[Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus].

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

5-Hydroxytryptophan inhibits ß-casein biosynthesis and promotes goat mammary epithelial cell apoptosis through the JAK2/STAT5a axis and the HTR7.

5-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of 5-hydroxytryptophan (5-HTP) into l-tryptophan, which is then converted into 5-HT by monoamine-oxidase (MAO-A). In the mammary gland, 5-HT has been shown to have a variety of paracrine-autocrine actions, including suppressing lactation, controlling the destiny of mammary epithelial cells, and maintaining calcium homeostasis throughout the transition from pregnancy to lactation. To examine the effects of 5-HT on the composition of colostrum and milk, a total of 30 transition Guan Zhong dairy goats were intramuscularly injected with 5-HTP (1.0 mg/kg) every morning before feeding from 10 d before the projected parturition date to the day of parturition. The average number of days animals received injections was 8.2 ± 3.2 d. 5-HTP treatment increased serum 5-HT concentration from days 5 to 2 relative to parturition (P < 0.05), and decreased the casein concentration of colostrum (P < 0.05). In the in vitro experiment, mammary epithelial cells isolated from three individual goats' mammary glands were separately treated with 200 µM 5-HTP, 30 µM PCPA (the specific inhibitor of TPH1), or 200 µM 5-HTP + 50 µM SB269970 (the selective antagonist of 5-HTR7). The results showed that 200 µM 5-HTP inhibited the expression of ß-casein, downregulated the activity of the JAK2/ STAT5a signaling pathway, and promoted the apoptosis of goat mammary epithelial cells (GMECs) (P < 0.05). When GMECs were treated with 30 µM Four-chloro-dl-phenylalanine (PCPA), a specific inhibitor of 5-HT synthesis, the mRNA expression of STAT5a and the phosphorylated STAT5a protein level were upregulated. The 50 µM SB269970 treatment rescued the effects of 5-HTP on GMECs (P < 0.05). Taken together, the results indicated that 5-HTP exerted an inhibitory effect on ß-casein synthesis and a proapoptotic effect in GMECs via HTR7 and the JAK2/STAT5a axis.

5-Hydroxytryptamine (5-HT), which is produced in both the mammary gland and the central nervous system, is a recognized important regulator of mammary gland homeostasis. Casein is the major protein in the milk of mammals including cows, goats, and humans, and is a crucial source of high-quality amino acids for humans. In this study, prenatal intramuscular injection of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, not only increased the level of 5-HT in the serum of goats before delivery but also decreased the concentration of casein in colostrum. Furthermore, in goat mammary epithelial cells which are responsible for milk synthesis, it was found that 5-HTP blocked genes and signal pathways related to casein synthesis, and also promoted cell apoptosis. Additional results demonstrated that the type 7 5-HT receptor (HTR7) mediated the impacts of 5-HT, which provided a potential reliable target for improving milk quality.

[Identification and quality evaluation of germplasm resources of commercial Scutellaria baicalensis based on DNA barcode and HPLC].

Scutellaria baicalensis is a commonly used Chinese medicinal herb. In this study, we identified the germplasm resources of commercial S. baicalensis samples based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences according to the available chloroplast genome sequencing results, and measured the content of baicalin by HPLC. Through the above means we determined the best DNA barcode that can be used to detect the germplasm resources and evaluate the quality of commercial S. baicalensis samples. A total of 104 samples were collected from 24 provinces, from which DNA was extracted for PCR amplification. The amplification efficiencies of trnH-psbA, petA-psbJ, and ycf4-cemA sequences were 100%, 59.62%, and 25.96%, respectively. The results of sequence analysis showed that 5, 4, and 2 haplotypes were identified based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences, respectively. However, the sequences of haplotypes in commercial samples were different from that of the wild type, and the joint analysis of three fragments of S. baicalensis only identified 6 haplotypes. Furthermore, the phylogenetic analysis and genetic distance analysis indicated that trnH-psbA could be used to identify S. baicalensis from adulterants. The above analysis showed that trnH-psbA was the best fragment for identifying the germplasm resources of commercial S. baicalensis samples. We then analyzed the haplotypes(THap1-THap5) of commercial S. baicalensis samples based on trnH-psbA and found that THap2 was the main circulating haplotype of the commercial samples, accounting for 86.55% of the total samples, which indicated the scarce germplasm resources of commercial S. baicalensis samples. The content of baicalin in all the collected commercial S. baicalensis samples exceeded the standard in Chinese Pharmacopoeia and had significant differences(maximum of 12.21%) among samples, suggesting that the quality of commercial S. baicalensis samples varied considerably. However, there was no significant difference in baicalin content between different provinces or between different haplotypes. This study facilitates the establishment of the standard identification system for S. baicalensis, and can guide the commercial circulation and reasonable medication of S. baicalensis.

