A Systems Biology-Based Approach to Uncovering the Molecular Mechanisms Underlying the Effects of Dragon's Blood Tablet in Colitis,Involving the Integration of Chemical Analysis,ADME Prediction,and Network Pharmacology

Traditional Chinese medicine (TCM) is one of the oldest East Asian medical systems. The present study adopted a systems biology-based approach to provide new insights relating to the active constituents and molecular mechanisms underlying the effects of dragon''s blood (DB) tablets for the treatment of colitis. This study integrated chemical analysis, prediction of absorption, distribution, metabolism, and excretion (ADME), and network pharmacology. Firstly, a rapid, reliable, and accurate ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was employed to identify 48 components of DB tablets. In silico prediction of the passive absorption of these compounds, based on Caco-2 cell permeability, and their P450 metabolism enabled the identification of 22 potentially absorbed components and 8 metabolites. Finally, networks were constructed to analyze interactions between these DB components/metabolites absorbed and their putative targets, and between the putative DB targets and known therapeutic targets for colitis. This study provided a great opportunity to deepen the understanding of the complex pharmacological mechanisms underlying the effects of DB in colitis treatment.     

Aging Increases the Susceptivity of MSCs to Reactive Oxygen Species and Impairs Their Therapeutic Potency for Myocardial Infarction

Myocardial infarction (MI) is one of the leading causes of death worldwide and Mesenchymal Stem Cells (MSCs) transplantation has been considered a promising therapy. Recently, it was reported that the therapeutic effectiveness of MSCs is dependent on the age of the donor, yet the underlying mechanism has not been thoroughly investigated. This study was designed to investigate whether this impaired therapeutic potency is caused by an increased susceptivity of MSCs from old donors to reactive oxygen species (ROS). The MSCs were isolated from the subcutaneous inguinal region of young (8–10 weeks) and old (18 months) Sprague–Dawley (SD) rats. By exposing these MSCs to H₂O₂, we found that the adhesion of MSCs from old donors was damaged more severely. Specifically, decreased expression of integrin and reduced phosphorylation of focal adhesion kinase Src and FAK were observed. Furthemore, H₂O₂ triggered an increased apoptosis of MSCs from old donors. To study the viability and therapeutic potency of MSCs from young and old donors in vivo, these MSCs were transplanted into acute MI model rats. We observed a more rapidly decreased survival rate of the old MSCs in the infarct region, which may be caused by their increased susceptivity to the micro-environmental ROS, as transplantation of the old MSCs with N-acetyl-L-cysteine (NAC), a ROS scavenger, protected them. The low viability of engrafted old MSCs consequently impaired their therapeutic effectiveness, judging by the histology and function of heart. Our study may help to understand the mechanism of MSCs-host interaction during MI, as well as shed light on the design of therapeutic strategy in clinic.     

Genetic Polymorphism of Cytochrome P450 4F2, Vitamin E Level and Histological Response in Adults and Children with Nonalcoholic Fatty Liver Disease Who Participated in PIVENS and TONIC Clinical Trials

Vitamin E improved liver histology in children and adults with NAFLD who participated in TONIC and PIVENS clinical trials, but with significant inter-individual variability in its efficacy. Cytochrome P450 4F2 (CYP4F2) is the major enzyme metabolizing Vit E, with two common genetic variants (V433M, rs2108622 and W12G, rs3093105) found to alter its activity. We investigated the relationship between CYP4F2 genotypes, α-tocopherol levels and histological improvement in these two trials. V433M and W12G variants were genotyped in TONIC (n = 155) and PIVENS (n = 213) DNA samples. The relationships between CYP4F2 genotypes, plasma α-tocopherol levels at baseline and weeks 48 (w48) and 96 (w96) and histological end points (overall improvement in liver histology and resolution of NASH) were investigated. As a result, the V433M genotype was significantly associated with baseline plasma α-tocopherol in the TONIC trial (p = 0.004), but not in PIVENS. Among those receiving Vit E treatment, CYP4F2 V433M genotype was associated with significantly decreased plasma α-tocopherol levels at w48 (p = 0.003 for PIVENS and p = 0.026 for TONIC) but not at w96. The w96 α-tocopherol level was significantly associated with resolution of NASH (p = 0.006) and overall histology improvement (p = 0.021)in the PIVENS, but not in the TONIC trial. There was no significant association between CYP4F2 genotypes and histological end points in either trial. Our study suggested the a moderate role of CYP4F2 polymorphisms in affecting the pharmacokinetics of Vit E as a therapeutic agent. In addition, there may be age-dependent relationship between CYP4F2 genetic variability and Vit E pharmacokinetics in NAFLD.     

Curvilinear Effects of Invasive Plants on Plant Diversity: Plant Community Invaded by Sphagneticola trilobata

The effects of invasive plants on the species diversity of plant communities are controversial, showing either a positive or negative linear relationship. Based on community data collected from forty 5 m×5 m plots invaded by Sphagneticola trilobata in eight cities across Hainan Island, China, we found S. trilobata decreased plant community diversity once its cover was beyond 10%. We demonstrated that the effects of invasive/native plants on the plant diversity of communities invaded by S. trilobata were curvilinear. These effects, which showed peaks under different degrees of vegetation cover, appeared not only for S. trilobata and all invasive plants, but also for all native plants. Invasive plants primarily had negative effects on plant diversity when they became abundant at a much lower cover level (less than 35%), compared with the native plants (over 60%). Thus, it is necessary to distinguish a range for assessing the effects of plants, especially invasive plants. Our results also confirmed that the invasion intensity of invasive alien plants increased with the intensity of local economic development. We highlight and further discuss the critical importance of curvilinear effects of biological invasion to provide ideas regarding the conservation of local biodiversity and the management of invasive plants.     

