Abstract
Laboratory Automation and High-Throughput Chemistry
Systematic Evaluation of Immune Regulation and Modulation
Cancer immunotherapies are showing promising clinical results in a variety of malignancies. Monitoring the immune as well as the tumor response following these therapies has led to significant advancements in the field. Moreover, the identification and assessment of both predictive and prognostic biomarkers have become key components to advancing these therapies. Thus, it is critical to develop systematic approaches to monitor the immune response and to interpret the data obtained from these assays.
To address these issues and make recommendations to the field, the Society for Immunotherapy of Cancer reconvened its Immune Biomarkers Task Force. As a part of this Task Force, Working Group 3 consisting of multidisciplinary experts from industry, academia, and government focused on the systematic assessment of immune regulation and modulation.
In this review, the tumor microenvironment, microbiome, bone marrow, and adoptively transferred T cells are used as examples to discuss the type and timing of sample collection. In addition, potential types of measurements, assays, and analyses are discussed for each sample. Specifically, these recommendations focus on the unique collection and assay requirements for the analysis of various samples as well as the high-throughput assays to evaluate potential biomarkers. (Stroncek, D. F.; et al. J. Immunother. Cancer.
Development of a Novel Automated Screening Method for Detection of FVIII Inhibitors
Factor VIII activity is routinely determined by measuring the activated partial thromboplastin time of a patient plasma sample and determining percentage activity from a standard curve. To maximize the detection of a clotting factor inhibitor, a subjective assessment of parallelism of a patient curve compared with a standard curve is performed. Evans et al. develop and validate an automated objective method to assess parallelism as a rapid screening tool for detection of an inhibitor to factor VIII during routine FVIII assays.
The authors perform FVIII assays on a subset of FVIII-deficient patients with hemophilia A with and without inhibitors. Using a ratio of the slopes from parallelism curves obtained by an independent Microsoft Excel program in patients compared with a normal standard curve, the authors determine a cutoff ratio predictive of the presence of an inhibitor.
A cutoff ratio of patient to control slopes of <0.45 for the detection of an inhibitor to FVIII is 100% sensitive and 91.6% specific, with a positive predictive value of 92.3% and a negative predictive value of 100%.
Using a ratio of the slopes from parallelism curves in patients with and without an inhibitor, Evans et al. develop and validate a rapid, automated, and objective method to assess parallelism as an added screening tool for detection of an inhibitor to factor VIII during routine FVIII assays on a STAGO-based coagulation platform. This simple automated method has the potential to detect inhibitors to other clotting factors. (Evans, M. S. Int. J. Lab. Haematol.
Fully Automated Two-Step Assay for Detection of Metallothionein through Magnetic Isolation Using Functionalized γ-Fe2O3 Particles
Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play a substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. To increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study is to develop a fully automated and high-throughput assay for the estimation of MT levels.
Merlos Rodrigo et al. report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition, this study describes a two-step assay, which starts with an isolation of the protein using functionalized paramagnetic particles and finishes with their electrochemical analysis. The easy-to-use, cost-effective, error-free, and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors. (Merlos Rodrigo, M. A.; et al. J. Chromatogr. B.
High-Throughput System-Wide Engineering and Screening for Microbial Biotechnology
Genetic engineering and screening of large numbers of cells or populations can create bottlenecks in today’s systems biology and applied (micro) biology. Instead of using standard methods in bottles, flasks, or 96-well plates, scientists are increasingly relying on high-throughput strategies that miniaturize their experiments to the nanoliter and picoliter scale and the single-cell level.
In this review, Vervoort et al. summarize different high-throughput system-wide genome engineering and screening strategies for microbes. More specifically, the authors emphasize the use of multiplex automated genome evolution and CRISPR/Cas systems for high-throughput genome engineering and the application of (lab-on-chip) nanoreactors for high-throughput single-cell or population screening. (Vervoort, Y.; et al. Curr. Opin. Biotechnol.
A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity
Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target.
To facilitate quantitative high-throughput screening campaigns for the discovery, characterization, and structure-activity relationship studies of small-molecule ALDH1A1 inhibitors with cellular activity, Yasgar et al. show the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. The authors demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, the authors establish a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of more than 1000 small molecules (Yasgar, A.; et al. Plos One.
The More the Merrier: High-Throughput Single-Molecule Techniques
The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviors will be observed, representing a multitude of pathways. This realization necessitates that an adequate number of observations are recorded to fully characterize this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput.
This review discusses many developing techniques that address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing the throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments but have also made improvements to ease-of-use, accessibility, and automation of data analysis. (Hill, F. R.; et al. Biochem. Soc. Trans.
Microfluidics and Microbioreactors
Multisensor-Integrated Organs-on-Chips Platform for Automated and Continual In Situ Monitoring of Organoid Behaviors
Organ-on-a-chip systems are miniaturized microfluidic three-dimensional human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body.
Multiple organoid models may be further connected through microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in the precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capabilities are strongly desired for long-term monitoring.
Here, Zhang et al. report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. The authors believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters. (Zhang, Y. S.; et al. Proc. Natl. Acad. Sci. U.S.A.
Microfluidic Screening Reveals Heparan Sulfate Enhances Human Mesenchymal Stem Cell Growth by Modulating Fibroblast Growth Factor-2 Transport
Cost-effective expansion of human mesenchymal stem/stromal cells (hMSCs) remains a key challenge for their widespread clinical deployment. Fibroblast growth factor-2 (FGF-2) is a key hMSC mitogen that is often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF-2, which critically interacts with cell surface heparin sulfate (HS).
Titmarsh et al. assess the interplay of FGF-2 with a heparin sulfate variant (HS8) engineered to bind FGF-2 and potentiate its activity. Bone marrow–derived hMSCs are screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 µg/mL) increases hMSC proliferation and cell numbers after 3 days, with an effect equivalent to FGF-2 (50 ng/mL). In combination, the effects of HS8 and FGF-2 are additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provide insights into modulation of FGF-2 transport by HS8. HS8 treatment induces proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF-2 accumulation, whereas responses to FGF-2 occur primarily in the upstream chambers.
Adding HS8 along with FGF-2, however, maximizes the range of FGF-2 effectiveness. Measurements of FGF-2 in static cultures reveal that this is because HS8 causes increased endogenous FGF-2 production and liberates FGF-2 from the cell surface into the supernatant. HS8 also sustains the levels of supplemented FGF-2 available over 3 days. These results suggest the HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF-2 production and maximizing the effect of supplemented FGF-2. This is an exciting strategy for cost-effective expansion of hMSCs. (Titmarsh, D. M.; et al. Stem Cells Transl. Med.
Analytical Chemistry
Selection of Group-Specific PAE-Binding DNA Aptamers via Rational Designed Target Immobilization and Applications for Ultrasensitive and Highly Selective Detection of PAEs
Phthalic acid esters (PAEs) are ubiquitous in the environment, and some of them are recognized as endocrine disruptors that cause concerns for ecosystem functioning and public health. Because of the diversity of PAEs in the environment, there is a vital need to detect the total concentration of PAEs in a timely and low-cost way. To fulfill this requirement, it is highly desired to obtain group-specific PAE binders that are specific to the basic PAE skeleton.
In this study, for the first time, Han et al. identify the group-specific PAE-binding aptamers via rational designed target immobilization. The two target immobilization strategies are adopted to respectively display either the phthalic ester group or the alkyl chain at the surface of the immobilization matrix. The former enables the rapid enrichment of aptamers after four rounds of selection.
The top 100 sequences are cytosine-rich (44.7%) and differentiate from each other by only one to four nucleotides at limited locations. The top two aptamers all display the nanomolar dissociation constants to both the immobilized target and the free PAEs (DBP, BBP, DEHP). The authors further demonstrate the applications of the aptamers in the development of high-throughput PAE assays and DEHP electrochemical biosensors with exceptional sensitivity (LOD: 10 pM) and selectivity (>105-fold). PAE aptamers targeting one of the most sought after targets thus offers the promise of convenient, low-cost detection of total PAEs. This study also provides insights on the aptamer selection and sensor development of highly hydrophobic small molecules. (Han, Y.; et al. Anal. Chem.
Thiamine Assays: Advances, Challenges, and Caveats
Thiamine (vitamin B1) is essential to the health of all living organisms, and deficiency has long been associated with diseases in animals such as fish, birds, alligators, and domesticated ruminant mammals. Thiamine is also implicated in several human diseases including Alzheimer’s, diabetes, dementia, depression, and, most notably, Wernicke-Korsakoff syndrome and Beriberi disease. Yet highly sensitive and specific detection of thiamine remains an analytical challenge, as pM to nm levels of thiamine need to be detected in environmental and human samples, respectively, various phosphorylated variants need to be discriminated, and rapid on-site detection would be highly desirable. Furthermore, appropriate sample preparation is mandatory, owing to the complexity of the relevant sample matrices including fish tissues, ocean water, and body fluids.
This review has two objectives. First, it provides a thorough overview of analytical techniques published for thiamine detection over the past 15 years. Second, it describes the principles of analytical approaches that are based on biorecognition and may open up new avenues for rapid and high-throughput thiamine analysis. Most notably, periplasmic binding proteins, ribozymes, and aptamers are of particular interest, as they function as bioaffinity recognition elements that can fill an important assay technology gap, owing to the unavailability of thiamine-specific commercial antibodies. Finally, the authors provide brief evaluations of key outcomes of the major assay concepts and suggest how innovative techniques could help develop sensitive and specific thiamine analytical test systems. (Edwards, K. A.; et al.
An Assay System for Point-of-Care Diagnosis of Tuberculosis Using Commercially Manufactured PCB Technology
Rapid advances in clinical technologies, detection sensitivity, and analytical throughput have delivered a significant expansion in our knowledge of prognostic and diagnostic biomarkers in many common infectious diseases, such as tuberculosis (TB). During the past decade, a significant number of approaches to TB diagnosis have been attempted at point-of-pare (PoC), exploiting a large variation of techniques and materials.
