Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Design,synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Nicotinamide adenine dinucleotide (NAD⁺) synthetase catalyzes the last step in NAD⁺ biosynthesis. Depletion of NAD⁺ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD⁺ synthetase (NadE) from Mtb, we expect to eliminate NAD⁺ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC₅₀ = 1000 µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC₅₀ value of 90 µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100 µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.     

艳山姜果实的生药学及气相色谱研究

艳山姜为姜科山姜属植物,艳山姜(Alpinia zerumbet)的干燥成熟果实,是贵州少数民族地区的传统习用药物,艳山姜在贵州省的种植面积已超过130 hm~2,是贵州"南药"的第一大宗产品,也是治理石漠化的重要经济植物,其自然资源非常丰富,亦是民间常用的香料植物资源。该研究采用性状、显微及理化鉴定方法,对艳山姜果实进行了系统的生药学研究,并对α-蒎烯、莰烯、β-蒎烯及1,8-桉叶油醇四个主要心血管药理活性成分进行含量分析。结果表明:艳山姜果实性状鉴别特征为果皮见12~20条纵棱隆起,顶端具花被残基突起,种子团由白色隔膜分为3瓣,每瓣具种子8~20粒不等,较易散落。显微鉴别特征为:种子横切面具1~2列油细胞,外胚乳细胞含淀粉粒,并可见细小草酸钙方晶;果实粉末可见螺纹导管、草酸钙方晶、淀粉粒、石细胞等。气相色谱法测得艳山姜果实挥发油中α-蒎烯、莰烯、β-蒎烯及1,8-桉叶油醇的平均含量分别为4.292%、3.966%、9.703%、27.171%。该研究的性状、显微鉴别方法准确、简单、易行,可作为艳山姜药材的鉴别依据;气相色谱含量测定方法重现性好,测定结果精确可靠,可用于艳山姜果实挥发油的含量测定。该研究结果为艳山姜药材的鉴定及进一步开发利用提供科学参考。     

Effects of early experience with low-quality roughage on liver metabolome in lambs

Introduction

Early experience with low-quality roughage has the potential to improve its utilization in ruminants.

Objectives

This study determined the effects of early experience with low-quality roughage on dry matter intake (DMI), body weight (BW) and the hepatic metabolism of lambs.

Methods

Ten lambs (3 month old) were randomly allocated to two treatments: the low-quality roughage group (LR) or the control group (C). The study lasted 7 months. In the first 4 months, LR was fed low-quality roughage, whereas C was fed normal roughage. In the last 3 months, both groups were fed low-quality roughage. Dry matter intake (DMI) was determined at the 1st, 3rd, 5th and 7th months (P1, P2, P3 and P4). Analysis of liver tissue metabolomics were carried out in P2 and P3.

Results

DMI was greater in LR than C in P1 (p?<?0.05), but did not differ in P2. In P3 and P4, DMI was greater in LR than in C (p?<?0.01). The concentration of five glycolysis/gluconeogenesis intermediates, some lipid metabolism-related metabolites, six amino acids and several amino acid metabolism-related metabolites were different between treatments in P2. From P2 to P3, concentrations of these metabolites changed in LR. However, no difference was found between treatments in P3.

Conclusion

Feeding low-quality roughage to lambs early in life influenced hepatic glycolysis/gluconeogenesis, fatty acid oxidation and amino acid metabolism. However, for lambs who had no early experience with low-quality roughage, similar alterations were induced in the liver after 1 month of eating low-quality roughage.

     

Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

兴安落叶松种群的稳定性与火干扰关系的研究

从种群生态学和干扰生态学的角度出发,采用火史重建的方法,研究了大兴安岭北部兴安落叶松种群的稳定性与火干扰的关系。结果表明：研究区内历史上火干扰频繁,平均间隔期为２８．９年,火烧强度较低。兴安落叶松依靠较强的耐火力、火力恢复力及自我恢复力具一定的稳定性。种群耐火力与长期火状况密切相关,表现为：低频类〈中频类〈高频类,高强类〈中强类〈低强类。火后恢复力与自我恢复力和最近一次火烧强度及距今时间密切相关：     

选择性支气管动脉插管转铁蛋白受体单克隆抗体灌注治疗中晚期肺癌(附68例临床报告)

本文对68例中晚期肺癌进行选择性支气管动脉插管采用转铁蛋白受体单克隆抗体与表阿霉素、顺伯等药物制成偶联物灌注。结果显示,肿瘤明显消退24例(35.2％),部分消退36例(52.5％),无变化8例(12.3％),总有效率为87.7％。通过对支气管动脉插管,导向灌注疗法结果显示,本疗法优于周围静脉供药,且无严重并发症,副作用轻,不失为一种可供选择的治疗方法。     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.