Transcriptome analysis of genes associated with autolysis of Coprinus comatus.

Coprinus comatus, widely known as "Jituigu", is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of the fruiting bodies after harvest. To gain insight into the molecular mechanism underlying this autolysis, we divided the growth of C. comatus fruiting bodies into four stages: infant stage (I), mature stage (M), discolored stage (D), and autolysis stage (A). We then subjected these stages to de novo transcriptomic analysis using high-throughput Illumina sequencing. A total of 12,946 unigenes were annotated and analyzed with the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). We analyzed the differentially expressed genes (DEGs) between stages I and M, M and D, and D and A. Because the changes from M to D are thought to be related to autolysis, we focused on the DEGs between these two stages. We found that the pathways related to metabolic activity began to vary in the transition from M to D, including pathways named as autophagy-yeast, peroxisome, and starch and sucrose metabolism. This study also speculates the possible process of the autolysis of Coprinus comatus. In addition, 20 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study, which is the first to describe the transcriptome of C. comatus, provides a foundation for future studies concerning the molecular basis of the autolysis of its fruiting bodies.

Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway.

Background: Follistatin-like 1 (FSTL1) is a novel profibrogenic factor that induces pulmonary fibrosis (PF) through the transforming growth factor-beta 1 (TGF-ß1)/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase (MAPK) pathway. Therefore, this study aimed to investigate the role of FSTL1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods: PF was induced in Fstl1+/-and wild-type (WT) C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTL1 and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fstl1 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fstl1+/- and WT fibroblasts treated with recombinant human FSTL1 protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student's t-test was used to compare the differences between two groups. Results: Fstl1 deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury (0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07 ± 0.07, t = 8.92, P = 0.0009; respectively), compared with WT lungs at the same time and in primary lung fibroblasts (0.82 ± 0.01 vs. 1.01 ± 0.04, t = 4.06, P = 0.0150; 1.04 ± 0.03 vs. 1.24 ± 0.03, t = 4.44, P = 0.0100; and 0.76 ± 0.05 vs. 0.99 ± 0.05, t = 4.48, P = 0.0100; respectively), compared with TGF-ß1-stimulated WT group. Recombinant human FSTL1 protein in lung fibroblasts enhanced TGF-ß1-mediated phosphorylation of the ERK (1.19 ± 0.08 vs. 0.55 ± 0.04, t = 6.99, P = 0.0020), p38 (1.18 ± 0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020), and JNK (1.11 ± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030), compared with the TGF-ß1-stimulated WT group. Fstl1-deficient fibroblasts showed reduced alpha-smooth muscle actin (α-SMA) expression (0.70 ± 0.06 vs. 1.28 ± 0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76 ± 0.02, t = 6.31, P = 0.0007; compared with the TGF-ß1-treated WT group). Compared with the corresponding condition in the control group, the TGF-ß1/FSTL1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 (0.73 ± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078) and JNK (0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40, P = 0.0046) signaling. The proliferation of mouse lung fibroblast cells (MLgs) significantly decreased after treatment of an inhibitor of p38 (0.30 ± 0.01 vs. 0.46 ± 0.03, t = 4.64, P = 0.0009), JNK (0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001), and Smad2/3 (0.18 ± 0.02 vs. 0.46 ± 0.02, t = 12.69, P = 0.0001) signaling compared with the dimethylsulfoxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 (70.17 ± 3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 ± 0.95, t = 10.14, P = 0.0005 for the invasive cells), JNK (72.30 ± 3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells), and Smad2/3 (64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 13.29, P = 0.0002 for the invasive cells) signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion: FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.

