The effects of tea flower saponin, which was extracted and purified by macroporous resin and preparative liquid chromatography, on the proliferation and traits of human ovarian cancer stem like cells (OCSLCs) and its mechanism were studied. The stem cell marker aldehyde dehydrogenase (ALDH) activity, cell vitality, colony formation capacity, tumor sphere formation capacity and the expression of stemness-related proteins were measured to investigate the effects of tea flower saponin on OCSLCs of A2780/CP70 cells obtained by serum-free suspension culture. Western blot assay was used to investigate the effects of tea flower saponin on Wnt/β-catenin signaling pathway proteins. The results showed that tea flower saponin could decrease the proliferation of OCSLCs by suppressing their cell activity and colony formation ability. Tea flower saponin inhibited the formation of tumor sphere and reduced cell self-renewal ability. The ALDH＋ cell proportion and expressions of Oct-4 and Nanog proteins were decreased in OCSLCs treated with tea flower saponin. In addition, tea flower saponin treatment could down-regulate the expression of P-AKT, P-GSK-3β, β-catenin, c-Myc and up-regulate the expression of P-β-catenin to inhibit the Wnt/β-catenin signaling pathway. These results indicate that suppression of the Wnt/β-catenin signaling pathway might be one of the mechanisms by which tea flower saponin inhibits the proliferation of OCSLCs and cancer stem cell traits.
In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence of single-guide RNA (sgRNA) of goose SCD gene; second, synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays; finally, prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids. Results showed that the lentiviral plasmid was successfully constructed, and the strong double positive cell groups co-expressing the red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP) were screened out when the lentiviral plasmid infected the Chinese hamster ovary (CHO) cells. The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene.
Transgelin-like protein (TLP) is a novel shell matrix protein identified previously from the myostracum layer of Mytilus coruscus shell. For exploring its function in the shell formation of mussel, the TLP was recombinantly expressed by Escherichia coli expression system based on the sequence analysis and codon optimization. The functions of recombinant TLP (rTLP) on calcite- and aragonite-type calcium carbonate crystals were then investigated, including morphology, polymorph, crystallization rate, and binding ability of calcium carbonate crystals. Sequence analysis showed that the TLP contained a calponin homology (CH) domain, and the spatial structure of TLP predicted by SWISS-MODEL presented a conformation formed predominately by α-helices. Functional analyses showed that the rTLP had significant effects on the morphological change of aragonite-type calcium carbonate crystal and polymorph change of calcite-type calcium carbonate crystal, suggesting the function of this protein in the transformation of calcite to aragonite. In addition, the rTLP showed inhibition of calcite-type calcium carbonate crystallization rate in vitro, and promotion of aragonite-type calcium carbonate crystallization rate under high protein concentration. Moreover, the rTLP presented binding abilities to the calcite-type calcium carbonate crystal rather than the aragonite-type calcium carbonate crystal. Considering the myostracum layer is composed of aragonite-type calcium carbonate crystal, we speculate that the TLP may play important roles in the shell biomineralization and the formation of myostracum layer.
In order to develop a rapid, specific, and accurate detection method for the concentration of recombinant human bone morphogenetic protein-2 (rhBMP-2) through Escherichia coli-based expression systems, enzyme-linked immunosorbent assay (ELISA) was combined with microfluidic chip. The specific first antibody for rhBMP-2 was screened from several commercial products. Based on the regulation strategy of antibody orientation for enhancing detection signal, plasma-protein A method was then used to modify the detection microwells of the microfluidic chip. After tuning the conditions of plasma treatment on the detection microwells, adsorption efficiency of the first antibody and strength of the final detection signal were evaluated. It was found that the better capture efficiency of the first antibody could be obtained by using the higher power in the plasma treatment process. The best plasma condition was the power of 100 W and treatment time of 30 s. After the optimized modification conditions were applied for the microfluidic chip, the dilute concentrations of rhBMP-2 in a range of 0-2 000 pg/mL were achieved. In comparison with the standard assay carried out in the 96-well microtiter plate, the microwells of microfluidic chip exhibited a broader linear detection range (0-2 000 pg/mL vs. 0-250 pg/mL) and a much less reagent consumption (Each sample needed 600 μL reagent consumption in the standard assay, while about 16 μL in the microwell assay, which was 97.3% reduction in dosage). Clearly, this plasma-protein A immobilization strategy holds a great potential for polymeric microfluidic chip based assay in biomedical application, food safety, and environment monitoring.
Previous work proved that a strain of Meyerozyma guilliermondii isolated from the Xisha islands of Hainan Province had good salt-tolerant ability which could grow under 12% NaCl stress culture condition. Based on the above results, the transcriptome sequencing of the straincultured under salt stress and non-salt stress for 24 h was constructed by Illumina HiSeqTM in this study. The results were as follows: the two samples yielded 1 027 significantly differential expression genes, of which 458 genes were up-regulated and 569 genes were down-regulated. According to the gene ontology (GO) functional annotations, the differential expression genes of M. guilliermondii treated with salt stress were mainly concentrated on the classification of biological process, in which nucleotide metabolism, sugar metabolism and coenzyme metabolism genes were greatly different. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differential expression genes showed that the most of the enrichment pathways were related to cell division and metabolism, which were corresponding to the GO enrichment results. The above results can provide scientific basis for further biological research on the effect of the growth of M. guilliermondii under salt stress.
