Exploring the mechanism of hirudin in the treatment of diabetic kidney disease using network pharmacology combined with molecular docking verification.

OBJECTIVE: To explore the mechanism of hirudin in the treatment of diabetic kidney disease (DKD). METHOD: Cytoscape software was used to analyze the network between hirudin targets and active components in the treatment of DKD. The biological function and mechanism of effective targets of hirudin for DKD treatment were analyzed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. Molecular docking technology was used to simulate the docking of key targets, and the DKD rat model was used to verify the first 4 key targets with high "Hydrogen number" among the top 10 targets verified by molecular docking. RESULTS: Total of 12334 DKD targets were screened in GeneCards, OMIM and other databases, Hirudin and DKD had 247 common target genes, and the protein interaction network got 2115 edges. The DAVID database was used for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, confirming that hirudin in treatment of DKD involves multiple signaling pathways such as the forkhead box O signaling pathway, the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, the vascular endothelial-derived growth factor signaling pathway and other signaling pathways. The top ten key targets of hirudin in treatment of DKD were verified by molecular docking. Animal experiments showed that hirudin could decrease the expression of caspase-3 in renal tissue of DKD rats, and increase the expression of RAC-alpha serine/threonine-protein kinase, Catalase, and Heat shock protein HSP 90-alpha in renal tissue of DKD rats. CONCLUSION: This study preliminarily reveals that hirudin treats DKD through multiple targets and pathways, and molecular docking and animal experiments indicates the feasibility of this study. Hirudin may be directly or indirectly involved in the regulation of cell metabolism, oxidative stress and other mechanisms in the treatment of DKD, which will lay the foundation for future molecular biological experiments of hirudin in the treatment of DKD.

Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Image quality assessment of computed tomography pulmonary angiography performed using a 70 kV/sn150 kV model combined with the advanced modeled iterative reconstruction method in overweight patients / 中华放射医学与防护杂志

Objective:To evaluate the image quality of dual-source computed tomography pulmonary angiography (DE-CTPA) with low-dose contrast agent using the advanced modeled iterative reconstruction (ADMIRE) method with 70 kVp and non-linear blending in overweight patients.Methods:Seventy patients (normal BMI, 35; overweight, 35) with suspected pulmonary embolization who underwent DE-CTPA between October 2018 and March 2019 were included in this study. The imaging protocol included assessments at 70 kV/sn150 kV with 30 ml of contrast agent, and images were obtained at 70 kVp and 150 kVp with and without linear blending. The CT value, SD value, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) of pulmonary arteries were compared and analyzed among groups 1 (70 kVp, normal BMI), 2 (non-linear blending, normal BMI), 3 (70 kVp, overweight), and group 4 (non-linear blending, overweight). The radiation dose parameters included CT volume dose index (CTDI vol), dose length product (DLP), and effective dose ( E). Results:The CT values for the pulmonary artery did not show significant differences among the four groups ( P>0.05). The SD value of the segmental artery in group 1 was higher than that in group 4 ( t=2.69, P<0.05). The SNR values of the pulmonary artery trunk and sub-segmental artery in group 2 were higher than those in group 3 ( t=1.44, 5.40, P<0.05), while the corresponding value of the left pulmonary artery trunk in group 2 was higher than those in groups 3 and 4 ( t=1.52, 1.52, P<0.05). The CNR values of the pulmonary artery trunk and sub-segmental artery in group 2 were higher than those in group 3 ( t=1.45, 5.01, P<0.05) and that of the left pulmonary artery trunk in group 2 was higher than those in groups 3 and 4 ( t=1.50, 1.50, P<0.05). The E values for normal BMI and overweight patients were(1.60±0.54)mSv and(1.88±0.45)mSv, respectively. Conclusions:For overweight patients, the CTPA protocol using ADMIRE with a 70 kV/sn150 kV scanning mode could yield diagnostic image quality with significantly lower radiation and contrast material doses.