Abstract
Laboratory Automation and High-Throughput Biology
Design of an Automated Reagent-Dispensing System for Reaction Screening and Validation with DNA-Tagged Substrates
Laboratory automation strategies have vast potential for accelerating discovery processes. They enable higher efficiency and throughput for time-consuming screening procedures and reduce error-prone manual steps. Automating repetitive procedures can, for instance, support chemists in optimizing chemical reactions. Particularly, the technology of DNA-encoded libraries (DELs) may benefit from automation techniques, since translation of chemical reactions to DNA-tagged reactants often requires screening of multiple reaction parameters and evaluation of large numbers of reactants. Here, the authors describe a portable, automated system for reagent dispensing that was designed from open source materials. The system was validated by performing amide coupling of carboxylic acids to DNA-linked amines and a micelle-mediated Povarov reaction to DNA-tagged hexahydropyrroloquinolines. The latter reaction required accurate pipetting of multiple components, including different solvents and a surface-active reagent. Analysis of reactions demonstrated that the robotic system achieved high accuracy comparable to experimentation by an experienced chemist with the potential of higher throughput (Bobers, J. et al., ACS Comb. Sci.
Recent Advances in Robotic Protein Sample Preparation for Clinical Analysis and Other Biomedical Applications
Discovery of new protein biomarker candidates has become a major research goal in the areas of clinical chemistry, analytical chemistry, and biomedicine. These important species constitute the molecular target when it comes to diagnosis, prognosis, and further monitoring of disease. Their analysis, however, requires powerful, selective, and high-throughput sample preparation and product (analyte) characterization approaches. In general, manual sample processing is tedious, complex, and time-consuming, especially when large numbers of samples have to be processed (e.g., in clinical studies). Automation via microtiter-plate platforms involving robotics has brought improvements in high-throughput performance, while comparable or even better precision and repeatability (intraday and interday) were achieved. At the same time, waste production and exposure of laboratory personnel to hazards were reduced. In comprehensive protein analysis workflows (e.g., liquid chromatography–tandem mass spectrometry analysis), sample preparation is an unavoidable step. This review surveys the recent achievements in automation of bottom-up and top-down protein and/or proteomics approaches. Emphasis is put on high-end multiwell-plate robotic platforms developed for clinical analysis and other biomedical applications. The literature from 2013 to date has been covered (Alexovič, M. et al., Clin. Chim. Acta
Microfluidics
Microfluidics in Hemostasis: A Review
Hemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. Because they are leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of hemostatic disorders. The field of microfluidic and lab-on-a-chip (LOC) technologies is rapidly advancing, and the important role of miniaturized diagnostics is becoming more evident in the healthcare system, with particular importance in near-patient testing (NPT) and point-of-care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges that have the knock-on effect of improving healthcare and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of hemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing microchannels are reviewed in the context of application to the field of hemostasis (Panchal, H. J., Molecules
Emerging Trends in Microfluidics-Based Devices
One of the major challenges for scientists and engineers today is to develop technologies for the improvement of human health in both developed and developing countries. The need for cost-effective, high-performance diagnostic techniques is very crucial for providing accessible, affordable, and high-quality healthcare devices. In this context, microfluidic-based devices (MFDs) offer powerful platforms for automation and integration of complex tasks onto a single chip. The distinct advantage of MFDs lies in precise control of the sample quantities and the flow rate of samples and reagents, which enables quantification and detection of analytes with high resolution and sensitivity. With these excellent properties, microfluidics (MFs) have been used for various applications in healthcare, along with other biological and medical areas. This review focuses on the emerging demand for MFs in different fields, such as biomedical diagnostics, environmental analysis, food and agriculture research, and so on, in the past 3 or so years. It also aims to reveal new opportunities in these areas and future prospects of commercial MFDs (Solanki, S. et al., Biotechnol. J.
