Life Sciences Discovery and Technology Highlights

High-Throughput Biology and Chemistry

Immobilized Enzyme-Based Analytical Tools in the -Omics Era: Recent Advances

Protein analysis is a field under rapid development mainly thanks to technological advances that have granted miniaturization of analytical devices, automation, and higher detection sensitivity. The interest in the field has paralleled the expansion of the -omics era, laying down the bases for the current applications in proteomics and glycomics. Advances in protein sample transformation prior to analysis have led to reduction of sample consumption and contamination, enhancing throughput. Within this context, and thanks to the availability of new high-performing materials and technologies, increasingly more efficient and miniaturized enzyme-based analytical tools have been proposed to overcome shortcomings encountered in the in-solution enzymatic reactions (protein digestion and protein deglycosylation, for proteomics and glycomics, respectively). In this context, immobilized enzyme reactors (IMERs) and IMER-based platforms have been developed as promising approaches toward automation and higher analysis throughput. The scenario is in continuous development as underlined by 34 papers published in the last 5 years. This review encompasses recent advances in the design and operational setups of IMERs purposely developed for the analysis of proteins and glycoproteins. Recently developed dual IMERs, which integrate more than one processing step into a single IMER, and analytical platforms exploiting tandem IMERs are also reviewed and commented on. (Naldi, M., et al. J. Pharm. Biomed. Anal. 2018, 160, 222–237.)

Recent Applications of Click Chemistry in Drug Discovery

Click chemistry has been exploited widely in the past to expedite lead discovery and optimization. Indeed, copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry is a bioorthogonal reaction of widespread utility throughout medicinal chemistry and chemical biology. The authors review recent applications of CuAAC click chemistry to drug discovery based on the literature published since 2013. Furthermore, the authors provide the reader with their expert perspectives on the area, including their outlook on future developments. Click chemistry reactions are an important part of the medicinal chemistry toolbox and offer substantial advantages to medicinal chemists in terms of overcoming the limitations of useful chemical synthesis, increasing throughput, and improving the quality of compound libraries. To explore new chemical spaces for drug-like molecules containing a high degree of structural diversity, it may be useful to merge the diversity-oriented synthesis and “privileged” substructure-based strategy with bioorthogonal reactions using sophisticated automation and flow systems to improve productivity. Large compound libraries obtained in this way should be of great value for the discovery of bioactive compounds and therapeutic agents. (Jiang, X.; et al. Expert Opin. Drug Discov. 2019, 14, 1–11.)

The Potential of Circulating Cell-Free RNA as a Biomarker in Cancer

It is now clear that circulating cell-free ribonucleic acids (ccfRNAs), including messenger RNA (mRNA) and microRNA (miRNA), are potential cancer biomarkers. As ccfmiRNA is relatively more stable than ccfmRNA, research should concentrate on developing novel methods to preserve the stability of ccfmRNA and standardization of the protocol, which includes extraction, detection, and multicenter validation. This literature review concentrates on the potential of ccfRNA being used as a biomarker in cancer, with a special focus on mRNAs and miRNAs. With the advancement of high-throughput technologies such as RNA sequencing, a panel of biomarkers will be used for the diagnosis, prognosis, and therapeutic monitoring of cancer patients. In order to achieve this important target, bioinformatics education to pathologists, scientists, and technologists in molecular diagnostic laboratories is essential. Moreover, the panel of these new ccfRNA biomarkers has to obtain approval or clearance from an authority such as the US Food and Drug Administration (FDA), and the standard of utilizing these new protocols has to be recognized via accreditation exercises. Therefore, there is still a long way to go before an extensive use of ccfRNA biomarkers in cancer patients can be realized. (Cheung, K. W. E.; et al. Expert Rev. Mol. Diagn. 2019, 19, 579–590.)

