Despite worldwide applications of the modified live vaccines (MLV), canine distemper virus (CDV) remains a significant threat to highly susceptible wildlife animals and domestic dogs. Thus, to develop a novel completely safe vaccine is necessary. In this study, CDV hemagglutinin (H) protein as antigen target was expressed in a transient transfected Expi293F cells sandwiched with signal peptides and Twin strep II or Fc tag. The signal peptides HSA and mouse Ig kappa significantly produced the most secretory expressions of CDV H protein compared to the signal peptides TRY, AP, IL-2 and INS. Fc tag impaired CDV H protein secretion by dimer formation. The Expi293F cell pools harboring site-specific integration of the CDV H gene and GFP-T2A-BleoR gene in AAVS1 safe-harbor locus were generated by a dual- or single- sgRNA guided dCas9-piggyBac system with Zeocin screening. The dual-sgRNA provided higher transposition efficiency with 10.59 % or 10.32 % GFP+ cells than the single-sgRNA. Two monoclonal Expi293F cell lines E6 and G10 sorted by flow cytometry could stably produce 2.8 g per liter of CDV H protein in supernatant with a purity over 95 %. The secreted CDV H protein induced robust, dose-dependent neutralizing antibody responses in both mice and dogs. A 2 mg/kg dose of CDV H protein protected dogs 100 % survival against the virulent CDV HBF-1 challenge. This establishment of a transposon-mediated platform for stable expression of CDV H antigen protein in Expi293F cells demonstrates the incredible plasticity and provides a versatile framework for future subunit vaccine development.
Whole-genome sequencing (WGS) enables comprehensive analysis of tumour genomes, but its use in formalin-fixed paraffin-embedded (FFPE) samples is limited by DNA fragmentation and low yields. Whole-genome amplification (WGA) methods such as multiple displacement amplification (MDA) can boost DNA availability but distort copy-number alteration (CNA) profiles. DNA ligation-mediated MDA (DLMDA) mitigates this bias by reconstituting fragmented templates, yet its performance in FFPE-derived DNA remains uncertain. We compared paired DLMDA pre-amplified (2 h, 8 h) and non-pre-amplified FFPE prostate tumour samples from 15 archival blocks (5, 15 and 20 years old). DLMDA increased DNA yield by 7.3–15.4-fold, with global CNA patterns largely preserved. However, DLMDA significantly reduced the detected total CNA burden from 10.4 % (baseline) to 4.6 % after 2 h and 7.6 % after 8 h pre-amplification, independently of FFPE sample age. CNA dropouts were randomly distributed across the genome, indicating that DLMDA does not introduce regional bias. Our results show that DLMDA enables robust DNA yield recovery and avoids false-positive CNA artefacts, suggesting DLMDA can be used as rescue strategy for poor quality, low yield, FFPE-derived DNA, but at the cost of reduced CNA sensitivity. While suitable for CNA screening pipelines through WGS, further improvements are required to minimise the false-negative risk and improve the technique’s sensitivity for FFPE-based genomics.
Population aging drives a parallel increase in muscle atrophy and cognitive decline, two major causes of disability in older adults. Mitochondrial dysfunction and chronic inflammation are core drivers of aging, with pyroptosis serving as a critical link between them. However, conventional single‑technique approaches capture only fragmented, static information and fail to bridge molecular events with whole‑organism phenotypes. This review introduces a unique integrative perspective that weaves established multimodal technologies ranging from molecular interaction analysis and absolute quantification to cellular metabolic and electrophysiological recordings, tissue clearing and laser microdissection, and finally to in vivo imaging and body composition analysis into a coherent, cross‑scale chain of evidence. By applying this paradigm to the mitochondria‑pyroptosis axis, we reveal how oxidative mitochondrial DNA enhances NLRP3 binding, how aged cells exhibit metabolic vulnerability and electrophysiological susceptibility, how pyroptotic hotspots form with spatial heterogeneity in tissues, and how pyroptosis signals quantitatively correlate with the decline in muscle mass and cognitive function. The core contribution of this review is the methodological framework of multimodal integration that enables causal tracing from molecular triggers to functional outcomes. This paradigm offers a transferable strategy for dissecting complex aging mechanisms and provides a theoretical foundation for targeting the mitochondria‑pyroptosis axis in precision anti‑aging interventions.
Ribosome profiling is a powerful technique to discover translation events, and to reveal translational dynamics at gene and codon level. The procedure includes laborious ribosome isolation steps using density gradient ultracentrifugation. However, ultracentrifuges are not available in every laboratory, and ultracentrifugation is not suitable for high-throughput experimentation. PEG precipitation has been used to purify ribosomes and in this study we have tested whether PEG precipitation can be used as an alternative method to isolate ribosomes for ribosome profiling. We have benchmarked PEG precipitation against spin column-based size-exclusion and standard sucrose density ultracentrifugation methods for ribosome isolation. The methods were tested using the bacterial model system Bacillus subtilis treated with mupirocin, which stalls ribosomes at isoleucine codons, providing a testable reference for ribosome profile comparison. We found that the use of PEG precipitation, size-exclusion spin columns, and a combination of both, yielded ribosome profiles that were comparable to the standard procedure. With these methods, ultracentrifugation can be omitted, facilitating ribosome profiling and enabling high-throughput analyses.
