An informatic pipeline for managing high-throughput screening experiments and analyzing data from stereochemically diverse libraries

Integration of flexible data-analysis tools with cheminformatics methods is a prerequisite for successful identification and validation of “hits” in high-throughput screening (HTS) campaigns. We have designed, developed, and implemented a suite of robust yet flexible cheminformatics tools to support HTS activities at the Broad Institute, three of which are described herein. The “hit-calling” tool allows a researcher to set a hit threshold that can be varied during downstream analysis. The results from the hit-calling exercise are reported to a database for record keeping and further data analysis. The “cherry-picking” tool enables creation of an optimized list of hits for confirmatory and follow-up assays from an HTS hit list. This tool allows filtering by computed chemical property and by substructure. In addition, similarity searches can be performed on hits of interest and sets of related compounds can be selected. The third tool, an “S/SAR viewer,” has been designed specifically for the Broad Institute’s diversity-oriented synthesis (DOS) collection. The compounds in this collection are rich in chiral centers and the full complement of all possible stereoisomers of a given compound are present in the collection. The S/SAR viewer allows rapid identification of both structure/activity relationships and stereo-structure/activity relationships present in HTS data from the DOS collection. Together, these tools enable the prioritization and analysis of hits from diverse compound collections, and enable informed decisions for follow-up biology and chemistry efforts.     

3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry

In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self-organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long-term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ-on-a-chip” systems. Organ-on-a-chip technology allows a broad range of “on-chip” and “off-chip” analytical techniques, whereby “on-chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on-chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.     

Diosgenin aus Yams als Hormonvorstufe

Diosgenin as a sapogenin is the aglycone of the saponine dioscin. It is occuring in plants such as yam, from which it is isolated on industrial scale since the 1940ies. Plant diosgenin is a starting substance for the synthesis of human, i.e. animal, hormones and “the pill”, which as a hormonal contraception represents a revolution in the control of human sexuality. History, isolation and spectroscopy of this complex sapogenin are reported. This article extends the briskly discussed feature “50 years pill in Germany” (Streller & Roth, ChiuZ, 2011 [( 1 ) ]) by a particular compound example. It belongs to the series on isolation and spectroscopy of natural products in this journal.     

Gold Nanoparticle-based “Mix and Measure” Fluorimetric Assays to Quantify Antibody Titer

Monoclonal antibodies (mAbs) for treatment of human diseases are typically human or humanized Immunoglobulin G (IgG) produced in mammalian cell lines. A rapid, less tedious, and high throughput method to quantify mAbs is in demand to accelerate mAb production efficiency. To quantify mAb titer, we developed gold nanoparticle (AuNPs)-based “mix and measure” fluorimetric assays by exploiting AuNPs’ fluorescence quenching ability. The AuNPs are functionalized by an Fc binding protein, i. e. protein G, which binds human IgG and fluorescently labeled rat IgG (Alexa Fluor 488-rat IgG) with differential affinity. The assays can be in competition or displacement format. The competitive binding of human IgG drug and the labelled rat IgG to protein G-coated AuNP lead to varied fluorescent intensity that is proportional to the amount of human IgG analte; or the displacement of the labelled rat IgG from protein G-coated AuNP by human IgG can lead to fluorescent recovery that is also proportionally related to human IgG concentration. The assays can quantify therapeutic mAbs in the range of 10–1,000 mg/L, demonstrated for Herceptin, Avastin, and Humira in cell culture media. The assays have fast turn over time (within 15 min). They can be performed in microplates and are suitable for high throughput “on-line” or “at-line” measurement in mAbs production lines.     

Green Synthesis of Red‐Emitting Carbon Nanodots as a Novel “Turn‐on” Nanothermometer in Living Cells

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel “turn‐on” carbon‐dot‐based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave‐assisted method and exhibit red fluorescence (λ_em=615 nm) with high quantum yields (15 %). Then, an on–off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation‐induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs–GSH mixture could behave as an off–on fluorescent probe for temperature. Thus, red‐emitting CNDs can be utilized for “turn‐on” fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3‐E1 cells as an example model to demonstrate the red‐emitting CNDs can function as “non‐contact” tools for the accurate measurement of temperature and its gradient inside a living cell.     

Kinetic analysis of cellulose pyrolysis: a short review

Since the 1950s, cellulose pyrolysis has been the subject of intense study, with kinetic analyses forming a major part of these studies. They represent useful tools for a better understanding of the physicochemical process and for the proper design of industrial pyrolysis units. Until recently, the methods most frequently used in these analyses were based on model-fitting, i.e. the fitting of the experimental data to a number of mathematical models. Nowadays, other methods, so-called “model-free” methods, are considered to be more suited. These are based on the principle that, at constant conversion, the reaction rate depends only on temperature. In its first part, this short review presents the particularities and drawbacks of the traditional model-fitting models. Subsequently, several main contributions in this field are listed and discussed. Finally, the more suited “model-free” (isoconversional) methods are explained and several main studies presented, as well as a comparison of this method with the model-fitting ones.     