Polymorphisms of the stem cell marker gene CD133 are associated the clinical outcome in a cohort of Chinese non-small cell lung cancer patients.

OBJECTIVES: To evaluate the prognostic relevance of four functional single nucleotide polymorphisms (SNPs) in CD133 (rs2240688A>C, rs10022537T>A, rs7686732C>G, and rs3130C>T) on overall survival (OS) of non-small cell lung cancer (NSCLC) patients. DESIGN: Retrospective cohort study. SETTING: Department of General Surgery, in a general hospital, Henan Province, China. PARTICIPANTS: NSCLC patients aged ≥18 years, who were not receiving preoperative neoadjuvant therapies and had a blood sample available for genotyping, were eligible for inclusion. Those participants who were pregnant or breastfeeding, had a previous history of cancer, had other primary tumours, or who had had primary tumours of the skin and nasopharynx, were excluded from the study. OUTCOME MEASURES: The primary endpoint was OS, which was calculated from the date of enrolment until the date of death or date of last follow-up. RESULTS: There was a total of 1383 participants, with a median age of 63 years; 726 (52.5%) were male. Compared with thers2240688 AA genotype, the variant AC/CC genotypes were independently associated with OS (HR 1.27, 95% CI 1.12 to 1.45 for AC genotype; HR 2.32, 95% CI 1.91 to 2.80 for CC genotype). Higher hazard ratios for associations between CD133 rs2240688 polymorphism and OS were observed in patients with adjuvant chemotherapy (HR 1.86, 95% CI 1.52 to 2.26) and radiotherapy for curative intent (HR 1.90, 95% CI 1.55 to 2.33). CONCLUSIONS: The study confirmed the significant association between the SNP rs2240688 A>C of CD133 and OS of NSCLC patients. Larger population-based studies in different ethnic groups are necessary to further validate the role and mechanisms of CD133 in NSCLC.

Polymorphisms of the Stem Cell Marker Gene CD133 and the Risk of Lung Cancer in Chinese Population.

OBJECTIVE: To explore the association of functional single-nucleotide polymorphisms (SNPs) of CD133 with the risk of lung cancer. METHODS: We conducted a hospital-based, case-control study of 1017 lung cancer patients and 1035 cancer-free controls frequency-matched by age and sex. Four functional CD133 SNPs (rs2240688 A > C, rs10022537 T > A, rs7686732 C > G, and rs3130 C > T) were selected and genotyped. Unconditional univariate and multivariate logistic regression analyses were carried out to evaluate the associations of genotypes of CD133 SNPs with lung cancer risk. RESULTS: Compared with rs2240688 AA genotype, the variant AC/CC genotypes were associated with a statistically increased risk of lung cancer under a recessive model (adjusted odds ratio 1.19; 95 % confidence interval 1.01-1.42). The risk remained in patients with other histology types, but not with adenocarcinoma and squamous cell cancers. CONCLUSIONS: These findings suggest that SNP rs2240688 A > C of CD133 may be a potential biomarker for genetic susceptibility to lung cancer, but require further research with larger populations.

The distinguishing cellular features of pulmonary artery smooth muscle cells from chronic thromboembolic pulmonary hypertension patients.

In chronic thromboembolic pulmonary hypertension (CTEPH), central thrombi are the most likely disease initiators, and progressive pulmonary vascular remodeling, which is characterized by marked proliferation of pulmonary artery smooth muscle cells (PASMCs), may also contribute to the long-term progression of CTEPH. This study was designed to investigate the cellular characteristics of PASMCs isolated from the organized thrombotic tissues of CTEPH. In the present study, analysis of PASMCs isolated from five CTEPH patients and three control subjects showed that cells from CTEPH patients had certain characteristics that distinguished them from control cells, including inferior or no cell-cell contact inhibition growth, increased sensitivity to hypoxia-induced proliferation, resistance to serum starvation-induced apoptosis, and mitochondrial metabolism disorder. These differences in the PASMCs in endarterectomized tissue of CTEPH patients may prove useful in understanding the pathobiology of CTEPH.

[Investigation of ion channel mechanism for the regulatory volume decrease in lung adenocarcinoma cells].

