RESUMEN
Pheretima,also called"earthworms",is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edi-tion).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing al-gorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.
RESUMEN
Objective:To provide experimental evidence for genetic counseling and prenatal diagnosis by analyzing the clinical characteristics, screening and identification of the function of suspicious variants in a X-1inked spondyloepiphyseal dysplasia tarda (SEDT) family.Methods:The family members' medical history, general physical examination, femur, spine X-ray examination were collected. Peripheral blood samples of the family members were collected and DNA was extracted from these samples. Sequencing clinical whole exons of proband DNA by targeted gene high-throughput sequencing method, then analysis sequencing data. The suspicious mutation was confirmed in pedigree members by PCR and Sanger sequencing. Reverse transcription polymerase chain reaction (RT-PCR) experiments of total RNA from blood lymphocytes were performed. The amplification of exons 3 and 4 of the pathogenic gene were amplified and identified by agarose gel. The expression of the pathogenic gene was also detected.Results:Three affected males of the family were diagnosed with SEDT according to their clinical and radiological features. A nonsense mutation in the transport protein particle complex subunit 2 ( TRAPPC2) gene NM_001011658: c.91A>T (p.K31*) was found in the proband using whole exome sequencing. This variation was also detected in his cousin, but not in non-phenotypic members of the family. The RT-PCR result for amplification of exon 3 and 4 of peripheral blood lymphocytes was the same as those of normal controls, indicating that the mutation did not affect the splicing of transcripts. qPCR results showed that the transcriptional expression of TRAPPC2 in patients was significantly lower than that in family normal controls and normal people controls. Conclusion:Identification of the novel nonsense mutation (c.91A>T) in the SEDT family enables early patients screening, carrier detection, genetic counseling, prenatal diagnosis, and clinical prevention and treatment. The detailed genotype/phenotype descriptions contribute to the SEDT mutation spectrum. The study of the function of TRAPPC2 mutation will help to further elucidate the role of sedlin in cartilage.