Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

厌氧消化过程抑制因素的研究进展

厌氧消化是一种高效的废物处理方法。但厌氧消化过程中经常出现诸如甲烷产量低、工序不稳定等问题,使得这项技术不能被广泛应用。废弃物中种类繁多的抑制物质是导致厌氧消化不稳定或失败的主要原因。概述了厌氧消化过程中经常出现的抑制因素的抑制机制和控制因子。     

In Vivo and In Vitro Comparison of the DPP-IV Inhibitory Potential of Food Proteins from Different Origins after Gastrointestinal Digestion

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC₅₀) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC₅₀ of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity.     

Structure-Activity Studies Reveal Scope for Optimisation of Ebselen-Type Inhibition of SARS-CoV-2 Main Protease

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of M^pro, particularly with respect to improved selectivity.     

蛋白酶产生菌LN02发酵玉米黄粉蛋白的条件优化

通过单因素实验和正交实验初步优化了蛋白酶产生菌LN02发酵玉米黄粉蛋白的条件。结果表明,菌株LN02发酵玉米黄粉蛋白的最佳条件为：初始pH值5.5、装液量60 mL/250 mL三角瓶、发酵温度35℃、发酵时间5 d。     

Activity‐Based Probes for Monitoring Postproline Protease Activity

Postproline proteases constitute a subset of serine proteases involved in the regulation of many signaling events and are emerging as promising therapeutic targets for prevalent diseases, such as diabetes and cancer. Therefore, monitoring their activity in different tissues and diverse physiological states would certainly facilitate the elucidation of their physiological role and the establishment of new therapeutic targets. Here, we have synthesized a dipeptidyl phosphonate activity‐based probe that has proved to be highly selective for a specific postproline protease, prolyl oligopeptidase (POP). Its high sensitivity allows the detection of the endogenous activity of POP both by in‐gel analysis and mass spectrometry. The evidence provided by mass spectrometry for the high selectivity of the synthesized probe opens the possibility of using dipeptidyl phosphonates not only for activity‐based profiling (ABP), but also for other ABP applications like substrate‐based protease identification.     

Production of Alkaline Protease by Bacillus thuringiensis H 14 in Aqueous Two-Phase Systems

Alkaline protease production by Bacillus thuringiensis H 14 was studied in aqueous two-phase systems composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The alkaline protease produced during fermentation, partitioned into the upper phase (about 80%) and total protease produced were about 2·80 and 2·26 times higher than that of homogeneous fermentation when the fermentations were carried out in aqueous two-phase systems from the beginning and made after 45 h of inoculation respectively. © 1997 SCI.     

Mialostatin,a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.     

D2蛋白酶间接ELISA检测方法的建立

目的建立D2蛋白酶间接ELISA检测方法,为D2蛋白酶的进一步研究及应用奠定基础。方法以纯化的D2蛋白酶为抗原,免疫新西兰白兔,制备D2蛋白酶多克隆抗体,建立D2蛋白酶间接ELISA检测方法,并进行验证及初步应用。结果D2蛋白酶间接ELISA检测方法的最佳反应条件为:一抗稀释度为1∶64000,二抗稀释度为1∶80000,抗原稀释度为1∶100,饱和包被浓度为1160ng/ml,封闭剂为10%胎牛血清。标准曲线的线性检测范围为18～1160ng/ml。该方法准确性、重复性良好,特异性强、敏感性高,与琼脂扩散试验检测结果的符合率为95.5%。结论已建立了D2蛋白酶间接ELISA检测方法,可用于D2蛋白酶含量的检测。     

废水中总磷测定消解方法的比较

对测定总磷含量不同消解方法从标准曲线绘制、样品测定等方面进行对比实验。结果表明,经实验证明不同消解方法精密度和准确度都符合要求。     

Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors

Since the outbreak of SARS-CoV-2 in December 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (M^Pro) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential M^Pro inhibitors we developed a luminescent assay using a peptide based probe containing a cleavage site specific for M^Pro. This assay was validated showing IC₅₀ values similar to those reported in the literature for known M^Pro inhibitors and can be used to screen new inhibitors.     

