Background: The BRAF V600E gene encodes for the mutant BRAF V600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAF V600E mutations in tumor DNA, there is limited information about the level of BRAF V600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. Methods: Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAF V600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAF V600E DNA was performed in parallel for comparison and normalization of BRAF V600E mRNA expression levels. Results: The mRNA-based mutation detection assay enables detection of the BRAF V600E mRNA transcripts in a 10, 000-fold excess of wildtype BRAF counterparts. While BRAF V600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAF V600E mRNA in FFPE samples of thyroid cancer tissue. Conclusions: The expression levels of BRAF V600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAF V600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

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Abstract. Nguyen HU, Nguyen TH, Chau NQK, Le VV, Tran VH. 2020. Biology, morphology and damage of the lesser coconut weevil, Diocalandra frumenti (Coleoptera: Curculionidae) in southern Vietnam. Biodiversitas 21: 4686-4694. The lesser coconut weevil, Diocalandra frumenti, is an emerging pest of coconut trees in Vietnam. To help develop control options for D. frumenti, this study investigated its morphological and biological characteristics, and quantified damage levels on coconut trees. Results from the study showed that attack by D. frumenti on coconut trees is correlated with characteristic damage symptoms (e.g., oozing sap) on all maturity stages of coconut fruits throughout the year (24.7% infestation), with higher damage levels on young fruits (57.6%). Results also showed that infestation levels on trees (58.9%), coconut bunches (19.4%), and fruits (7.77%) varied greatly. Adults have four different morphologies, but genetic study showed that they are all one species. The life cycle from egg to adult averaged 167± 34.3 days. The average development time for eggs, larvae, female pupae, male pupae, male adults, and female adults was 5.62 ± 0.62, 142.3 ± 34.3, 9.79 ± 0.86, 10.2 ± 1.26, 81.5 ± 34.7, and 81.8 ± 37.2 days, respectively. In conclusion, D. frumenti is an important pest in coconut trees with significant fluctuations in the duration of its development stages, and variation in shape, color, and size of the eggs, larvae, pupae, and adults. They can attack any coconut orchard and have harmful impacts on coconut trees, coconut bunches, and fruits throughout the year.