OBJECTIVE: This study was designed to observe the regulatory volume decrease (RVD) process in human lung adenocarcinoma cells (A549) and to investigate its ion channel mechanism. METHODS: Electric measurement system of cell volume was used to detect the cell volume changes following exposure to hypotonic solution. Whole-cell patch clamp recordings were applied to investigate the characteristics of the volume-sensitive Cl(-) channel in A549 cells. RESULTS: Extracellular hypotonicity induced cell swelling followed by a typical RVD process, which can be inhibited by Cl(-) channel blocker (NPPB 100 micromol/L) and K(+) channel blocker (CsCl 5 mmol/L). Meanwhile, a outward-rectifying chloride currents which was sensitive to NPPB and DIDs was recorded in A549 using the whole cell patch clamp. CONCLUSIONS: The human lung adenocarcinoma cells has RVD process which is dependent on the parallel activation of Cl(-) channel and K(+) channel. The volume-sensitive Cl(-) channel is involved in volume regulation of lung adenocarcinoma cells.

[Arrhythmia triggered by stretching rabbit left ventricles and the block effect of streptomysin].

AIM: To observe the effect of stretching left ventricles in the end of action potential on rabbit cardiac activity, and to investigate its possible mechanisms. METHODS: Stretch (120 mmHg, 50 ms) was applied in the end of action potential by the pressure-clamp technique to observe if there would be any changes in rabbit cardiac activity and streptomycin (500 micromol/L) was used to identify the mechanism. RESULTS: Stretch in the end of action potential caused arrhythmia (P < 0.05) and streptomycin blocked the above effect (P < 0.05). CONCLUSION: Streptomycin could block the effect of stretching left ventricles in the end of action potential on rabbit cardiac activity, which indicates that stretch-activated ion channels involve it.

[Discrimination among different brands of coffee by using vis-near infrared spectra].

Near infrared spectroscopy technology was used to distinguish three different brands of coffee bought from the supermarket. Two models, PCA-BP and WT-BP, were employed for the analysis and prediction of the samples. The discrimination among the different brands of coffee was based on the combination of the function of data compression in the PCA and WT technology and the ability of learning and prediction of the artificial neural network. In the experiment, 60 samples were used for model calibration and 30 for brand prediction. The result showed that both the PCA-BP and WT-BP models achieved 100% discrimination rate, and the wavelet transforms technology is superior to the principal component analysis both in time-consuming and the capability of data compression. It is indicated that the model set up by the combination of WT technology and BP neural network in the present study is rapid in analysis and precise in pattern discrimination. It can be concluded that a new approach to distinguishing pure coffee was of fered and the result of this experiment established the foundation for the determination of the raw material (coffee bean) of different brands of coffee in the market.

A ventricular pressure-clamping system for the study of mechano-electrical feedback.

It is necessary to control the mechanical stimuli precisely in the studies of cardiac mechano-electrical feedback (MEF). In the present study a ventricular pressure-clamping system has been developed, which can be applied to isolated-perfused rabbit hearts. Controlled by a computer, this system not only can make the left ventricle follow a command defining the same pressure wave as that during a beating cycle under physiological condition, but also deliver mechanical stimuli with a proper waveform to the ventricle at a particular time phase. This system integrates multiple functions, including perfusing, pacing, recording of electrocardiogram and monophasic action potentials, and clamping and measuring of ventricular pressures in isolated-perfused hearts. Thus, it is a distinct system for investigating the phenomena and mechanisms of cardiac MEF at organ level.

[Studies on fingerprints of Bupleurum chinense from Hebei Province with HPLC-UV].

OBJECTIVE: To establish a HPLC fingerprint analysis method for identification of Bupleurum chinense of Hebei Province and compare the fingerprints of Radix Bupleuri collected from different habitats so as to establish a sensitive and specific method for controlling the quality of Radix Bupleuri. METHODS: The HPLC-UV fingerprints of Radix Bupleuri from different habitats were obtained from Waters 1525 instruments. The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and water at a flow rate of 1.0 ml/min with UV detector at 203 nm. The temperature of column was 30 degrees C. RESULTS: The mutual mode of HPLC-UV fingerprints was set up, and the similar degrees to the crude drugs of different habitats were compared. CONCLUSION: It is simple and quick to differ Bupleurum chinense from different habitats with the method that can be used as a quality control item for Radix Bupleuri.