MVA titration by plaque assay using crystal violet staining in DF-1 cells

Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.

Characterization and Genomic Analysis of a Plaque Purified Strain of Cyanophage PP

Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.

Establishing an animal model of unstable atherosclerotic plaques

Background Atherosclerotic plaque rupture and coronary thrombosis are the main causes of acute coronary syndromes. However, there is no animal model of unstable atherosclerotic plaques. The presence of the p53 gene in advanced atherosclerotic plaques and the sensitivity to p53-induced apoptosis of smooth muscle cells isolated from these plaques prompted us to build an animal model of unstable atherosclerotic plaques using p53 gene transfer. Methods Sixty-four New Zealand white rabbits were randomly divided into two groups: group A (n=54) and group B (n=10). Rabbits in group A underwent balloon-induced abdominal aortic wall injury and were then given a diet of 1% cholesterol, while rabbits in group B were given a diet of 1% cholesterol without the induction of aortic wall injury. At the end of the eighth week, rabbits in group A were randomly divided into two subgroups: group A 1 (n=27) and group A 2 (n=27). Recombinant adenovirus carrying p53 or β-galactosidase (LacZ) genes were injected through a catheter into the aortic segments rich in plaques in groups A 1 and A 2, respectively. Two weeks later, 10 rabbits each from groups A 1 and A 2 were killed to observe the occurrence of spontaneous plaque ruptures, and the remaining rabbits in groups A 1, A 2, and B all underwent pharmacological triggering with an injection of Chinese Russell’s viper venom (CRVV) and histamine. Results The over expression of p53 in group A 1 [(32.4±10.2)% vs (15.8±3.6)% in group A 2 and (16.2±6.7)% in group B, P<0.001, respectively] resulted in a marked increase in cellular apoptosis [(2.5±0.8)% vs (1.0±0.3)% in group A 2 and (0.9±0.4)% in group B, P<0.01, respectively], an accumulation of inflammatory cells within the plaques, and a significant decrease in vascular smooth muscle cells (VSMCs) and in the thickness of the fibrous caps. Although spontaneous plaque rupture was rare in group A 1, plaque ruptures and thrombosis occurred in 12 rabbits with a total of 20 lesions after pharmacological triggering. By contrast, pharmacological triggering led to plaque rupture and thrombosis in only 5 rabbits for a total of 7 lesions in group A 2 and in none of the rabbits in group B. Conclusion After transfection with human wild-type p53 gene and pharmacological triggering, plaque rupture and thrombosis occur in most atherosclerotic lesions in rabbits, thus offering a reliable model for the further study of unstable atherosclerotic plaques.