A pilot study to screen the trisomy 13 from the amniotic fluid puncture.

Trisomy 13 (Patau syndrome) is a kind of congenital chromosomal abnormality disease. Trisomy 13 has high occurrence in fetuses or infants from the old aged pregnant women. Screening out the fetus with trisomy 13 early and avoiding the infant with trisomy 13 to be born is the main strategy in the care of delivery women with the fetus with trisomy 13. The current screening method is not perfect and has room to strengthen. In this study, we aimed to establish a method to strengthen the current screening methods, which would be cheap, fast and convenient. Technically, we obtained the commercially available genomic DNA extracted from the amniotic fluid puncture of the pregnant woman with the trisomy 13 fetus, 2 genomic DNA extracted from 2 healthy male (one adult and one teenager) and 1 genomic DNA extracted from 1 healthy adult female as the qPCR template DNAs and the commercially available Sybr green qPCR mater mix as the qPCR reaction liquid; we also designed and synthesized 5 pairs of qPCR primers, respectively, corresponding to IL-10 gene on 1# chromosome, STAT1 gene on 2# chromosome, CXCR3 gene on X chromosome, TSPY1 gene on Y chromosome and LINC00458 gene on 13# chromosome. We then performed Sybr green qPCR measurement. Further, we used the qPCR data to perform the mathematical calculation and finally formed a new algorithm. Using this new algorithm, we easily distinguished the trisomy 13 sample out of the normal samples. The method established this study could strengthen and complement the current methods. In conclusion, our study initiated a pilot study to screen the trisomy 13 and prospected some new directions for efforts.

A qPCR method to characterize the sex type of the cell strains from rats.

A simple and fast method was established to identify the sex types of the rat-derived cell strains. The single copy X-chromosome-linked gene AR and the single copy Y-chromosome-linked gene Sry were both detected with qPCR for the rat genomic DNA sample and the AR/Sry ratio was calculated. According to the law of the AR/Sry ratio, a new method to identify the sex types of the rat-derived cell strains was developed. The new assay was proved effective. The new assay showed advantages over the traditional sex type identification PCR methods, which detected only the Sry gene. Moreover, the new method was used to identify the sex types of two rat-derived cell strains unknown for the sex types and the results were confirmed with the in situ hybridization. Finally, the problem of the cross contamination between the female and the male samples was addressed and discussed extensively.

Study of oleanolic acid on the estrodiol production and the fat production of mouse preadipocyte 3T3-L1 in vitro.

The women during the menopause period have an increased tendency for the obesity, which represents the more fat production than during the premenopausal period. Although this is not beneficial overall, it could provide a compensatory source for the estrogen production for the menopausal women. So it would be meaningful to find an agent that could inhibit the fat production while does not disturb the total estrogen production by fat tissues. In the present study, the effect of oleanolic acid (OA) on the fat production and the total estrogen production of the differentiating mouse preadipocyte 3T3-L1 as well as the mechanisms behind those effects were preliminarily investigated. The cell line 3T3-L1 was chosen as the model cell because it is usually used for the research about the obesity. During the induced differentiation of 3T3-L1 cells, cells were intervened continuously with OA. The fat production was determined with the oil red staining assay and the total estrogen production was measured with the ELISA assay. Finally, the expression patterns for important genes of the fat production and the estrogen production were studied, respectively with the real-time fluorescence quantitative PCR (qPCR). The results showed that for the differentiating 3T3-L1 cells, OA could significantly inhibit the fat production and did not disturb the total estrogen production significantly. In the mechanism studies, OA was found to significantly down-regulate ACC, the key gene for fat synthesis, which could explain the inhibitory effect of OA on the fat production; OA was also found to significantly up-regulate CYP11A1, CYP17, CYP19, the key genes for the estrogen synthesis and significantly down-regulate CYP1A1, the key gene for the estrogen decomposition, which preliminarily explained the lack of the effect of OA on the total estrogen production. In conclusion, OA was found able to inhibit the fat production while maintaining the total estrogen level and the mechanisms for the above findings were preliminarily clarified, which suggests that OA may be useful to treat the menopausal obesity.

