Metabolomics Study on the Toxicity of Aconite Root and Its Processed Products Using Ultraperformance Liquid-Chromatography/Electrospray-Ionization Synapt High-Definition Mass Spectrometry Coupled with Pattern Recognition Approach and Ingenuity Pathways Analysis

The mother and lateral root of <i>Aconitum carmichaelii Debx</i>, named “Chuanwu” (CW) and “Fuzi”, respectively, has been used to relieve joint pain and treat rheumatic diseases for over 2000 years. However, it has a very narrow therapeutic range, and the toxicological risk of its usage remains very high. The traditional Chinese processing approach, <i>Paozhi</i> (detoxifying measure),can decompose poisonous Aconitum alkaloids into less or nontoxic derivatives and plays an important role in detoxification. The difference in metabolomic characters among the crude and processed preparations is still unclear, limited by the lack of sensitive and reliable biomarkers. Therefore, this paper was designed to investigate comprehensive metabolomic characters of the crude and its processed products by UPLC-Q-TOF-HDMS combined with pattern recognition methods and ingenuity pathway analysis (IPA). The significant difference in metabolic profiles and changes of metabolite biomarkers of interest between the crude and processed preparations were well observed. The underlying regulations of <i>Paozhi</i>-perturbed metabolic pathways are discussed according to the identified metabolites, and four metabolic pathways are identified using IPA. The present study demonstrates that metabolomic analysis could greatly facilitate and provide useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Aconite roots in the clinic

Proteomics and phosphoproteomics analysis of tissues for the reoccurrence prediction of colorectal cancer

Many stage II/III colorectal cancer (CRC) patients may relapse after routine treatments. Aberrant phosphorylation can regulate pathophysiological processes of tumors, and finding characteristic protein phosphorylation is an efficient approach for the prediction of CRC relapse. We compared the tissue proteome and phosphoproteome of stage II/III CRC patients between the relapsed group (n = 5) and the non-relapsed group (n = 5). Phosphopeptides were enriched with Ti4+-IMAC material. We utilized label-free quantification-based proteomics to screen differentially expressed proteins and phosphopeptides between the two groups. Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were used for bioinformatics analysis. The immune response of the relapsed group (Z-score −2.229) was relatively poorer than that of the non-relapsed group (Z-score 1.982), while viability of tumor was more activated (Z-score 2.895) in the relapsed group, which might cause increased relapse risk. The phosphorylation degrees of three phosphosites (phosphosite 1362 of TP53BP1, phosphosite 809 of VCL and phosphosite 438 of STK10) might be reliable prognostic biomarkers. Some promising proteins and phosphopeptides were discovered to predict the relapse risk in postoperative follow-ups.</p

Purification of cellular interaction partner of influenza strains A/Beijing/501/2009(H1N1) NS1.

<p>A, The purified protein complexes were detected by SDS-PAGE. Left panel: the protein complexes purified from A549 cells transfected with pnTAP-NS1 plasmids. Right panel: the protein complexes purified from A549 cells transfected with pnTAP vector. M represents protein marker. The bait protein bands and its cellular interaction proteins identified by mass spectrometry are indicated by an arrow and an asterisk, respectively. B, The purified protein complexes were detected by immunoblotting with antibodies against CBP. Left panel: the protein complexes purified from A549 cells transfected with pnTAP-NS1 plasmids. Right panel: the protein complexes purified from A549 cells transfected with pnTAP vector.</p

Co-localization of NS1 and β-tubulin in the nucleus, and Influenza virus A/Beijing/501/2009(H1N1) NS1 induce apoptosis.

<p>(A), (B), (C), (E), (F), (G). A549 cells were transfected with pCMV5-HA-NS1 and control vector pCMV5, respectively. NS1 was apparent from 24 h post-transfection, mainly in nucleus of A549 cells transfected with pCMV5-HA-NS1 (green color) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048340#pone-0048340-g003" target="_blank">Figure 3E</a>). On the other hand, β-tubulin was stained in nucleus and cytoplasm (red color) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048340#pone-0048340-g003" target="_blank">Figure 3F</a>).The signals of NS1 and β-tubulin clearly overlapped in nucleus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048340#pone-0048340-g003" target="_blank">Figure 3G</a>). (D), (H). The A549 cells transfected with pCMV5-HA-NS1 were stained with Hoechst 33342, and exhibited a stronger blue fluorescence and condensated and fragmented nuclear at 24 h post transfection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048340#pone-0048340-g003" target="_blank">Figure 3H</a>).</p

Identification of β-tubulin as a novel NS1-binding protein.

