Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring.

Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.

Evaluation of MEDAG gene expression in papillary thyroid microcarcinoma: associations with histological features, regional lymph node metastasis and prognosis.

Papillary thyroid microcarcinoma accounts for a large proportion of papillary thyroid carcinoma, especially among new cases. Many  PTMC patients have regional lymph node metastasis, with some experiencing recurrence and even death. However, the risk factors and mechanism by which PTMC relates to these factors are unknown. In this study, differentially expressed genes were identified with microarray from The Cancer Genome Atlas, followed by analysis using the Kyoto Encyclopedia of Genes and Genomes. Immunohistochemistry, immunofluorescence, western blot and Oil Red O staining were carried out to evaluate expression levels and functional alterations. Mesenteric Estrogen Dependent Adipogenesis expression was observed in almost all cases of papillary thyroid microcarcinomas, and the location of expression was associated with histological subtype. High expression was correlated with metastasis and poor disease-free survival. Furthermore, the enrichment analysis indicated that Mesenteric Estrogen Dependent Adipogenesis expression may be associated with metabolic reprogramming to influence metastasis and prognosis. These findings contribute to a better understanding of how Mesenteric Estrogen Dependent Adipogenesis affects metastasis and the prognosis of papillary thyroid microcarcinoma patients and suggest that Mesenteric Estrogen Dependent Adipogenesis expression may be a novel prognostic marker in these patients.