In this work, Evans et al. describe an electronics-based enzyme-linked immunosorbent assay (eELISA), using a lab-on-a-printed circuit board (LoPCB) approach, for TB diagnosis based on cytokine detection. The test relies on an electrochemical (amperometric) assay, comprising a high-precision bioinstrumentation board and amperometric sensors, produced exclusively using standard PCB manufacturing processes. Electrochemical detection uses standard Au and Ag electrodes together with a bespoke, low-power, multichannel, portable data-acquisition system.
The authors demonstrate high-performance assay chemistry performed at microfluidic volumes on Au pads directly at the PCB surface with improved limit of detection (~10 pg/mL) over standard colorimetric enzyme-linked immunosorbent assay methods. The assay also is implemented in plasma, showing the utility of the system for medical applications. This work is a significant step toward the development of a low-cost, portable, high-precision diagnostic and monitoring technology, which, once combined with appropriate PCB-based microfluidic networks, will provide complete LoPCB platforms. (Evans, D.; et al. Sci. Rep.
Detection of Enzyme Inhibitors in Crude Natural Extracts Using Droplet-Based Microfluidics Coupled to HPLC
Natural product screening for new bioactive compounds can greatly benefit from low reagents consumption and high-throughput capacity of droplet-based microfluidic systems. However, the creation of large droplet libraries in which each droplet carries a different compound is a challenging task. A possible solution is to use a high-performance liquid chromatograph (HPLC) coupled to a droplet-generating microfluidic device to sequentially encapsulate the eluting compounds.
In this work, Ochoa et al. demonstrate the feasibility of carrying out enzyme-inhibiting assays inside nanoliter droplets with the different components of a natural crude extract after being separated by a coupled HPLC column. In the droplet formation zone, the eluted components are mixed with an enzyme and a fluorogenic substrate that permits it to follow the enzymatic reaction in the presence of each chromatographic peak and identify those inhibiting the enzyme activity. Using a fractal shape channel design and automated image analysis, the authors are able to identify inhibitors of Clostridium perfringens neuraminidase present in a root extract of the Pelargonium sidoides plant. This work demonstrates the feasibility of bioprofiling a natural crude extract after being separated in HPLC using microfluidic droplets online and represents an advance in the miniaturization of natural products screening. (Ochoa, A.; et al. Anal. Chem.
Advances in CRISPR Technology
Enhancing the Throughput and Multiplexing Capabilities of Next-Generation Sequencing for Efficient Implementation of Pooled shRNA and CRISPR Screens
Next-generation sequencing is becoming the method of choice for functional genomic studies that use pooled shRNA or CRISPR libraries. A key challenge in sequencing these mixed-oligo libraries is that they are highly susceptible to hairpin and/or heteroduplex formation. This results in polyclonal, low-quality, and incomplete reads and reduces sequencing throughput. Unfortunately, this challenge is significantly magnified in low- to medium-throughput benchtop sequencers as failed reads that significantly perturb the maximization of sequence coverage and multiplexing capabilities.
Here, Islam et al. report a methodology that can be adapted to maximize the coverage on a benchtop, Ion PGM System for smaller shRNA libraries with high efficiency. This ligation-based, half-shRNA sequencing strategy minimizes failed sequences and is equally amenable to high-throughput sequencers for increased multiplexing. Toward this, the authors also demonstrate that their strategy to reduce heteroduplex formation improves multiplexing capabilities of pooled CRISPR screens using Illumina NextSeq 500. Overall, the method will facilitate sequencing of pooled shRNA or CRISPR libraries from genomic DNA and maximize sequence coverage. (Islam, M.F.; et al. Sci. Rep.
Applications of Genome Editing Tools in Precision Medicine Research
The emergence of genome editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, have enabled researchers to achieve somatic and germline genomic manipulations in cell lines and model organisms. Within a couple of years, genome editing is now being rapidly developed for multiple applications and is widely used in biomedical researches, including creation of disease models with desired genetic mutations, screening in a high-throughput manner for drug resistance genes, and making appropriate editions to genes in vivo for disease treatment. All these applications have been facilitating the development of precision medicine research.
In this review, Shuang et al. describe the use of genome editing technologies for a variety of research and translational applications in the precision medicine field. The authors also highlight some existing limitations or challenges as well as future directions. (Shuang, L.; et al. Yi chuan = Hereditas.
An Inducible CRISPR-ON System for Controllable Gene Activation in Human Pluripotent Stem Cells
Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging, and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed.
Here, Guo et al. construct a CRISPR-ON system to efficiently up-regulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette are knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. The authors show that the dCas9-VPR level can be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, the authors are able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promotes naïve pluripotent gene expression, enhances cell survival and clonogenicity, and enables hESCs to integrate with the inner cell mass of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs. (Guo, J.; et al. Protein Cell.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.