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Objective: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze–thaw cycles on qPCR reagents for eDNA analysis across six assays. Results: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of − 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations. [ABSTRACT FROM AUTHOR]

Background After completion of embryogenesis, many organisms experience an additional obligatory developmental transition to attain a substantially different juvenile or adult form. During anuran metamorphosis, the aquatic tadpole undergoes drastic morphological changes and remodelling of tissues and organs to become a froglet. Thyroid hormones are required to initiate the process, but the mechanism whereby the many requisite changes are coordinated between organs and tissues is poorly understood. Metabolites are often highly conserved biomolecules between species and are the closest reflection of phenotype. Due to the extensive distribution of blood throughout the organism, examination of the metabolites contained therein provides a system-wide overview of the coordinated changes experienced during metamorphosis. We performed an untargeted metabolomic analysis on serum samples from naturally-metamorphosing Rana catesbeiana from tadpoles to froglets using ultraperformance liquid chromatography coupled to a mass spectrometer. Total and aqueous metabolite extracts were obtained from each serum sample to select for nonpolar and polar metabolites, respectively, and selected metabolites were validated by running authentic compounds. Results The majority of the detected metabolites (74%) showed statistically significant abundance changes (padj < 0.001) between metamorphic stages. We observed extensive remodelling of five core metabolic pathways: arginine and purine/pyrimidine, cysteine/methionine, sphingolipid, and eicosanoid metabolism and the urea cycle, and found evidence for a major role for lipids during this postembryonic process. Metabolites traditionally linked to human disease states were found to have biological linkages to the system-wide changes occuring during the events leading up to overt morphological change. Conclusions To our knowledge, this is the first wide-scale metabolomic study of vertebrate metamorphosis identifying fundamental pathways involved in the coordination of this important developmental process and paves the way for metabolomic studies on other metamorphic systems including fish and insects. [ABSTRACT FROM AUTHOR]

Background: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues., Results: We report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution., Conclusions: Our biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.

Background: Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action., Results: Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport., Conclusion: We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone.