Classification of Chinese herbs based on the cluster analysis of delayed luminescence

Traditional Chinese material medica are an important component of the Chinese pharmacopeia. According to the traditional Chinese medicinal concept, Chinese herbal medicines are classified into different categories based on their therapeutic effects, however, the bioactive principles cannot be solely explained by chemical analysis. The aim of this study is to classify different Chinese herbs based on their therapeutic effects by using delayed luminescence (DL). The DL of 56 Chinese herbs was measured using an ultra‐sensitive luminescence detection system. The different DL parameters were used to classify Chinese herbs according to a hierarchical cluster analysis. The samples were divided into two groups based on their DL kinetic parameters. Interestingly, the DL classification results were quite consistent with classification according to the Chinese medicinal concepts of ‘cold’ and ‘heat’ properties. In this paper, we show for the first time that by using DL technology, it is possible to classify Chinese herbs according to the Chinese medicinal concept and it may even be possible to predict their therapeutic properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification,Quantification, and System Analysis of Protein N‐ε Lysine Methylation in Anucleate Blood Platelets

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N‐ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl‐lysine (meK) proteome of anucleate blood platelets is characterized. With high‐resolution, multiplex MS methods, 190 mono‐, di‐, and tri‐meK modifications are identified on 150 different platelet proteins—including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin‐like protein (DBNL, Hip‐55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.     

Computer-aided lead optimization: improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i) (app) of 7+/-2.4 microM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i) (app) of 3.8+/-0.8 microM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

Recent advances in TMEM16A: Structure,function, and disease

TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca²⁺ and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.     

MicroRNA-323-3p promotes myogenesis by targeting Smad2

Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3′-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis.     

MiR-323a regulates ErbB3/EGFR and blocks gefitinib resistance acquisition in colorectal cancer

The rapid onset of resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) limits its clinical utility in colorectal cancer (CRC) patients, and pan-erb-b2 receptor tyrosine kinase (ErbB) treatment strategy may be the alternative solution. The aim of this study was to develop a possible microRNA multi-ErbB treatment strategy to overcome EGFR-TKI resistance. We detect the receptor tyrosine kinase activity in gefitinib-resistant colorectal cancer cells, ErbB3/EGFR is significantly activated and provides a potential multi-ErbB treatment target. MiR-323a-3p, a tumor suppressor, could target both ErbB3 and EGFR directly. Apoptosis is the miR-323a-3p inducing main biological process by functional enrichment analysis, and The EGFR and ErbB signaling are the miR-323a-3p inducing main pathway by KEGG analysis. MiR-323a-3p promotes CRC cells apoptosis by targeting ErbB3-phosphoinositide 3‐kinases (PI3K)/PKB protein kinase (Akt)/glycogen synthase kinase 3 beta (GSK3β)/EGFR-extracellular regulated MAP kinase (Erk1/2) signaling directly. And miR-323a-3p, as a multi-ErbBs inhibitor, increase gefitinib sensitivity of the primary cell culture from combination miR-323a-3p and gefitinib treated subcutaneous tumors. MiR-323a-3p reverses ErbB3/EGFR signaling activation in gefitinib-resistant CRC cell lines and blocks acquired gefitinib resistance.Subject terms: Colorectal cancer, Cancer therapeutic resistance     

Analysis of 5-methoxytryptamine at the femtomole level in the rat and quail brain by gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry

A sensitive method for the measurement of endogenous 5-methoxytryptamine in brain tissue has been developed using capillary column gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry. 5-Methoxytryptamine was first converted to N-[²H₃]acetyl-5-methoxytryptamine by reaction with hexa-deuterated acetic anhydride, followed by reaction with pentafluoropropionic anhydride to yield the highly electron-capturing 3,3′-spirocyclic pentafluoropropionyl indolenine derivative. Quantitative analysis was carried out by selected-ion monitoring of the [M-HF] and [M-HF-DF] ion intensity of the 3,3′-spirocyclic pentafluoropropionyl indolenine derivative, using 5-methoxy-[α,α,β,β-²H₄]tryptamine as the internal standard. The presence of 5-methoxytryptamine in the brain tissue was demonstrated. In the absence of a monoamine oxidase inhibitor, the mean±S.D. levels of 5-methoxytryptamine in the rat and quail whole brain were found to be 30±6 and 347±52 pg/g, respectively. The possible physiological functions of 5-methoxytryptamine as a neuromodulator and/or neurotransmitter have to be considered.     

Genetic association study of intron variants in the forkhead box protein P3 gene in Chinese patients diagnosed with cervical cancer

The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high‐risk human papilloma virus (HR‐HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR‐HPV infection and CC malignant lesions (TC vs. TT: OR = 0.71, 95% CI = 0.51–0.99); the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions (TC+CC vs. TT: OR = 0.69, 95% CI = 0.50–0.95; C vs. T: OR = 0.78, 95% CI = 0.63–0.97). The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR‐HPV infection and CC malignant lesions (GA vs. GG: OR = 0.70, 95% CI = 0.51–0.96; GA+AA vs. GG: OR = 0.69, 95% CI = 0.51–0.94; A vs. G: OR = 0.75, 95% CI = 0.58–0.96). Patients with CC and HR‐HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T‐C‐A decreased the risk of HR‐HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR‐HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC.