Native Mass Spectrometry for the Study of PROTAC GNE-987-Containing Ternary Complexes

PRO teolysis TA rgeting C himeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous binding of both the target protein and an E3-Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE-987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3-Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high-throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.     

A Strategy for Discovering Heterochiral Bioactive Peptides by Using the OB2nP Library and SPOTs Method

d -Amino acid containing peptides are promising as drug lead compounds because of their expected higher stability in vivo. A heterochiral random peptide library called the one-bead–2ⁿ-peptide (OB2ⁿP) library, which can display 2ⁿ peptide diastereomers per bead, has been developed. Through screening of the OB2ⁿP library and subsequent binding assay among the peptide diastereomers synthesized in parallel by means of the SPOTs method, new heterochiral mimotopes for the anti-β-endorphin monoclonal antibody have been obtained. One mimotope was a new ligand for the μ-opioid receptor. The screening strategy enabled d -amino acid containing drug leads to be obtained efficiently by expanding searchable chemical space without increasing the experimental scale.     

A high-throughput chaotic advection microreactor for preparation of uniform and aggregated barium sulfate nanoparticles

A high-throughput (105.5 g/h) passive four-stage asymmetric oscillating feedback microreactor using chaotic mixing mechanism was developed to prepare aggregated Barium sulfate (BaSO₄) particles of high primary nanoparticle size uniformity. Three-dimensional unsteady simulations showed that chaotic mixing could be induced by three unique secondary flows (i.e., vortex, recirculation, and oscillation), and the fluid oscillation mechanism was examined in detail. Simulations and Villermaux–Dushman experiments indicate that almost complete mixing down to molecular level can be achieved and the prepared BaSO₄ nanoparticles were with narrow primary particle size distribution (PSD) having geometric standard deviation, σ_g, less than 1.43 when the total volumetric flow rate Q_total was larger than 10 ml/min. By selecting Q_total and reactant concentrations, average primary particle size can be controlled from 23 to 109 nm as determined by microscopy. An average size of 26 nm with narrow primary PSD (σ_g = 1.22) could be achieved at Q_total of 160 ml/min.     

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10⁴ copies per library member are used as the input.     

汉江上游干支流沉积物细菌群落多样性和影响因素

为了解汉江上游干支流沉积物细菌多样性以及确定性过程和随机性过程在沉积物细菌群落构建过程中的相对重要性,基于Illumina高通量测序技术,分析了环境因子对细菌群落组成的影响,采用非度量多维尺度(NMDS)排序探究了季节之间沉积物细菌群落的差异,并结合中性群落模型和标准化随机率量化了确定性过程和随机过程对群落构建的影响。结果表明:汉江上游及其支流细菌群落主要由变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、蓝藻门(Cyanophyta)、浮霉菌门(Planctomycetes)和酸杆菌门(Acidobacteria)等组成；细菌群落在不同季节有显著差异；地理距离和环境因子对细菌群落结构影响较小,确定性过程并未在细菌群落组成中起到主导作用；随机过程很大程度上影响了群落在秋季和春季的组成,是沉积物细菌群落构建的主导因素。     

茅台镇酱香型白酒酿造环境中真菌菌群多样性分析

采用高通量测序技术结合数理统计软件分析方法，系统研究茅台镇酱香型白酒不同酿造轮次生产环境中的真菌菌群结构及特征。7 个轮次中共检测到4 个真菌门、212 个真菌属。根据各菌门、属的相对丰度，子囊菌门（Ascomycota）和担子菌门（Basidiomycota）是茅台镇酱香型白酒酿造环节中各酿造轮次的优势菌门，节担菌属（Wallemia）、复膜孢酵母属（Saccharomycopsis）为各轮次绝对优势真菌属。不同轮次环境样品中真菌组成结构的相似度较高，但其各轮次标志性真菌属存在差异。7 个酿造轮次共有相同真菌属56 个，在各轮次占主导地位，特征性真菌属在各轮次当中相对丰度和占比较小但种类丰富。Ascomycota最强节点为德巴利酵母属（Debaryomyces），Basidiomycota最强节点为红酵母属（Rhodotorula）。本研究揭示了茅台镇酱香型白酒不同酿造轮次环境中真菌菌群结构及其特征，为深入研究酱香型白酒酿造机理以及对酿造环境中微生物结构的解析提供了参考依据。     

