Abstract
Laboratory Automation and High-Throughput Chemistry
Online Solid Phase Extraction and Liquid Chromatography–Mass Spectrometric Determination of Nucleoside Drugs in Plasma
The bioanalysis and especially the sample preparation of nucleoside drugs in complex media, such as human plasma, has been challenging due to the high polarity and high solubility of these drugs in water. Online solid phase extraction (SPE) offers significant advantages, such as automation and timesaving. Thus, several types of SPE columns have been developed for compounds with different polarities.
In this study, SPE is applied to overcome the issue of sample pretreatment of nucleoside drugs in human plasma, with the final aim of establishing a robust analytical platform for drugs with similar structures. A simple, easy-to-use, and efficient method is described for the simultaneous determination of lamivudine, zidovudine, didanosine, and emtricitabine in human plasma via online SPE and high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS).
Following a simple centrifugation step, a 10-µL plasma sample is injected directly onto the HPLC system. The Oasis MCX (Waters Corporation, Milford, MA, USA) cartridge is washed, and the analytes are removed by back-flushing directly onto the analytical column. The analytes are quantified using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Similarly, with the development and application of a Bond Elut (Agilent Technologies, Santa Clara, CA, USA) phenylboronic acid (PBA) SPE cartridge, a fully automated online SPE-HPLC-MS/MS method is established for the simultaneous determination of ribavirin and taribavirin in human plasma. Linear calibration curves are obtained over the range of 0.5 to 2000 ngmL–1, and the limit of quantification ranges from 0.5 to 10 ng mL–1, which is sensitive enough for clinical drug monitoring. The intra- and interday precisions are in the range of 0.2% to 8.9%, and the trueness ranges between 88.9% and 113.1%. Excellent recoveries from plasma are achieved with a range between 86.7% and 105.1%.
This procedure is easier to perform and requires less sample handling compared to methods previously described in the literature. This high-throughput method involving the direct injection of plasma samples may provide a practical solution for the analysis of multiple nucleoside drugs in clinical research. The method is tested in plasma samples from some patients and shows good performance (Wang, M.; et al. Talanta
Membrane Protein Production in E. coli for Applications in Drug Discovery
Producing high-quality purified membrane proteins for structure-based drug design and biophysical assays compatible with typical timelines in drug discovery is a significant challenge. Escherichia coli has been an expression host of the utmost importance for soluble proteins and has applications for membrane proteins as well. However, membrane protein overexpression in E. coli may lead to toxicity and low yields of functional product.
Here, the authors review the challenges encountered with heterologous overproduction of α-helical membrane proteins in E. coli and a range of strategies to overcome them. A detailed protocol is also provided for expression and screening of membrane proteins in E. coli using a His-specific fluorescent probe and fluorescent size-exclusion chromatography (Snijder, H. J.; Hakulinen, J. Adv. Exp. Med. Biol.
High-Throughput Automated Scoring of Ki67 in Breast Cancer Tissue Microarrays from the Breast Cancer Association Consortium
Automated methods are needed to facilitate high-throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large-scale studies. To address this need, the authors of this report present an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. Abubakar et al. used 166 TMAs containing 16,953 tumor cores representing 9059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs are stained for Ki67 using standard immunohistochemical procedures and scanned and digitized using the Ariol system. An automated algorithm is developed for the scoring of Ki67, and scores are compared to computer-assisted visual (CAV) scores in a subset of 15 TMAs in a training set.
The authors also assess the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, the authors observe good discriminatory accuracy (area under the curve [AUC] = 85%) and good agreement (κ = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varies by TMA (κ range = 0.37–0.87) and study (κ range = 0.39–0.69). The automated method performs better in satisfactory cores (κ = 0.68) than suboptimal (κ = 0.51) cores (p for comparison = 0.005) and among cores with higher total nuclei counted by the machine (4000–4500 cells: κ = 0.78) than those with lower counts (50–500 cells: κ = 0.41; p = 0.010).
Among the 9059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics are found to be in the expected directions. The authors’ findings indicate that automated scoring of Ki67 can be an efficient method to obtain good-quality data across large numbers of TMAs from multicenter studies. However, robust algorithm development and rigorous pre- and postanalytical quality control procedures are necessary to ensure satisfactory performance (Abubakar, M.; et al. J. Pathol. Clin. Res.