A novel multiple target plasmid (MTP) molecule that satisfies the identification of specific transgenic soybean transformants in China was constructed. Fourteen transformants approved for import into China and four ones unauthorized but have important application prospects, were chosen as the targets. The transformants’ specific sequences described in corresponding national detection standard and the detection sequence of the soybean endogenous reference gene Lectin were properly arranged and spliced, and then was inserted to linearized pUC18 plasmid to produce a multi-target plasmid pDDID-1905. Among the insertion sequences, plenty of restriction endonuclease recognition sites were dispersed uniquely, which could be used for later revision and/or update of the plasmid. Polymerase chain reaction (PCR) detection targeted on the 19 inserted sequences was performed to verify the utility of the plasmid. The results showed that all target sequences were successfully amplified and the products met the expected sizes, which indicated that the multi-target plasmid pDDID-1905 suitable for the identification of 18 transgenic transformants was constructed. Due to the widest target coverage of pDDID-1905, it will greatly simplify the cumbersome preparation of positive materials for identifying these transgenic soybean transformants and their derivates.
To study the effects of in vitro digestion products of royal jelly protein on the proliferation and apoptosis of gastric cancer cell SGC-7901 and to explore its possible mechanism so as to provide an experimental basis for the royal jelly protein in the treatment of gastric cancer, the royal jelly protein was extracted by alkali extraction and acid precipitation. The in vitro digestion products of royal jelly protein were obtained by in vitro simulated digestion experiments. The gastric cancer cells SGC-7901 were cultured in vitro with different concentrations of royal jelly protein in vitro digestion products, and the influence on gastric cancer cells were observed by an inverted microscope. The colony formation ability of gastric cancer cells after induction was tested. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect the inhibition against proliferation of the gastric cancer cells; the cell cycle and apoptosis were detected by flow cytometry; and the expression levels of p53 and PARP-1 proteins in the gastric cancer cells were detected by Western blotting. The results showed that the in vitro digestion products of royal jelly protein leaded to the shrinkage of gastric cancer cells, decreased cell density and colony formation ability (P＜0.05), and inhibited the proliferation of gastric cancer cell SGC-7901. When the in vitro digestion product concentration was 0.2 mg/mL, the inhibition rates at 24 h and 48 h were (57.58±3.48)% and (62.84±1.98)%, respectively (P＜0.05). Compared with the blank control group, the in vitro digestion products of royal jelly protein inhibited the proliferation of SGC-7901 cells in a dose-dependent manner, and the S phase cells decreased, which leading to the apoptosis. After induction by in vitro digestion products of royal jelly protein, the expression of p53 increased in the gastric cancer cells (P＜0.05), and the expression level of PARP-1 protein decreased (P＜0.05). The above results show that the in vitro digestion products of royal jelly protein can inhibit the proliferation of gastric cancer cell SGC-7901 and induce its apoptosis. The mechanism may be related to the up-regulation of p53 expression and the down-regulation of PARP-1 expression.
DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNA marker by an improved overlap extension polymerase chain reaction (PCR) was established. The DNA fragment, containing the identical sequence in its 5 ' and 3 ' ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR, two single-stranded DNA in the 3 ' end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories.
Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) markers were applied to analyze genetic diversity among 43 strawberry (Fragaria×ananassa Duch.) cultivars and effectivenesses of these two kinds of molecular markers were also compared. The results showed that there were 6.43 polymorphic sites per primer pair of 30 SSR primers, and the average polymorphism information content (PIC) of each site was 0.628 4. While there were 14.30 polymorphic sites per primer pair of 20 SRAP primers，and the average PIC of each site was 0.911 4. The correlation coefficient between clustering results based on SSR markers and SRAP markers was 0.817, which was significant. The correlation coefficients between SSR markers, SRAP markers and SSR+SRAP joint markers were 0.938 and 0.966, respectively, which reached extremely significant levels. Both SSR and SRAP markers can be used to analyze the genetic diversity of strawberry, but the effect of SRAP marker is better than that of SSR marker. The analysis of SSR+SRAP joint markers can better evaluate the genetic diversity and genetic relationship of strawberry germplasm.
Fungal pigments are the potential resources as a natural food colorant. An endophytic fungus RJL03, which is able to produce abundant soluble red pigments, was isolated from the medicinal plant Rehmannia glutinosa Libosch. For further research and utilization of this strain and its secondary metabolites, morphological and molecular characteristics of RJL03 were identified. For observing the colonial morphology, the strain was cultured on different substrates at 25 ℃ for seven days. The fungus was also observed through light microscope and scanning electron microscope. The colony of this fungus was white at first and turned red at later stage, villous, circular; the mycelium was composed of irregularly branched, septate; conidia had borne terminally on pedicels, single or 2-10 in chain, which was about (8.0-16.5) μm×(7.5-14.0) μm; the cleistothecia was globose, brown, arising terminally from the apex of short hyphae, whose diameter was 32-70 μm; the ascospore was ellipsoidal, smooth, colorless or red, which was about (6.0-7.5) μm×(4.0-5.0) μm. Sequence analysis showed that ITS and 18S of the strain had 100% and 93% similarities with Monascus sanguineus, respectively. In conclusion, morphological results and phylogenetic analysis provided great evidence that endophytic fungus RJL03 was M. sanguineus. This study first reported M. sanguineus isolates from Rehmannia glutinosa Libosch. in China . It is considered that M. sanguineus, unlike M. purpureus, should be an independent species.