Solid-Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo-based oligonucleotide binder” (HOB), a variant of Halo-tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.     

Statistical interpretation of the Amerithrax “morph” assay results

In the Amerithrax investigation PCR‐based “morph assays” were used to link the anthrax letters with the RMR‐1029 flask at USAMRIID. Quantitative data reported for several of these assays are not consistent with Poisson sampling statistics, but instead exhibit “Taylor's Law” behavior where the variance greatly exceeds the mean. A plausible statistical model for this behavior can explain the large number of observed negative and “inconclusive” findings, and implies a high likelihood that a repository sample could contain a “morph” mutant at concentrations well above the nominal detection limit but nonetheless give a negative or inconclusive test result. A Bayesian framework relates the assay results to the probability that a sample actually contains all four morph mutants, even though it tested negative for at least one. The analysis implies that the observed false negative rate actually does not significantly weaken the conclusion that the morph assays correctly excluded all but the stocks derived from RMR‐1029 as possible sources of the letter powders, at least when the test results were unambiguous. These findings expand upon and resolve some of the issues cited in recent reviews, and indicate the importance of developing a rigorous statistical framework for interpreting “morph” assay data.     

Preparation of Porous Protein-Based Hydogel for Highly Sensitive Protein Chips

Summary: Protein chips are important tools for high-throughput analysis of biological events. We have developed a novel method to prepare a protein-based hydrogel, that is, a “Three-Dimensional Nano-structured Protein Hydrogel” (3-D NPH), which is composed of protein and polymer nano-particles. The 3-D NPH could be easily prepared by dispensing a protein and polymer mixture on a substrate. Surprisingly, gold particles conjugated with protein A diffused into the 3-D NPH which was made of mouse IgG through the pores. We have shown that the protein chips made with our 3-D NPH method has tremendously improved sensitivity in detecting protein-protein interactions compared with that of direct protein immobilization methods.     

中药质量评价关键问题与分析方法探讨

该文针对我国目前中药质量评价体系现状,总结了中药质量评价的关键科学问题,并围绕“发现”与“控制”有效成分的中药质量评价方法进行探讨。笔者结合多年中药质量分析研究工作经验,分析了中药“谱-效”关系研究、全时段等基线多波长融合指纹图谱、生物活性测定法3种质量评价与检测方法的研究内容、手段、适用范围等,为丰富、完善中药质量控制方法和中药质量标准体系的提升提供了借鉴。     

“Surface water” and “strong-bonded water” in cyclodextrins: a Karl Fischer titration approach

Cyclodextrins are some of the most used carriers for bioactive compounds (as host–guest complex) and many factors influence the association–dissociation of this complex, some of them being related to hydrophobicity. In the solid state, cyclodextrins contain two types of water molecules: “surface” water molecules (especially close to the crystal surface) and “strong-bonded” water molecules (especially from the cyclodextrin cavity), but the classification is hard to do, and the concentration of these water molecules are relatively difficult to estimate by simple methods. In the present study we used the volumetric Karl Fischer titration to estimate these types of water molecules in cyclodextrins by means of the rate of water reaction (related to diffusion from cyclodextrin crystals). “Surface” water molecules are titrated with rates between 1.8–2.8 mM/s for α-cyclodextrin, while for β-cyclodextrin these rates are little bit higher (2.9–3.4 mM/s). The rates corresponding to “strong-bonded” water molecules are approximately tens fold lower (0.05–0.3 mM/s for α-cyclodextrin and 0.15–0.33 mM/s for β-cyclodextrin). The approximate ratio between “surface” and “strong-bonded” water molecules could also be estimated by this simple and rapid method.     

Pending templates imprinted polymers—hypothesis,synthesis, adsorption,and chromatographic properties

This is the first time when protein‐imprinted polymers are prepared with “pending templates.” The polymers were synthesized in the presence of a real sample (chicken egg white), rather than any known commercial proteins. Compared with a simultaneously synthesized nonimprinted control polymer, the polymers show higher adsorption capacity for abundant components (as “pending templates”) in the original sample. Chromatography experiments indicated that the columns made of the imprinted polymers could retain abundant species (imprinted) and separate them from those not imprinted. Thus, the sample could be split into dimidiate subfractions with reduced complexities. “Pending template imprinting” suggests a new way to investigate molecular imprinting, especially to dissect, simplify, and analyze complicated samples through a series of polymers just imprinted by the samples per se.     