Accurate Detection for a Wide Range of Mutation and Editing Sites of MicroRNAs from Small RNA High-Throughput Sequencing Profiles
Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false-positive rate (FPR) are both required.
Zheng et al. propose a novel method named MiRME for accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach that refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3′-editing sites, 57 A-to-I editing sites of which 32 are novel, and some putative noncanonical editing sites.
The authors demonstrate that a few noncanonical editing sites do not result from mutations in the genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME shows much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles (Zheng, Y. Nucleic Acids Res.
Microfluidics and Microbioreactor
A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection
Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression offers potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease.
This report presents a multilayer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel, and colchicine) with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231) are lysed on chip, and the released mRNA templates are captured and reversely transcribed into single-strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclin-dependent kinase inhibitor 1A (CDKN1A), and aurora kinase A (AURKA) genes from single cells are amplified and quantified in real time through multiplex PCR. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling and providing precision detection of drug-induced genotoxic stress. Throughput is set at 18 and can be further increased following the same approach. Numerical simulation of on-chip single-cell trapping and heat transfer is employed to evaluate the chip design and operation (Dong, H.; Sun, H. Sensors (Basel).
Microfluidic Devices for Automation of Assays on Drosophila melanogaster for Applications in Drug Discovery and Biological Studies
Drug discovery is a long and expensive process that usually takes 12 to 15 years and can cost up to ~$1 billion. A conventional drug discovery process starts with high-throughput screening and selection of drug candidates that bind to specific targets associated with a disease condition. However, this process does not consider whether the chosen candidate is optimal not only for binding but also for ease of administration, distribution in the body, effect of metabolism, and associated toxicity, if any.
A holistic approach, using model organisms early in the drug discovery process to select drug candidates that are optimal not only in binding but also suitable for administration, distribution, and not toxic, is now considered a viable way to lower the cost and time associated with the drug discovery process. Since the manual assays are manually performed and required skilled operators, they are not readily amenable to high-throughput scale. Recently, microfluidics has been used to automate many of the operations (e.g., sorting, positioning, drug delivery) associated with Drosophila drug discovery assays to increase their throughput.
This review highlights recent microfluidic devices that have been developed for Drosophila assays with primary application toward drug discovery for human diseases. The microfluidic devices that are reviewed in this article are categorized by the stage of the Drosophila that are used. In each category, the microfluidic technologies behind each device are described and their potential biological applications are discussed (Ghaemi, R.; Selvaganapathy, P. R. Curr. Pharm. Biotechnol.
Scaling and Automation of a High-Throughput Single-Cell-Derived Tumor Sphere Assay Chip
Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1%–5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies.
In this study, Cheng et al. present the scaling and automation of high-throughput single-cell–derived tumor sphere assay chips, facilitating the tracking of up to ∼10,000 cells on a chip with ∼76.5% capture rates. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single cells with a variety of fluorescent intensities, a highly adaptable analysis program is developed for cell/sphere counting and size measurement.
Using a Pluronic F108 (Sigma Aldrich, St. Louis, MO, USA) (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment is created to test a controversial hypothesis: whether larger or smaller cells are more stem-like, defined by the capability to form single-cell–derived spheres. Different cell lines show different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, the authors identify heterogeneity in sphere growth dynamics, indicating cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high throughput and automation in CSC studies (Cheng, Y. H.; et al. Lab Chip
Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High-Density Microbioreactor Arrays
Inducing cardiomyocyte proliferation in postmitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signaling pathways, but it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signaling pathway penetrance and interplay.
To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, the authors of this report present a high-density microbioreactor array (HDMA)—a microfluidic cell culture array containing 8100 culture chambers. Titmarsh et al. use HDMAs to combinatorially screen Wnt, Hedgehog, IGF, and FGF pathway agonists. The Wnt activator CHIR99021 is identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67 and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration (Titmarsh, D. M.; et al. Sci. Rep.
Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy
Since the 1960s, combination chemotherapy has been widely used as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low-throughput screening, laborious and time-consuming procedures, and ethical concerns.
In this article, Ding et al. present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform that integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, that can potentially speed up the entire discovery cycle of potent drug combinations.