Genomics
FACT Caught in the Act of Manipulating the Nucleosome
The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as “facilitates chromatin transcription” (FACT), consisting of subunits SPT16 and SSRP1, promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and DNA repair. The mechanism by which FACT causes these opposing outcomes is, however, unknown. Here, Yang et al. report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen–deuterium exchange data. The authors find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A–H2B, allowing FACT–H2A–H2B to dock onto a complex containing DNA and histones H3 and H4. SPT16 binds nucleosomal DNA and tethers H2A–H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding and can assume two conformations, depending on whether a second H2A–H2B dimer is present. The authors’ data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage by facilitating removal of the H2A–H2B dimer, stabilizing intermediate subnucleosomal states, and promoting nucleosome reassembly. The authors’ findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes (Yang, L. et al., Nature
Recent Advances in Genetic Engineering Tools Based on Synthetic Biology
Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. During the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, Jun et al. discuss the current genome-scale engineering technologies used for microbial engineering. Recently developed strategies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, multiplex automated genome engineering (MAGE), promoter engineering, CRISPR-based regulations, and synthetic small regulatory RNA (sRNA)-based knockdown, are considered as powerful tools for genome-scale engineering in microbiological systems. MAGE, which modifies specific nucleotides of the genome sequence, is used as a genome-editing tool. Contrastingly, synthetic sRNA, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) are mainly used to regulate gene expression without modifying the genome sequence. This review introduces the recent genome-scale editing and regulating technologies and their applications in metabolic engineering (Jun, R. et al., J. Microbiol.
A Brief History of Human Disease Genetics
A primary goal of human genetics is to identify DNA sequence variants that influence biomedical traits, particularly those related to the onset and progression of human disease. During the past 25 years, progress in realizing this objective has been transformed by advances in technology, foundational genomic resources, and analytical tools, and by access to vast amounts of genotype and phenotype data. Genetic discoveries have substantially improved our understanding of the mechanisms responsible for many rare and common diseases and driven development of novel preventative and therapeutic strategies. Medical innovation will increasingly focus on delivering care tailored to individual patterns of genetic predisposition (Melina, C. et al., Nature 2020, 577(7789), 179–189).
Automation in COVID-19 Research
Evaluation of Commercial and Automated SARS-CoV-2 IgG and IgA ELISAs Using Coronavirus Disease (COVID-19) Patient Samples
Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. The authors evaluated SARS-CoV-2 immunoglobulin G and A (IgG and IgA) enzyme-linked immunosorbent assays (ELISAs) in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset (Jääskeläinen, A. et al., J. Euro. Surveill., 2020, 25(18), 2000603. doi: 10.2807/1560-7917.ES.2020.25.18.2000603).
Validation of a Chemiluminescent Assay for Specific SARS-CoV-2 Antibody
Objectives: Faced with the coronavirus disease 2019 (COVID-19) pandemic and its impact on the availability and quality of both therapeutic and diagnostic methods, the Belgian authorities have decided to launch a procedure for additional evaluation of the performance of serological tests offered for sale in the national territory. This has been proposed with a double aim: (1) an in-depth verification of the analytical and clinical performances presented by the manufacturer, and (2) an economy of scale in terms of centralized validation for all the laboratories using the tests subject to evaluation. Methods: A retrospective validation study was conducted including the serum of 125 patients to determine the analytical and clinical performances of the LIAISON®SARS-CoV-2 from DiaSorin detecting anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) immunoglobulin G (IgG) and to compare its clinical performance with that of the enzyme-linked immunosorbent assay (ELISA) test from Euroimmun, one of the first commercially available tests allowing the detection of anti-SARS-CoV-2 IgA and IgG. Results: The performances of the LIAISON®SARS-CoV-2 satisfied all the acceptance criteria and provided “real-world” analytical and clinical performances very close to the ones reported by the manufacturer in its insert kit. Comparison between the LIAISON®SARS-CoV-2 and the ELISA method did not reveal any difference between the two techniques in terms of sensitivities and specificities regarding the determination of the IgG. Conclusions: This study reports the validation of the LIAISON®SARS-CoV-2 in detecting IgG antibodies specifically directed against SARS-CoV-2. The analytical and clinical performances are excellent, and the automation of the test offers important rates, ideal for absorbing an extension of testing (Tré-Hardy, M. et al., Clin. Chem. Lab Med.
Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the etiological agent responsible for the 2019–2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19). Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here, Zhenming et al. describe the results of a program that aimed to rapidly discover lead compounds for clinical use by combining structure-assisted drug design, virtual drug screening, and high-throughput screening. This program focused on identifying drug leads that target the main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2. The authors identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, the authors assayed more than 10,000 compounds—including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds—as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration (IC50) values that ranged from 0.67 to 21.4 μM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. The authors’ results demonstrate the efficacy of their screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available (Zhenming, J. et al., Nature
SARS-CoV-2 Infection Virological Diagnosis
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has to be confirmed by virological diagnosis. Multiple diagnostic tests are available without enough perspective on their reliability. Therefore, it is important to choose the most suitable test according to its sensitivity and specificity but also to the stage of the disease. Currently, reverse transcription PCR (RT-PCR) detection of the viral genome in respiratory samples is the most reliable test to confirm the diagnosis of an acute SARS-CoV-2 infection. It has to be done in a Class II biological safety laboratory. It may lack sensitivity, however, particularly in the advanced phase of infection, and this depends closely on the samples' quality. Rapid PCR by cartridge system reduces response times but is not suitable for laboratories with a high throughput of requests. Detection of virus antigens on respiratory samples is a quick and easy-to-use technique; however, it does not have good specificity and sensitivity and cannot be used for diagnosis and patient management. The detection of specific antibodies against SARS-CoV-2 is better used for epidemiological analyses. Research should be encouraged to overcome the limits of the currently available diagnostic tests (Thabet, L. et al., Tunis. Med.
A Fully Automatic Deep Learning System for COVID-19 Diagnostic and Prognostic Analysis
Coronavirus disease 2019 (COVID-19) has spread globally, and medical resources have become insufficient in many regions. Fast diagnosis of COVID-19, and finding high-risk patients with worse prognosis for early prevention and medical resources optimization, are important. Here, Shuo et al. proposed a fully automatic deep learning (DL) system for COVID-19 diagnostic and prognostic analysis by routinely used computed tomography. The authors retrospectively collected 5372 patients with computed tomography images from seven cities or provinces. Firstly, 4106 patients with computed tomography images were used to pre-train the DL system, making it learn lung features. Afterward, 1266 patients (924 with COVID-19, of whom 471 had follow-up for 5+ days; and 342 with other pneumonia) from six cities or provinces were enrolled to train and externally validate the performance of the DL system. In the four external validation sets, the DL system achieved good performance in identifying COVID-19 from other pneumonia [area under the curve (AUC) = 0.87 and 0.88] and viral pneumonia (AUC = 0.86). Moreover, the DL system succeeded in stratifying patients into high-risk and low-risk groups, whose hospital stay times have significant differences (p = 0.013 and 0.014). Without human assistance, the DL system automatically focused on abnormal areas that showed consistent characteristics with reported radiological findings. DL provides a convenient tool for fast screening of COVID-19 and finding potentially high-risk patients, which may be helpful for medical resource optimization and early prevention before patients show severe symptoms (Shuo, W. et al., Eur. Respir. J.,
In Vitro Diagnostic Assays for COVID-19: Recent Advances and Emerging Trends
There have been tremendous advances in in vitro diagnostic (IVD) assays for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main IVD assays used for COVID-19 use real-time reverse transcriptase PCR (RT-PCR) that takes a few hours. But the assay duration has been shortened to 45 min by Cepheid. Of interest is the point-of-care (POC) molecular assay by Abbott that decreased the assay duration to just 5 min. Most molecular tests have been approved by the US Food and Drug Administration (FDA) under emergency use authorization (EUA) and are Conformité Européenne (CE) marked. A wide range of serology immunoassays (IAs) have also been developed that complement the molecular assays for the diagnosis of COVID-19. The most prominent IAs are automated chemiluminescent IA (CLIA), manual enzyme-linked immunosorbent assay (ELISA), and rapid lateral flow IA (LFIA), which detect the immunoglobulin M (IgM) and immunoglobulin G (IgG) produced in persons in response to SARS-CoV-2 infection. The ongoing research efforts and advances in complementary technologies will pave the way to new POC IVD assays in the coming months. The performance of IVD assays needs to be critically evaluated, however, before they are used for the clinical diagnosis of COVID-19 (Vashist, S. K. et al., Diagnostics (Basel)
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.