Mdb1, a Fission Yeast Homolog of Human MDC1, Modulates DNA Damage Response and Mitotic Spindle Function

During eukaryotic DNA damage response (DDR), one of the earliest events is the phosphorylation of the C-terminal SQ motif of histone H2AX (H2A in yeasts). In human cells, phosphorylated H2AX (γH2AX) is recognized by MDC1, which serves as a binding platform for the accumulation of a myriad of DDR factors on chromatin regions surrounding DNA lesions. Despite its important role in DDR, no homolog of MDC1 outside of metazoans has been described. Here, we report the characterization of Mdb1, a protein from the fission yeast Schizosaccharomyces pombe, which shares significant sequence homology with human MDC1 in their C-terminal tandem BRCT (tBRCT) domains. We show that in vitro, recombinant Mdb1 protein binds a phosphorylated H2A (γH2A) peptide, and the phospho-specific binding requires two conserved phospho-binding residues in the tBRCT domain of Mdb1. In vivo, Mdb1 forms nuclear foci at DNA double strand breaks (DSBs) induced by the HO endonuclease and ionizing radiation (IR). IR-induced Mdb1 focus formation depends on γH2A and the phospho-binding residues of Mdb1. Deleting the mdb1 gene does not overtly affect DNA damage sensitivity in a wild type background, but alters the DNA damage sensitivity of cells lacking another γH2A binder Crb2. Overexpression of Mdb1 causes severe DNA damage sensitivity in a manner that requires the interaction between Mdb1 and γH2A. During mitosis, Mdb1 localizes to spindles and concentrates at spindle midzones at late mitosis. The spindle midzone localization of Mdb1 requires its phospho-binding residues, but is independent of γH2A. Loss of Mdb1 or mutating its phospho-binding residues makes cells more resistant to the microtubule depolymerizing drug thiabendazole. We propose that Mdb1 performs dual roles in DDR and mitotic spindle regulation.     

Effects of Feedstock and Pyrolysis Temperature on Biochar Adsorption of Ammonium and Nitrate

Biochar produced by pyrolysis of biomass can be used to counter nitrogen (N) pollution. The present study investigated the effects of feedstock and temperature on characteristics of biochars and their adsorption ability for ammonium N (NH₄ ⁺-N) and nitrate N (NO₃ ⁻-N). Twelve biochars were produced from wheat-straw (W-BC), corn-straw (C-BC) and peanut-shell (P-BC) at pyrolysis temperatures of 400, 500, 600 and 700°C. Biochar physical and chemical properties were determined and the biochars were used for N sorption experiments. The results showed that biochar yield and contents of N, hydrogen and oxygen decreased as pyrolysis temperature increased from 400°C to 700°C, whereas contents of ash, pH and carbon increased with greater pyrolysis temperature. All biochars could sorb substantial amounts of NH₄ ⁺-N, and the sorption characteristics were well fitted to the Freundlich isotherm model. The ability of biochars to adsorb NH₄ ⁺-N followed: C-BC>P-BC>W-BC, and the adsorption amount decreased with higher pyrolysis temperature. The ability of C-BC to sorb NH₄ ⁺-N was the highest because it had the largest cation exchange capacity (CEC) among all biochars (e.g., C-BC400 with a CEC of 38.3 cmol kg⁻¹ adsorbed 2.3 mg NH₄ ⁺-N g⁻¹ in solutions with 50 mg NH₄ ⁺ L⁻¹). Compared with NH₄ ⁺-N, none of NO₃ ⁻-N was adsorbed to biochars at different NO₃ ⁻ concentrations. Instead, some NO₃ ⁻-N was even released from the biochar materials. We conclude that biochars can be used under conditions where NH₄ ⁺-N (or NH₃) pollution is a concern, but further research is needed in terms of applying biochars to reduce NO₃ ⁻-N pollution.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Functional Genetic Variants of TNFSF15 and Their Association with Gastric Adenocarcinoma: A Case-Control Study

The purpose of this study was to identify functional genetic variants in the promoter of tumor necrosis factor superfamily member 15 (TNFSF15) and evaluate their effects on the risk of developing gastric adenocarcinoma. Forty DNA samples from healthy volunteers were sequenced to identify single nucleotide polymorphisms (SNPs) in the TNFSF15 promoter. Two TNFSF15 SNPs (−358T>C and −638A>G) were identified by direct sequencing. Next, genotypes and haplotypes of 470 gastric adenocarcinoma patients and 470 cancer-free controls were analyzed. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Serologic tests for Helicobacter pylori infection were measured by enzyme-linked immuno-sorbent assay (ELISA). Subjects carrying the TNFSF15 −358CC genotype were at an elevated risk for developing gastric adenocarcinoma, compared with those with the −358TT genotype (OR 1.42, 95% CI, 1.10 to 2.03). H. pylori infection was a risk factor for developing gastric adenocarcinoma (OR 2.31, 95% CI, 1.76 to 3.04). In the H. pylori infected group, subjects with TNFSF15 −358CC genotype were at higher risks for gastric adenocarcinoma compared with those carrying −358TT genotype (OR: 2.01, 95%CI: 1.65 to 4.25), indicating that H. pylori infection further influenced gastric adenocarcinoma susceptibility. The −358 T>C polymorphism eliminates a nuclear factor Y (NF-Y) binding site and the −358C containing haplotypes showed significantly decreased luciferase expression compared with −358T containing haplotypes. Collectively these findings indicate that functional genetic variants in TNFSF15 may play a role in increasing susceptibility to gastric adenocarcinoma.     

Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy

Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1^st, 4^th and 6^th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.