Microfluidic impact printing using plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment that can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery (Ding, Y.; et al. Anal. Chem.
Analytical Chemistry
A Portable Device for Measuring Breath Acetone Based on Sample Preconcentration and Cavity Enhanced Spectroscopy
In this report, a portable and compact device measures acetone in breath samples. The device features a 7-cm-long high-finesse optical cavity as an optical sensor that is coupled to a miniature adsorption preconcentrator containing 0.5 g of polymer material. Acetone is trapped out of breath and released into the optical cavity, where it is probed by a near-infrared diode laser operating at ~1670 nm. With an optical cavity mirror reflectivity of 99.994%, a limit of detection of 159 ppbv (1σ) is demonstrated on samples from breath bags. Initial results on direct breath sampling are presented with a precision of 100 ppbv. The method is validated with measurements made using an ion-molecule reaction mass spectrometer. Data are presented on elevated breath acetone from two individuals following an overnight fast and exercise and from a third individual during several days of routine behavior (Blaikie, T. P.; et al. Anal. Chem., in press).
Advances in Genomics
The Potential of Radiomic-Based Phenotyping in Precision Medicine: A Review
Advances in genomics have led to the recognition that tumors are populated by distinct genotypic subgroups that drive tumor development and progression. The spatial and temporal heterogeneity of solid tumors has been a critical barrier to the development of precision medicine approaches because the standard approach to tumor sampling, often invasive needle biopsy, is unable to fully capture the spatial state of the tumor. Image-based phenotyping that represents quantification of the tumor phenotype through medical imaging is a promising development for precision medicine.
Medical imaging can provide a comprehensive macroscopic picture of the tumor phenotype and its environment that is ideally suited to quantifying the development of the tumor phenotype before, during, and after treatment. As a noninvasive technique, medical imaging can be performed at low risk and inconvenience to the patient. The semantic features approach to tumor phenotyping, accomplished by visual assessment of radiologists, is compared with a computational radiomic approach that relies on automated processing of imaging assays. Together, these approaches capture important information for diagnostic, prognostic, and predictive purposes.
Although imaging technology is already embedded in clinical practice for diagnosis, staging, treatment planning, and response assessment, the transition of these computational methods to the clinic has been surprisingly slow. This review outlines the promise of these novel technologies for precision medicine and the obstacles to clinical application (Aerts, H. J. JAMA Oncol., in press. doi: 10.1001/jamaoncol.2016.2631).
The Genetics of Blood Pressure Regulation and Its Target Organs from Association Studies in 342,415 Individuals
To dissect the genetic architecture of blood pressure and assess effects on target organ damage, Ehret et al. analyze 128,272 single-nucleotide polymorphisms (SNPs) from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals are used for validation. The authors identify 66 blood pressure–associated loci, of which 17 are new; 15 harbored multiple distinct association signals. The 66 index SNPs are enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combine in a risk score that shows comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score is significantly associated with target organ damage in multiple tissues but with minor effects in the kidney. These findings of Ehret et al. expand current knowledge of blood pressure–related pathways and highlight tissues beyond the classical renal system in blood pressure regulation (Ehret, G. B.; et al. Nat. Genet.
Whole Genome-Based Population Biology and Epidemiological Surveillance of Listeria monocytogenes
Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with high fatality rates, reinforcing the need for strongly coordinated surveillance and outbreak control.
Moura et al. offer a universally applicable genome-wide strain genotyping approach and investigate the population diversity of Lm using 1696 isolates from diverse sources and geographical locations. The authors define, with unprecedented precision, the population structure of Lm; demonstrate the occurrence of international circulation of strains; and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates.
Using historical isolates, the authors show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10–7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called epidemic clones) are estimated to be at least 50 to 150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology (Moura, A.; et al. Nat. Microbiol.
Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp
Genome sequencing projects in the past decade have revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining.
In this work, Xu et al. develop a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. The authors demonstrate mining from a strain of Streptomyces rochei that is known to produce streptothricins and borrelidin by expressing its BAC library in the surrogate host Streptomyces lividans SBT5 and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, the authors discover two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster, for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways (Xu, M.; et al. Appl. Environ. Microbiol.