Retinoic acid can induce mouse embryonic stem cell R1/E to differentiate toward female germ cells while oleanolic acid can induce R1/E to differentiate toward both types of germ cells.

Retinoic acid (RA) and oleanolic acid (OA) were studied about their potential to induce mouse embryonic stem cell R1/E (MESC-R1/E) to differentiate toward germ cells. Embryoid bodies (EBs) first formed from MESC-R1/E and EBs were allowed to attach to the bottoms of normal cell-culturing plate and grow. Then, different compounds including RA, OA and so on were respectively added to induce MESC-R1/E to differentiate. After 72 h, microscopy images were taken for all interventions, then total RNAs were extracted, cDNAs were synthesized and real-time fluorescence quantitative PCR (qPCR) was performed to detect the transcriptional expression patterns of 11 reproductive-differentiation-related genes for different compounds respectively. During the data analysis, it was found RA significantly up-regulated the expression levels of GDF-9, Stra8, SCP3, Mvh, ZP1, ZP2, and ZP3, while significantly down-regulated the levels of Itag6 and Itgb1, and the level of Oct-4 was down-regulated insignificantly, while the level of TP2 was up-regulated insignificantly; OA significantly up-regulated the expression levels of Stra8, SCP3, Mvh, ZP1, ZP2, Itgb1, and TP2, and the levels of Oct-4, GDF-9, ZP3, and Itga6 were up-regulated insignificantly. The data showed that RA can induce MESC-R1/E to differentiate toward female germ cells while OA can induce MESC-R1/E to differentiate toward male and female germ cells.

Oleanolic acid has similar effects as retinoic acid in inducing mouse embryonic stem cell 1B10 to differentiate towards germ cells.

This study investigated the effects of the compound oleanolic acid (OA) in inducing mouse embryonic stem cells (MESC) to differentiate towards germ cells (GC). MESC 1B10 was used as the model cell. 1B10 was cultured, and embryoid bodies (EBs) were produced from 1B10. The EBs were allowed to attach to the bottoms of culturing disks and grow. OA was added into the medium to induce the EBs to differentiate. Retinoic acid (RA) was used as the positive drug. After 72 h, total RNA was extracted, cDNA was synthesized, and real-time fluorescence quantitative PCR was performed to measure the transcriptional expression profiles of 11 reproduction-related genes affected by OA and RA, respectively. When the data were compared, it was found that OA up-regulated the transcriptional levels of Oct-4, GDF-9, Stra8, Mvh, ZP2, ZP3, Itga6, and TP2, and down-regulated transcriptional levels of SCP3, ZP1, and Itgb1; RA up-regulated the transcriptional levels of GDF-9, Stra8, Mvh, ZP2, ZP3, Itga6, Itgb1, and TP2, and down-regulated the transcriptional levels of Oct-4, SCP3, and ZP1. The data showed that OA and RA had similar effects in inducing differentiation of MESC towards GC.

A novel membrane-bound E3 ubiquitin ligase enhances the thermal resistance in plants.

High temperature stress disturbs cellular homoeostasis and results in a severe retardation in crop growth and development. Thus, it is important to reveal the mechanism of plants coping with heat stress. In this study, a novel gene that we identified from Brassica napus, referred to as BnTR1, was found to play a key role in heat stress response in planta. BnTR1 is a membrane-bound RINGv (C4HC3) protein that displays E3 ligase activity in vitro. We demonstrated that modest expression of BnTR1 is sufficient to minimize adverse environmental influence and confers thermal resistance on development without any detrimental effects in B. napus and Oryza sativa. Our investigation into the action mechanism indicates that BnTR1 is likely to be involved in mediating Ca²âº dynamics by regulating the activity of calcium channels, which further alters the transcripts of heat shock factors and heat shock proteins contributing to plant thermotolerance. Hence, our study identified BnTR1 as a novel key factor underlying a conserved mechanism conferring thermal resistance in plants.