<p>(A) Peptide mass fingerprinting of the 55 kDa protein. The protein was identified as β-tubulin using a program, MASCOT, and the peptides assigned to those of β-tubulin are shown. (B) Confirmation of the 55 kDa protein band in TAP purified protein complexes as β-tubulin. The protein complexes purified from A549 cells transfected with pnTAP-NS1 plasmids and that from pnTAP transfected cells were immunoblotted with the anti-β-tubulin antibody (top panel) or anti-CBP antibody (middle panel). The total cell lysate was also immunoblotted with the anti-CBP antibody (bottom panel). (C) Co-immunoprecipitation analysis of β-tubulin and NS1. The precipitates obtained were immunoblotted with the anti-β-tubulin antibody (top panel) or anti-CBP antibody (middle panel). The total cell lysate was immunoblotted with the anti-CBP antibody (bottom panel). (D) An illustration of the various NS1 truncations used to map the β-tubulin-binding domain in the NS1 protein. NS1 Full (NS1 full-length); NS1 N (NS1 N- terminal domain, that is the RNA binding domain); and NS1 C (NS C-terminal domain, that is the effector domain). (E–F) The N-terminal domain of NS1 interacts with β-tubulin. Left panel: the purified GST-fused NS1 proteins stained by coomassie brilliant blue. Right panel: the purified GST-fused NS1 proteins complexes obtained were detected by immunoblotting with anti-GST antibodies (top panel) or anti-β-tubulin antibodies (bottom panel).</p

Carboxybetaine Methacrylate-Modified Nylon Surface for Circulating Tumor Cell Capture

Conventional <i>in vitro</i> circulating tumor cell (CTC) detection methods are always limited by blood sample volume because of the requirement of a large amount of blood. The aim of this study was to overcome the limitation by designing and making an <i>in vivo</i> CTC capture device. In this study, we designed and prepared a kind of proper material to serve the purpose of intervention. A method employing 3-aminopropyltriethoxysilane (γ-APS) as the coupling reagent to graft carboxybetaine methacrylate (CBMA) and to immobilize an anti-epithelial cell adhesion molecular (EpCAM) antibody on Nylon was developed. The results of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy proved the successful graft of γ-APS and CBMA to Nylon. Furthermore, the predicted improvement in the biocompatibilities of our modified Nylon was confirmed by water contact angle measurement, bovine serum albumin adhesion, platelet adhesion, plasma recalcification time determination, and cytotoxicity tests. The tumor cells adhesion experiment revealed that Nylon with the antibody immobilized on it had an affinity for EpCAM positive tumor cells higher than that of pristine Nylon. Additionally, the capture ability of the CTCs was demonstrated in a nude mouse tumor model using the interventional device made of the modified Nylon wire. The positive results suggest that CBMA-grafted and anti-EpCAM antibody-immobilized Nylon is a promising new material for <i>in vivo</i> CTC capture devices

Facile Preparation of Paclitaxel Loaded Silk Fibroin Nanoparticles for Enhanced Antitumor Efficacy by Locoregional Drug Delivery

Non-toxic, safe materials and preparation methods are among the most important factors when designing nanoparticles (NPs) for future clinical application. Here we report a novel and facile method encapsulating anticancer drug paclitaxel (PTX) into silk fibroin (SF), a biocompatible and biodegradable natural polymer, without adding any toxic organic solvents, surfactants or other toxic agents. The paclitaxel loaded silk fibroin nanoparticles (PTX-SF-NPs) with a diameter of 130 nm were formed in an aqueous solution at room temperature by self-assembling of SF protein, which demonstrated mainly silk I conformation in the NPs. In cellular uptake experiments, coumarin-6 loaded SF NPs were taken up efficiently by two human gastric cancer cell lines BGC-823 and SGC-7901. In vitro cytotoxicity studies demonstrated that PTX kept its pharmacological activity when incorporating into PTX-SF-NPs, while SF showed no cytotoxicity to cells. The in vivo antitumor effects of PTX-SF-NPs were evaluated on gastric cancer nude mice exnograft model. We found that locoregional delivery of PTX-SF-NPs demonstrated superior antitumor efficacy by delaying tumor growth and reducing tumor weights compared with systemic administration. Furthermore, the organs of mice in NP treated groups didn’t show obvious toxicity, indicating the in vivo safety of SF NPs. These results suggest that SF NPs are promising drug delivery carriers, and locoregional delivery of SF NPs could be a potential future clinical cancer treatment regimen