[Multi-index evaluation of different parts of Amomum villosum].

To investigate the quality differences between the seeds and husks of Amomum villosum and explore the rationality of using the seeds without husks, this study determined the content of protocatechuic acid, vanillic acid, epicatechin, quercitrin, volatile oil, water extract, and ethanol extract. The 2,2-diphenyl-1-picrylhydrazyl(DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), and hydroxyl radical scavenging activities were determined to evaluate the antioxidant activities of seeds and husks. The quality differences between the seeds and husks were assessed through orthogonal partial least squares-discriminant analysis(OPLS-DA) and analytic hierarchy process(AHP) combined with the entropy weight method(EWM). Significant differences(P<0.05) were observed in all 10 indicators between the seeds and husks. The levels of epicatechin, quercetin, and volatile oil were higher in the seeds, whereas those of protocatechuic acid, vanillic acid, water extract, and ethanol extract were higher in the husks. The seeds showed stronger scavenging ability against DPPH and ABTS radicals, while the husks showed a stronger scavenging effect on hydroxyl radicals. OPLS-DA significantly discriminated between the seeds and husks. Furthermore, volatile oil, water extract, DPPH radical scavenging rate, quercitrin, ABTS radical scavenging rate, hydroxyl radical scavenging rate, and vanillic acid were selected as the main differential indicators by variable importance in projection(VIP). Comprehensive scores calculated by AHP combined with EWM indicated that the seeds were superior to husks in terms of overall quality. However, there are still some dominant components and a certain antioxidant effect in the husks. Therefore, it is suggested to using Amomi Fructus with a certain amount of husks or utilizing the husks for other purposes.

Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis.

BACKGROUND: Kazal-type serine protease inhibitors play a role in physiological processes such as blood coagulation and fibrinolysis. The amino acid residues at the P1 site are different, and they inhibit different types of proteases. The inhibitory mechanism of the protease in the salivary glands of Poecilobdella manillensis is still unclear. METHODS AND RESULTS: Based on cloning, prokaryotic expression and bioinformatics analysis, we studied the role of Kazal-type serine protease inhibitors in P. manillensis and analyzed their expression by quantitative real-time PCR. The results suggested that the recombinant protein was successfully expressed in the supernatant when a prokaryotic expression vector was constructed and induced with 0.2 mmol/L IPTG at 37 °C for 4 h, and the enzymatic activity was determined. The mature protein encodes 91 amino acids and has a relative molecular weight of 9929.32 Da, and after removing the signal peptide, the theoretical isoelectric point was 8.79. It is an unstable protein without a transmembrane domain. The mature protein contains two Kazal-type domains, in which all P1 residues are Lys, consisting of an α helix and three antiparallel ß sheets. The upregulated expression of the mRNA was induced after a meal was provided, and the results showed an increasing and then decreasing trend. CONCLUSIONS: Taken together, the results indicate that mature proteins from P. manillensis inhibit thrombin activity, laying the foundation for the subsequent in-depth study of the function of genes encoding Kazal-type serine protease inhibitors.

miR-331-3p Inhibits Tumor Cell Proliferation, Metastasis, Invasion by Targeting MLLT10 in Non-Small Cell Lung Cancer.

OBJECTIVE: Mounting research has established the role of microRNAs (miRNAs) as oncogenes or anti-oncogenes (tumor suppressors) in the development and progression of several cancers. The purpose of our current study is to delineate the roles and functional mechanisms of miR-331-3p and MLLT10 in non-small cell lung cancer (NSCLC) tumorigenesis. PATIENTS AND METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was employed to measure miR-331-3p expression levels in twenty-six matched tumor tissues and non-cancerous tissues collected from patients suffering from NSCLC, and from six NSCLC cell lines separately: A549, H1650, H292, H1299, H1944 and BEAS-2b. We employed the dual-luciferase activity assay to check whether the putative gene, MLLT10, was a downstream target of miR-331-3p in NSCLC pathogenesis and development. Western blot was conducted to analyze the protein expression levels of MLLT10 (AF10), E-cadherin, Vimentin, and GAPDH. CCK-8 assay, transwell migration assay, and transwell invasion assay were carried out to observe the functions of miR-331-3p and MLLT10 on NSCLC tumor cell proliferation, metastasis, and invasion, respectively. To identify whether the metastasis of NSCLC tumor cells was EMT-mediated, supplementary experiments involving E-cadherin and Vimentin were implemented. RESULTS: miR-331-3p was downregulated in NSCLC, which promoted tumor cell proliferation, whereas the overexpression of miR-331-3p inhibited tumor cell proliferation. Being a direct target of miR-331-3p, MLLT10 was negatively modulated by miR-331-3p, which suppressed tumor cell proliferation, migration, and invasion in NSCLC. However, MLLT10 overexpression alleviated the above inhibitory effects. Furthermore, EMT-mediated metastasis was proved to be present in NSCLC. CONCLUSION: miR-331-3p played a suppressor role in NSCLC tumor cell proliferation, EMT-mediated metastasis, and invasion by targeting MLLT10. Our findings highlighted that miR-331-3p/MLLT10 axis could be useful as a clinical diagnostic marker and therapeutic target in NSCLC patients.

Upregulation of hsa_circ_0136666 contributes to breast cancer progression by sponging miR-1299 and targeting CDK6.

