The usefulness of measuring n-butyric acid concentration as a new indicator of blood decomposition in forensic autopsy

In forensic medicine, although various alcohols have been reported as indicators of decomposition in collected blood, no studies have examined short-chain fatty acids as indicators. In this study, the blood n-butyric acid concentration was quantified, and the association between n-butyric acid and decomposition was investigated to determine whether the detection of n-butyric acid could be a new indicator of decomposition. Among the forensic autopsies performed from 2016 to 2018 in our laboratory, the cases were divided into decomposed (n = 20) and non-decomposed (n = 20) groups based on macroscopic findings. Blood samples collected at the time of autopsy were derivatized with 3-nitrophenylhydrazine hydrochloride after solid-phase extraction. The n-butyric acid concentration was measured using liquid chromatography–tandem mass spectrometry. In addition, ethanol and n-propanol were measured using a gas chromatography-flame ionization detector. There was a significant difference (p < 0.01) in the concentrations of n-butyric acid between the decomposed and non-decomposed groups (0.343 ± 0.259 [0.030–0.973] and 0.003 ± 0.002 [0.001–0.007] mg/mL, respectively). In the decomposed group, n-butyric acid was detected at high concentrations, even in cases where n-propanol was low. These results suggest that n-butyric acid is more likely to be an indicator of blood decomposition than n-propanol.     

Pan-cancer proteomic map of 949 human cell lines

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环境水样中痕量铜的浊点萃取-火焰原子吸收光谱测定法

目的建立环境水样中痕量铜的浊点萃取(cloud point extraction,CPE)-火焰原子吸收光谱(flame atomic absorption spectrometry,FAAS)测定法。方法样品在p H 9.5的条件下,加入0.4 ml的1 mmol/L 2-(5-溴-2-吡啶偶氮)-5-二乙氨基酚(5-Br-PADAP)溶液,0.1%氯化钙溶液0.1 ml,5%(W/V)Triton X-114溶液0.8 ml,40℃加热15 min后离心,采用火焰原子吸收光谱法进行检测。结果在2~240μg/L的线性范围内,所得回归方程为A=0.002 7c+0.024 6,r=0.995 8。以3倍信噪比计算,方法的检出限为0.62μg/L,富集倍数为36.58倍,平均加标回收率为96.28%~98.08%,RSD为1.67%~3.13%。结论该方法简单、灵敏,具有良好的重现性,适用于环境水样中痕量铜的测定。     

Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Investigation of non-linear Mate1-mediated efflux of trimethoprim in the mouse kidney as the mechanism underlying drug-drug interactions between trimethoprim and organic cations in the kidney

The purpose of this study was to elucidate the involvement of Mate1 in the tubular secretion of trimethoprim and saturation of Mate1-mediated efflux to address the mechanisms underlying the pharmacokinetic drug interactions with trimethoprim. Trimethoprim is a more potent inhibitor of MATE2-K than MATE1 with K_i values (μM) of 0.030–0.28 and 2.4–5.9, respectively. Trimethoprim is a substrate of human MATE1 and MATE2-K with K_m values of 2.3 ± 0.9 and 0.018 ± 0.004 μM, and mouse Mate1, but not human OCT2, mouse Oct1 and Oct2. Pyrimethamine significantly reduced the renal clearance (CL_R) of trimethoprim (mL/min/kg) from 40.0 ± 5.1 to 20.1 ± 3.7 (p < 0.05). Trimethoprim was given to mice at three infusion rates (150, 500, and 1500 nmol/min/kg). Together with an increase in the plasma concentrations of trimethoprim, the CL_R (mL/min/kg) of trimethoprim decreased to 25.9 ± 3.2, 13.5 ± 5.7, and 8.92 ± 1.50 at the respective rates. Trimethoprim decreased the CL_R of rhodamine 123 in an infusion rate-dependent manner: 11.5 ± 1.3 (control), 5.17 ± 1.55, 1.31 ± 0.50, and 0.532 ± 0.180. These results suggest that Mate1 mediates the tubular secretion of trimethoprim, and at therapeutic doses, MATEs-mediated efflux can be saturated, and thereby, cause drug interactions with other MATE substrates.     