Microfluidics

A Microfluidic Platform toward Automated Multiplexed In Situ Sequencing

Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into the spatial organization of tissues by enabling single-molecule quantification and submicron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle toward their routine use in laboratories. Here, we demonstrate an approach toward automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with a temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons, and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g., enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features (1) higher detection efficiency, (2) shorter protocol time, and (3) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potential, especially for cancer diagnostics, thus paving the way to rapid and effective therapies. (Maino, N.; et al. Sci Rep. 2019, 9, 3542.)

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, for example, pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, for example, finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites. (Mattes, D. S.; et al. Adv. Mater. 2019, doi: 10.1002/adma.201806656.)

The Usual Suspects 2019: Of Chips, Droplets, Synthesis, and Artificial Cells

Synthetic biology aims to understand fundamental biological processes in more detail than possible for actual living cells. Synthetic biology can combat decomposition and buildup of artificial experimental models under precisely controlled and defined environmental and biochemical conditions. Microfluidic systems can provide the tools to improve and refine existing synthetic systems because they allow control and manipulation of liquids on a micro- and nanoscale. In addition, chip-based approaches are predisposed for synthetic biology applications since they present an opportune technological toolkit capable of fully automated high throughput and content screening under low reagent consumption. This review critically highlights the latest updates in microfluidic cell-free and cell-based protein synthesis as well as the progress on chip-based artificial cells. Even though progress is slow for microfluidic synthetic biology, microfluidic systems are valuable tools for synthetic biology and may one day help to give answers to long asked questions of fundamental cell biology and life itself. (Eilenberger, C., et al.; Micromachines (Basel) 2019, doi: 10.3390/mi10050285.)

Electric and Electrochemical Microfluidic Devices for Cell Analysis

Microfluidic devices are widely used for cell analysis, including applications for single-cell analysis, healthcare, environmental monitoring, and organs-on-a-chip that mimic organs in microfluidics. Moreover, to enable high-throughput cell analysis, real-time monitoring, and noninvasive cell assays, electric and electrochemical systems have been incorporated into microfluidic devices. In this mini-review, Hiramoto et al. summarize recent advances in these systems, with applications from single cells to three-dimensional cultured cells and organs-on-a-chip. First, they summarize microfluidic devices combined with dielectrophoresis, electrophoresis, and electrowetting-on-a-dielectric for cell manipulation. Next, they review electric and electrochemical assays of cells to determine chemical section activity, and oxygen and glucose consumption activity, among other applications. In addition, they discuss recent devices designed for the electric and electrochemical collection of cell components from cells. Finally, they highlight the future directions of research in this field and their application prospects. (Hiramoto, K.; et al. Front. Chem. 2019, 7, 396.)

High-Throughput Single-Cell ChIP-seq Identifies Heterogeneity of Chromatin States in Breast Cancer

Modulation of chromatin structures via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here the authors describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, the authors found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development. (Grosselin, K.; et al. Nat. Genet. 2019, 51, 1060–1066.)

New Dimensions in Cancer and Synthetic Biology

Automated Single-Cell Analysis and Isolation System: A Paradigm Shift in Cell Screening Methods for Biomedicines

We have developed an automated robot that facilitates noninvasive isolation of a single cell with the most favorable properties from arrays containing >10⁵ cells, thus allowing the establishment of new cell screening methods for biomedicines. In this paper, an outline of the proposed automated single-cell analysis and isolation system (hereafter called “single-cell robot”) is reviewed by comparison with a conventional fluorescence-activated cell sorter (FACS). The single-cell robot could perform high-throughput screening for both mammalian cells secreting the highest amount of biomedicines (e.g., Chinese hamster ovary [CHO] cells or hybridomas) and stem cells with the highest pluripotency (e.g., embryonic stem [ES] cells), from a huge number of cell libraries based on the recently proposed concept of “single-cell-based breeding.” The rational screening method for the de novo agonist design could also be performed using yeast cells expressing functional mammalian cytokine receptors (e.g., epidermal growth factor receptor [EGFR], somatostatin G-protein-coupled receptor [SSTR5], and interleukin 5 receptor [IL5R]). Furthermore, the single-cell robot could comprehensively analyze the reaction between olfactory sensory neurons and specific odorants, which will shed light on how odorants are recognized by olfactory receptors. Taken together, these unique features of the proposed single-cell robot will contribute to the high-throughput development of forthcoming biomedicines. (Tatematsu, K.; Kuroda, S.; Adv. Exp. Med. Biol. 2018, 1068, 7–17.)