Circular RNAs (circRNAs) can participate in multiple cancers, including breast cancer. Increasing circRNAs are recognized in various cancers because of the high-throughput sequencing. However, the potential physiological effect of hsa_circ_0136666 in breast cancer progression is unknown. In our study, the biological role of hsa_circ_0136666 in breast cancer development was studied. It was displayed that hsa_circ_0136666 was greatly increased in breast cancer. In addition, overexpression of hsa_circ_0136666 was able to promote Michigan Cancer Foundation-7 (MCF7) and BT474 cell proliferation and triggered cell cycle in G2/M phase. microRNA plays critical role in tumor development and they can act as direct targets of circRNAs. miR-1299 has been implicated as a famous tumor suppressor in many cancers. Here, miR-1299 was predicted as the target of hsa_circ_0136666. Meanwhile, its Upregulation repressed breast cancer proliferation, migration and invasion capacity, which could be reversed by the increase of hsa_circ_0136666. Furthermore, Cyclin-dependent kinase 6 (CDK6) was speculated as the downstream target of miR-1299. In MCF7 and BT474 cells, CDK6 was greatly overexpressed and it was shown that CDK6 contributed a lot to breast cancer progression. Subsequently, it was implied that hsa_circ_0136666 could modulate CDK6 levels positively in vitro. In conclusion, it was revealed that Upregulation of hsa_circ_0136666 promoted breast cancer progression by sponging miR-1299 and targeting CDK6.

Enhanced biohydrogen production from sugarcane bagasse by Clostridium thermocellum supplemented with CaCO3.

Clostridium thermocellum ATCC 27405 was used to degrade sugarcane bagasse (SCB) directly for hydrogen production, which was significantly enhanced by supplementing medium with CaCO3. The effect of CaCO3 concentration on the hydrogen production was investigated. The hydrogen production was significantly enhanced with the CaCO3 concentration increased from 10mM to 20mM. However, with the CaCO3 concentration further increased from 20mM to 100mM, the hydrogen production didn't increase further. Under the optimal CaCO3 concentration of 20mM, the hydrogen production reached 97.83±5.19mmol/L from 2% sodium hydroxide-pretreated SCB, a 116.72% increase over the control (45.14±1.03mmol/L), and the yield of hydrogen production reached 4.89mmol H2/g SCBadded. Additionally, CaCO3 promoted the biodegradation of SCB and the growth of C. thermocellum. The stimulatory effects of CaCO3 on biohydrogen production are mainly attributed to the buffering capacity of carbonate. The study provides a novel strategy to enhance biohydrogen production from lignocellulose.

[Effect of culture supernatant of Toxoplasma gondii on the proliferation and apoptosis of BGC-823 cells].

OBJECTIVE: To investigate the effect of culture supernatant of Toxoplasma gondii on the proliferation and Toxoplasma gondii tachyzoites with seed counts of 2 x 10(7)/ml, apoptosis of human gastric cancer BGC-823 cells. METHODS: 4 x 10(7)/ml, and 8 x 10(7)/ml harvested from infected mice were cultured for 24 h, and then the culture supernatant was collected. BGC-823 cells (5 x 10(4)/ml) at mid-exponential phase were incubated with different concentrations of culture supernatants of Toxoplasma gondii tachyzoites. While in control group, the same volume of DMEM was given. At 24, 48 and 72 h after incubation, the measurement of tumor cell growth inhibition rate was performed by using CCK-8 kit. Cell apoptosis was observed under a fluorescence microscope after 24 h incubation. The DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Flow cytometric analysis was used to analyze cell cycle for cell proliferation index and the expression of p53 and Bcl-2. RESULTS: The culture supernatant of Toxoplasma tachyzoites inhibited the proliferation of BGC-823 cells and the growth inhibition rates increased with the time and the concentration. The highest rate reached at the 72nd hour when the concentration of Toxoplasma tachyzoites was 8 x 10(7)/ml. Apoptotic bodies were found in experimental group. The amount of apoptotic body was positively associated with the tachyzoite concentration. DNA fragment in the treated cells after 24 h incubation was revealed by agarose electrophoresis. When the proportion of BGC-823 cells in G0/G1 phase increased, the proportion of cells in S phase decreased. Cell proliferation index decreased with the increase of the concentration of tachyzoites. The proliferation index (0.36) of the cells cultured with culture supernatant of 8 x 10(7)/ml tachyzoites was significantly lower than that of control group (0.6, P < 0.05). p53 protein expression was higher in experiment group than that of the control, whereas Bcl-2 protein expression in experiment group was lower than that of the control (P < 0.05). CONCLUSION: The culture supernatant of Toxoplasma gondii can inhibit the proliferation of BGC-823 cells and cause apoptosis of BGC-823 cells, which may be related with up-regulating p53 expression and down-regulating Bcl-2 expression.

[Rapid determination of illicit beta2-agonist additives in health foods and traditional Chinese patent medicines with DCBI-MS/MS method].

A novel rapid method for detection of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines was developed with the desorption corona beam ionization mass spectrometry (DCBI-MS) technique. The DCBI conditions including temperature and sample volume were optimized according to the resulting mass spectra intensity. Matrix effect on 9 beta2-agonists additives was not significant in the proposed rapid determination procedure. All of the 9 target molecules were detected within 1 min. Quantification was achieved based on the typical fragment ion in MS2 spectra of each analyte. The method showed good linear coefficients in the range of 1-100 mg x L(-1) for all analytes. The relative deviation values were between 14.29% and 25.13%. Ten claimed antitussive and antiasthmatic health foods and traditional Chinese patent medicines from local pharmacies were analyzed. All of them were negative with the proposed DCBI-MS method. Without tedious sample pretreatments, the developed DCBI-MS is simple, rapid and sensitive for rapid qualification and semi-quantification of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines.