Proteomic analysis of plasma proteins from patients with cardiac rupture after acute myocardial infarction using TMT-based quantitative proteomics approach.

BACKGROUND: Cardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of CR has yet to be elucidated. METHODS: In the present study, a quantitative approach with tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry was used to characterize the differential protein expression profiles of patients with CR. Plasma samples were collected from patients with CR (n = 37), patients with AMI (n = 47), and healthy controls (n = 47). Candidate proteins were selected for validation by multiple reaction monitoring (MRM) and enzyme-linked immunosorbent assay (ELISA). RESULTS: In total, 1208 proteins were quantified and 958 differentially expressed proteins (DEPs) were identified. The difference in the expression levels of the DEPs was more noticeable between the CR and Con groups than between the AMI and Con groups. Bioinformatics analysis showed most of the DEPs to be involved in numerous crucial biological processes and signaling pathways, such as RNA transport, ribosome, proteasome, and protein processing in the endoplasmic reticulum, as well as necroptosis and leukocyte transendothelial migration, which might play essential roles in the complex pathological processes associated with CR. MRM analysis confirmed the accuracy of the proteomic analysis results. Four proteins i.e., C-reactive protein (CRP), heat shock protein beta-1 (HSPB1), vinculin (VINC) and growth/differentiation factor 15 (GDF15), were further validated via ELISA. By receiver operating characteristic (ROC) analysis, combinations of these four proteins distinguished CR patients from AMI patients with a high area under the curve (AUC) value (0.895, 95% CI, 0.802-0.988, p < 0.001). CONCLUSIONS: Our study highlights the value of comprehensive proteomic characterization for identifying plasma proteome changes in patients with CR. This pilot study could serve as a valid foundation and initiation point for elucidation of the mechanisms of CR, which might aid in identifying effective diagnostic biomarkers in the future.

Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

Fusarium Wilt Invasion Results in a Strong Impact on Strawberry Microbiomes.

Plant-endophytic microbes affect plant growth, development, nutrition, and resistance to pathogens. However, how endophytic microbial communities change in different strawberry plant compartments after Fusarium pathogen infection has remained elusive. In this study, 16S and internal transcribed spacer rRNA amplicon sequencing were used to systematically investigate changes in the bacterial and fungal diversity and composition in the endophytic compartments (roots, stems, and leaves) of healthy strawberries and strawberries with Fusarium wilt, respectively. The analysis of the diversity, structure, and composition of the bacterial and fungal communities revealed a strong effect of pathogen invasion on the endophytic communities. The bacterial and fungal community diversity was lower in the Fusarium-infected endophytic compartments than in the healthy samples. The relative abundance of certain bacterial and fungal genera also changed after Fusarium wilt infection. The relative abundance of the beneficial bacterial genera Bacillus, Bradyrhizobium, Methylophilus, Sphingobium, Lactobacillus, and Streptomyces, as well as fungal genera Acremonium, Penicillium, Talaromyces, and Trichoderma, were higher in the healthy samples than in the Fusarium wilt samples. The relative abundance of Fusarium in the infected samples was significantly higher than that in the healthy samples, consistent with the field observations and culture isolation results for strawberry wilt. Our findings provide a theoretical basis for the isolation, identification, and control of strawberry wilt disease.