基于UPLC-LTQ-Orbitrap-MS分析刺果番荔枝叶的化学成分

目的利用UPLC-LTQ-Orbitrap-MS技术对刺果番荔枝叶的化学成分进行定性分析。方法采用Waters Acquity UPLC HSS T 3色谱柱(2.1 mm×100 mm,1.8μm),流动相为0.1%甲酸水(A)和乙腈(B)梯度洗脱,流速为0.3 mL/min,进样量2μL,在电喷雾正负离子模式下采集数据。经Reaxys数据库检索番荔枝属类化合物信息,通过质谱信息比对各化合物的m/z值、保留时间、质谱特征碎片等,并结合文献数据对鉴定的化合物进行验证。结果根据各化合物的特征裂解规律,从刺果番荔枝叶中共鉴定出45个化合物,包括16个生物碱类,14个番荔枝内酯类,7个黄酮类和8个其他类化合物,其中以番荔枝内酯类和生物碱类成分居多,与文献报道番荔枝内酯与生物碱类化合物是发挥抗癌的主要活性成分一致。结论利用UPLC-LTQ-Orbitrap-MS技术对刺果番荔枝叶中的化学成分进行了快速、准确的定性分析,为刺果番荔枝叶的提取分离与药效物质基础的研究提供依据。     

Erythrocyte sphingolipid species as biomarkers of Alzheimer's disease

Diagnosing Alzheimer's disease (AD) in the early stage is challenging. Informative biomarkers can be of great value for population-based screening. Metabolomics studies have been used to find potential biomarkers, but commonly used tissue sources can be difficult to obtain. The objective of this study was to determine the potential utility of erythrocyte metabolite profiles in screening for AD. Unlike some commonly-used sources such as cerebrospinal fluid and brain tissue, erythrocytes are plentiful and easily accessed. Moreover, erythrocytes are metabolically active, a feature that distinguishes this sample source from other bodily fluids like plasma and urine. In this preliminary pilot study, the erythrocyte metabolomes of 10 histopathologically confirmed AD patients and 10 patients without AD (control (CTRL)) were compared. Whole blood was collected post-mortem and erythrocytes were analyzed using ultra-performance liquid chromatography tandem mass spectrometry. Over 750 metabolites were identified in AD and CTRL erythrocytes. Seven were increased in AD while 24 were decreased (P<0.05). The majority of the metabolites increased in AD were associated with amino acid metabolism and all of the decreased metabolites were associated with lipid metabolism. Prominent among the potential biomarkers were 10 sphingolipid or sphingolipid-related species that were consistently decreased in AD patients. Sphingolipids have been previously implicated in AD and other neurological conditions. Furthermore, previous studies have shown that erythrocyte sphingolipid concentrations vary widely in normal, healthy adults. Together, these observations suggest that certain erythrocyte lipid phenotypes could be markers of risk for development of AD.     

Platinum levels in various tissues of a patient who died 181 days after cisplatin overdosing determined by electrospray ionization mass spectrometry

Platinum (Pt) levels were determined in various tissues and body fluids obtained from a patient who died 181 days after cisplatin overdosing. The symptoms of cisplatin overdose, however, might have almost disappeared by day 40, and the patient’s death was ascribed to the recurrence of malignant lymphoma. Determination of Pt derived from cisplatin was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as internal standard. Pt and Ag complexed with diethyldithiocarbamate (DDC) in wetashed blood, and tissue solutions were extracted into isoamyl alcohol, and then acidified with oxalic acid. By injecting an aliquot of the isoamyl alcohol layer into a mass spectrometer in the direct flow injection mode, the quantitation was performed using the signals of Pt(DDC)₃ ⁺ and Ag(DDC)₂ ⁺ at m/z 639 and 403, respectively. The Pt levels ranged from 25ng/ml in blood to 2050ng/g wet weight in the liver of the patient, indicating that Pt remained at high levels in tissues, even after a period as long as 181 days after cisplatin overdosing.     

双波长紫外分光光度法测定贝母中腺苷和胸苷的含量

将4种贝母的甲醇提取物经薄层色谱法粗分离后,直接用双波长紫外分光光度法测定其含量。结果表明,该方法线性关系好,腺苷和胸苷标准曲线的相关系数均为0.9999,同时也发现平贝、炉贝和伊贝中腺苷都占核苷总量60%以上,而浙贝中仅占约40%,提示贝母生药的抗凝血活性可能与贝母中核苷类化合物的种类和含量的差别有关。