miR-31 and miR-222 was higher in NSCLC tissues than in adjacent tissues, while the expression of miR-145 and miR-126 was lower in NSCLC tissues than in adjacent tissues. The expression of miR-145 and miR-126 decreased with disease progression. The intraassay and interassay coefficients of variation were lower in xMAP than in RT-PCR. xMAP proved cheaper and more flexible in detecting multiple miRNAs of one sample. CONCLUSIONS: The Luminex xMAP bead-based suspension array for detecting miRNA has many advantages, such as allowing a smaller sample size (only 2 µL), no sample amplification, fast detection, high throughput, and flexible combination of multiple detection targets. The high throughput testing technology shows a great advantage in saving time and labor. We found that the Luminex xMAP bead-based suspension array is a good and feasible method for detecting miRNA expression with high-throughput technology.