Anatomical Adaptation—Early Clinical Evidence of Benefit and Future Needs in Lung Cancer

Definitive treatment of locally advanced non-small-cell lung cancer with radiation is challenging. During the course of treatment, anatomical changes such as tumor regression, tumor displacement/deformation, pleural effusion, and/or atelectasis can result in a deviation of the administered radiation dose from the intended prescribed treatment and thereby worsen local control and toxicity. Adaptive radiotherapy can help correct for these changes and can be generally categorized into three philosophical paradigms: (1) maintenance of prescribed dose to the initially defined target volume, (2) dose reduction to healthy organs while maintaining the initial prescribed dose to a regressing tumor volume, or (3) dose escalation to a regressing tumor volume with isotoxicity to healthy organs. Numerous single-institution studies have investigated these methods, and results from large prospective clinical trials will hopefully provide consensus on the method, utility, and efficacy of implementing adaptive radiation therapy (ART) in a clinical setting. Additional development into standardization and automation of the ART workflow, specifically in identifying when ART is warranted and in reducing the manual clinical effort needed to produce an adaptive plan, will be paramount to making ART feasible for the broader radiation therapy community. (Kavanaugh, J.; et al. Semin. Radiat. Oncol. 2019, 29, 274–283.)

Cell Extraction Automation in Single-Cell Surgery Using the Displacement Method

Micromanipulation is the precise in vitro handling and study of individual biological cells, where the smallest error can be disastrous. One such example is the extraction of cellular material from multicellular organisms, such as cells from early-stage embryos. In this paper, Wong and Mills propose automation methods for the extraction and retrieval of individual cells from a multicellular organism in vitro using the displacement method. Computer-controlled syringe pumps and micromanipulators combined with custom computer vision algorithms are used for automated cell extraction and retrieval. Automation feasibility is demonstrated through automated controlled extraction of one or two blastomeres from cleavage-stage embryos. Preliminary proof-of-concept blastomere extraction experiments involving mouse embryos obtained success rates ranging from 72% to 88% for the different extraction tasks: displacement, detection, and retrieval. These automated blastomere extraction experiments demonstrate that automated cell extraction is indeed feasible, but the process may still be improved. To the best of these authors’ knowledge, this paper is the first to report the automation of single-cell extraction from multicellular organisms using the displacement method, and especially for automated blastomere extraction from cleavage-stage embryos. These methods provide a set of tools for moving toward fully automated single-cell surgery procedures. (Wong, C. Y.; Mills, J. K. Biomed Microdevices 2019, 21, 52.)

Next-Generation Sequencing in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is lethal, and the majority of patients present with locally advanced or metastatic disease that is not amenable to cure. Thus, with surgical resection being the only curative modality, it is critical that disease is identified at an earlier stage to allow the appropriate therapy to be applied. Unfortunately, a specific biomarker for early diagnosis has not yet been identified; hence, no screening process exists. Recently, high-throughput screening and next-generation sequencing (NGS) have led to the identification of novel biomarkers for many disease processes, and work has commenced in PDAC. Genomic data generated by NGS not only have the potential to assist clinicians in early diagnosis and screening, especially in high-risk populations, but also may eventually allow the development of personalized treatment programs with targeted therapies, given the large number of gene mutations seen in PDAC. This review introduces the basic concepts of NGS and provides a comprehensive review of the current understanding of genetics in PDAC as related to discoveries made using NGS. (Shen, G. Q.; et al. Pancreas 2019, 48, 739–748.)