ICP-OES方法测定低合金钢中多元素两种消解方法比较

研究了用稀硝酸溶样,高氯酸发烟氧化[1]后,用盐酸硝酸酸化试液,在ICP光谱仪上进行元素测定,以及用稀硝酸溶样、加硫酸磷酸混合酸发烟后,加水溶解盐类测定试液,在ICP光谱仪上进行元素测定的两种方法。试验证明用硫磷混合酸发烟消解法测定低合金钢中多元素具有较高的灵敏度和精密度,方法的线性相关系数较好,相对标准偏差小于1.8%。与高氯酸法发烟消解法比较,硫酸磷酸混合酸发烟消解法具有操作简单,所需试剂较少,结果稳定等特点。     

石墨炉原子吸收分光光度法测定土壤中的镉-全消解法和微波消解法比对实验

用微波消解法与全消解法消解土壤标准样品,用石墨炉原子吸收光谱法测定消解液铅含量,比较两种消解方法的精密度和准确度。微波消解法的相对标准偏差为3.8%,全消解法为8.6%。与标准值的相对相差比较,微波消解法低于10%,电热板消解法高于19%。微波消解法的测出值均高于电热板消解法。     

低温厌氧消化废水处理的现状及展望

低温厌氧消化是一种低成本、低技术门槛的绿色生物废水处理方法,能够在经济和环境方面为社会带来巨大效益。本文介绍了低温厌氧消化技术的原理、优势和应用现状,并对附加后处理程序,结合深度测序或宏基因组学以及其他功能经济途径所获得的信息,发展更加稳定高效的低温厌氧消化反应器设备进行了展望。     

电子陶瓷材料元素分析的前处理方法研究

陶瓷材料,由于其耐高温、耐腐蚀、耐磨等性能优异,在各行各业应用相当广泛。但也正是因为它的这些特性,在使用化学方法进行元素分析时,会遇到样品难以消解的问题,导致测试无法进行。文章重点研究电子元件中常用的三氧化二铝、氧化锆类陶瓷材料的消解方法,并介绍使用ICP-OES对消解完全的样品溶液进行元素分析的注意事项。     

A Chemical Approach to Immunoprotein Engineering: Chemoselective Functionalization of Thioester Proteins in Their Native State

Less than 6 feet under : Serum proteins C3, C4, and α₂M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

     

消化用水的温度对纳米碳酸钙产品性能的影响

采用碳化法制备了纳米碳酸钙,考察了消化时的水温对石灰乳产率、制备的纳米碳酸钙产品的粒径分布、比表面积、吸油值和白度等物理性能的影响。结果表明：石灰经高温消化,能够提高石灰乳的产率,产率可达92%;高温消化有利于碳酸钙比表面积的升高、同时降低碳酸钙的颗粒粒度及吸油值,对碳酸钙的白度影响较小。当消化用水的温度为60℃时,制备的碳酸钙比表面积较高,而其粒度和吸油值较低。     

Effect of In Vitro Gastrointestinal Digestion on Amino Acids,Polyphenols and Antioxidant Capacity of Tamarillo Yoghurts

Laird’s Large tamarillo powder is high in protein (10%) essential amino acids (EAAs), gamma-aminobutyric acid (GABA) and polyphenols (0.6% phenolics plus anthocyanins) and fibre 25%. This study aimed to investigate, using a standardized static in vitro digestion model, the stability of amino acids and antioxidant capacity of polyphenols in yoghurt fortified with 5, 10 and 15% tamarillo powder either before (PRE) or after (POS) fermentation. Compared to plain yoghurt, the fruit polyphenols (rutinosides and glycosides) were retained and substantial increases in FEAAs (free essential amino acids), total phenolic content (TPC) and antioxidant activity were observed particularly at the end of intestinal phase of digestion. Together with SDS-PAGE results, peptides and proteins in tamarillo yoghurts were more easily digested and therefore may be better absorbed in the small intestine compared to the control. TPC and antioxidant activity of fortified yoghurts increased significantly after in vitro digestion. Relatively high bioaccessibilty of chlorogenic acid and kaempferol-3-rutinoside in digested PRE samples was observed. The results suggest that the yoghurt matrix might protect some compounds from degradation, increasing bioaccessibility and in the small intestine allow increased absorption and utilization possible. Fortification would deliver intact polyphenols and fibre to the large intestine and improve gut health. Further research of acceptability, shelf life, and then trials for health effects should be implemented.     

改善污泥厌氧消化性能的预处理技术

厌氧消化是污泥稳定化处理的主要技术之一。通过采用一定的预处理技术,破坏细胞结构,释放有机质,促进污泥的水解速率,从而改善污泥厌氧消化性能。该文综述了几种国内外目前研究较为广泛的预处理技术,主要包括热预处理法、转动球磨法、高压喷射法、超声波预处理法、酸预处理法、碱预处理法和氧化法等技术,并分别探讨了各种技术的作用原理、特点、处理效果及应用前景。