基于高通量测序的东湖蓝藻动态变化研究

采用高通量测序(High-throughput sequencing technology)和实时荧光定量PCR技术,结合水质参数的检测,研究了东湖两个不同富营养化程度湖区蓝藻整年丰度和群落结构的变化,并统计分析蓝藻与环境参数之间的关系。结果表明:(1)两区域微囊藻在6~7月达到全年最高值,但拷贝数相差近20倍。(2)两区域蓝藻门在属水平的变化也有显著差异,H区蓝藻在4~11月主要由浮丝藻属(Planktothrix)(4~6月)、微囊藻属(Microcystis)(6~9月)、聚球藻属(Synechococcus sp. PS717)(7~11月)交替演变,L区蓝藻主要是由束丝藻属(Aphanizomenon)(4~6月)和聚球藻属(Synechococcus sp. PS717)(4~10月)组成,并且与H区在相对丰度变化上比较,L区较平稳。(3) RDA分析表明,Tw、p H、NH4-N是影响H区蓝藻属水平分类差异的主要因素,影响L区蓝藻属水平差异的主要因素是Tw、TP、p H。(4)与以往传统藻类计数相比,还发现了以往显微观察难以发现的聚球藻属和拟柱孢藻属等,且聚球藻属在东湖蓝藻门中占优势地位,应在以后的研究中得到更多的关注。     

基于高通量测序技术研究不同品种桃果实的微生物多样性

目的 探究贵州不同品种桃果实附着的微生物菌群组成情况。方法 以镇远红桃、镇远艳红桃和玉屏黄桃为试材,利用高通量测序技术分析贵州不同品种桃果实微生物的多样性。结果 红桃的主要优势细菌属为泛菌属、鞘氨醇单孢菌属和假单胞菌属,优势真菌属为枝孢菌属、交链孢属和曲霉属;艳红桃的优势细菌属为拟杆菌属、布老特氏菌属和甲基杆菌属,优势真菌属为链核盘菌属、枝孢菌属和交链孢属;黄桃的优势细菌属为甲基杆菌属、鞘氨醇单孢菌属和棒状杆菌属,优势真菌属为枝孢菌属、链核盘菌属和交链孢属。此外,α多样性分析表明艳红桃的真菌多样性最高,而黄桃的真菌多样性次之;β多样性分析表明构成不同品种桃群落组成有明显差异,而黄桃与红桃的群落组成相似程度更高。结论 研究初步明确了贵州不同品种桃果实的微生物多样性,为不同品种桃果实的绿色生产和贮藏保鲜提供理论基础。     

Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin–NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin–NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.     

Scalable Production of Biomedical Microparticles via High-Throughput Microfluidic Step Emulsification

Drug microcarriers are widely used in disease treatment, and microfluidics is well established in the preparation of microcarrier particles. A proper design of the microfluidic platform toward scalable production of drug microcarriers can extend its application values in wound healing, where large numbers of microcarriers are required. Here, a microfluidic step emulsification method for the preparation of monodisperse droplets is presented. The droplet size depends primarily on the microchannel depth rather than flow rate, making the system robust for high-throughput production of droplets and hydrogel microparticles. Based on this platform, basic fibroblast growth factor (bFGF) is uniformly encapsulated in the microparticles, and black phosphorus (BP) is incorporated for controllable release via near-infrared (NIR) stimulation. The microparticles serve as drug carriers to be applied to the wound site, inducing angiogenesis and collagen deposition, thereby accelerating wound repair. These results indicate that the step emulsification technique provides a promising solution to scalable production of drug microcarriers for wound healing as well as tissue regeneration.