Parvalbumin is considered a major fish allergen. Here, we report the molecular evolution of the parvalbumin genes in bony fishes based on 19 whole genomes and 70 transcriptomes. We found unexpectedly high parvalbumin diversity in teleosts; three main gene types (pvalb-α, pvalb-β1, and pvalb-β2, including oncomodulins) originated at the onset of vertebrates. Teleosts have further multiplied the parvalbumin gene repertoire up to nine ancestral copies—two copies of pvalb-α, two copies of pvalb-β1, and five copies of pvalb-β2. This gene diversity is a result of teleost-specific whole-genome duplication. Two conserved parvalbumin genomic clusters carry pvalb-β1 and β2 copies, whereas pvalb-α genes are located separately in different linkage groups. Further, we investigated parvalbumin gene expression in 17 tissues of the common carp (Cyprinus carpio), a species with 21 parvalbumin genes in its genome. Two pvalb-α and eight pvalb-β2 copies are highly expressed in the muscle, while two alternative pvalb-α copies show expression in the brain and the testes, and pvalb-β1 is dominant in the retina and the kidney. The recent pairs of muscular pvalb-β2 genes show differential expression in this species. We provide robust genomic evidence of the complex evolution of the parvalbumin genes in fishes.

The real-time quantitative PCR (qPCR) calibration curves are highly reproducible and allow the generation of specific, sensitive, and reproducible data that can be used for gene quantification. However, it is important to rigorously validate the external calibration curve model in qPCR since absolute quantification is dependent on the standards used. We present a method for standardising qPCR-based quantification of the β-parvalbumin (β-pvalb) gene of Lophius piscatorius, a major fish allergen, using a plasmid DNA (pDNA) calibrator. In parallel experiments, standard curves were generated and compared from the genomic DNA (gDNA) isolated from L. piscatorius and pDNA carrying the target, pvalb. The commutability of pDNA and gDNA calibrators for the quantification of β-pvalb was assessed by employing a TaqMan qPCR, targeting the second intron of the pvalb gene of L. piscatorius. Higher PCR efficiencies, good linearity, and lower standard deviation (S.D.) values were observed with pDNA instead of gDNA calibrants. pDNA calibrants exhibited a lower bias in terms of closeness to the expected value of unknown samples than their genomic counterparts. The assay was specific and sensitive, where the limit of detection (LOD) and limit of quantification (LOQ) were five copies and ten copies per reaction. The short-term stability study of the pDNA calibrants indicated its stability for 60 days at − 20 °C and 30 days at 4 °C. The efficient results indicated a plasmid calibrator as a potential tool for absolute quantification of the pvalb gene and an alternative to conventional gDNA standards.

Background: Telomere shortening and mitochondrial dysfunction are well-known independent contributors to many diseases, but emerging evidence suggests a reciprocal relationship between the two processes. The role of the so-called telomere-mitochondrial axis in colorectal cancer (CRC) remains largely unknown. Methods: This prospective cohort study screened CRC patients who underwent surgery, from whom peripheral blood, intestinal mucosa, and tumor samples were collected. Colonoscopically confirmed cancer- and adenoma-free healthy individuals were screened as controls, from whom peripheral blood and intestinal mucosa samples were obtained. Relative mitochondrial DNA copy number (mtDNA-CN) and relative telomere length (RTL) were measured in all samples by real-time quantitative polymerase chain reaction and were further compared and correlated considering clinical data. Relative mtDNA-CN was quantified using both TaqMan probes and SYBR Green to compare both methods. Finally, multivariable analyses were conducted to investigate the association between both biomarkers and the risk of tumor recurrence and mortality. Results: A total of 166 CRC patients and 61 healthy individuals were included in the study. In TNM stage I patients, relative mtDNA-CN and RTL were negatively correlated with each other in intestinal mucosa (ρ = -0.77, p < 0.0001), tumor tissue (ρ = -0.41, p = 0.032), and the tumor-to-intestinal mucosa ratio (ρ = -0.39, p = 0.046). However, these associations disappeared with increasing TNM stage, suggesting a dysregulation of the telomere-mitochondrial axis in advanced disease. Higher relative mtDNA-CN in blood was associated with a lower risk of disease recurrence even after adjusting for multiple covariates (HR = 0.43, 95% CI 0.20–0.97, p = 0.041), highlighting its potential use as a prognostic tool. The quantification of mtDNA-CN performed by both methods -TaqMan probes and SYBR Green- was shown to be positively correlated ( p < 0.01). Relative mtDNA-CN and RTL were found to be tissue-dependent in both CRC patients and healthy controls. Conclusions: This study provides a novel contribution to the understanding of the almost unexplored telomere-mitochondrial axis in CRC, highlighting its potential role in disease progression and prognosis. Graphical Created with https://www.BioRender.com .

CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: relationships to tumor