Kinetic analysis of simple irreversible non-homogeneous reactions

Simple non-homogeneous irreversible reactions between a mobile reactant A, and immobile reactive groups R, in a solid yielding immobile product B, are considered. The possibilities are discussed of analysing such reactions when they are partially rate-controlled by the diffusion of A. It is shown that the effect of diffusion can be approximately described by a single parameter (“kinetic modulus”) up to a fairly marked degree of diffusion control; so that methods of kinetic analysis analogous to those used for reactions where the solid acts merely as a catalyst can be applied. The “effective reaction constant” previously defined for use as a kinetic modulus (analogous to the “Thiele modulus” of heterogeneous catalytic reactions) is considered further here. An improved, experimentally measurable kinetic modulus, based on the distribution of B across the solid, is also defined and studied.     

Discovery by serendipity: a new context for an old riddle

In the last years there has been a great improvement in the development of computational methods for combinatorial chemistry applied to drug discovery. This approach to drug discovery is sometimes called a “rational way” to manage a well known phenomenon in chemistry: serendipity discoveries. Traditionally, serendipity discoveries are understood as accidental findings made when the discoverer is in quest for something else. This ‘traditional’ pattern of serendipity appears to be a good characterization of discoveries where “luck” plays a key role. In this sense, some initial failures in combinatorial chemistry are frequently attributed to a naïf appropriation of a “serendipity model” for discovery (a “serendipity mistake”). In this paper we try to evaluate this statement by criticizing its foundations. It will be suggested that the notion of serendipity has different aspects and that the criticism to the first attempts could be understood as a “serendipity mistake.” We will suggest that “serendipity” strategies, a kind of blind search, can be seen sometimes as a “genuine part” of scientific practice. A discussion will ensue about how this characterization can give us a better understanding of some aspects of serendipity discoveries.     

Self-assembled structures and chemical reactions in room-temperature ionic liquids

Amphiphilic association in room-temperature ionic liquids (RT-ILs) — a “green” solvent shows analogies as well as clear differences from self-assembly in water. In this review, we summarize the known features of amphiphilic association structures in the form of micelles, microemulsions, vesicles and lyotropic liquid-crystalline phases in ionic liquids. Most of the methods making use of association to control reactivity could be developed also in RT-ILs and we give a few recently published examples of this strategy.     

Photochromic Fentanyl Derivatives for Controlled μ-Opioid Receptor Activation

Photoswitchable ligands as biological tools provide an opportunity to explore the kinetics and dynamics of the clinically relevant μ-opioid receptor. These ligands can potentially activate or deactivate the receptor when desired by using light. Spatial and temporal control of biological activity allows for application in a diverse range of biological investigations. Photoswitchable ligands have been developed in this work, modelled on the known agonist fentanyl, with the aim of expanding the current “toolbox” of fentanyl photoswitchable ligands. In doing so, ligands have been developed that change geometry (isomerize) upon exposure to light, with varying photophysical and biochemical properties. This variation in properties could be valuable in further studying the functional significance of the μ-opioid receptor.     

Use of data from environmental sampling for IAEA safeguards. Case study: uranium with near-natural 235U abundance

Environmental sampling (ES) is one of the measures applied in international nuclear safeguards. The detection capability of safeguards ES relies on a combination of highly sensitive analytical techniques and resourceful data evaluation. The evaluation process is dynamic, employing a variety of tools, information and analytical results. While the presence of uranium or plutonium may be a significant finding in itself, high quality isotopic measurements are essential to associate the material with a specific nuclear activity. This is illustrated in cases where the uranium detected appears to be “natural” or “near-natural”, but in fact can be identified with various nuclear processes.     

High-Performance Nucleic Acid Sensors for Liquid Biopsy Applications

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This “liquid biopsy” approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild-type sequences. This review discusses high-performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing- and amplification-based methods to next-generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis-based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present.     

Time-series analysis in analytical process control

Analysis of time series tries to extract tendencies from measured values dependent on time. For this purpose the cusum technique has proved to be a very sensitive tool for the evaluation of both current and completed time series. Even very weak tendencies can be detected at a high level of noise. Time-series analysis further tries to predict values to come from hitherto performed measurements. As a very flexible model exponential smoothing could be successfully used. Even for processes with a high extent of non-stationarity this model allowed a good prediction owing to the dynamics of the process. Three types of time-series analysis, i.e., evaluation of current measurements, retrospective evaluation and prediction of data (also known as “in vivo”, “post mortem” and “in futurum” time-series analysis) are demonstrated for problems stemming from analytical process control.     

Which Factors Determine the Reactivity and Regioselectivity of Free Radical Substitution and Addition Reactions?

The relative importance of bond strength, steric effects, and polarity in determining the rate and orientation of free radical subsitution and free radical addition reaction is considered. The factors which control substitution reaction (radical transfer reaction) are gathered together as five “rules”, and a similar five “rules” are proposed for addition rections. These “rules” are shown to be special cases of two “laws” which govern all free radical reactions.