Essence determines phenomenon: Assaying the material properties of biological condensates.

Intracellular spaces are partitioned into separate compartments to ensure that numerous biochemical reactions and cellular functions take place in a spatiotemporally controlled manner. Biomacromolecules including proteins and RNAs undergo liquid-liquid phase separation and subsequent phase transition to form biological condensates with diverse material states. The material/physical properties of biological condensates are crucial for fulfilling their distinct physiological functions, and abnormal material properties can cause deleterious effects under pathological conditions. Here, we review recent studies showing the role of the material properties of biological condensates in their physiological functions. We also summarize several classic methods as well as newly emerging techniques for characterization and/or measurement of the material properties of biological condensates.

Application of lateral flow and microfluidic bio-assay and biosensing towards identification of DNA-methylation and cancer detection: Recent progress and challenges in biomedicine.

DNA methylation is an important epigenetic alteration that results from the covalent transfer of a methyl group to the fifth carbon of a cytosine residue in CpG dinucleotides by DNA methyltransferase. This modification mostly happens in the promoter region and the first exon of most genes and suppresses gene expression. Therefore, aberrant DNA methylation cause tumor progression, metastasis, and resistance to current anti-cancer therapies. So, the detection of DNA methylation is an important issue in diagnosis and therapy of most diseases. Conventional methods for the assay of DNA methylation and activity of DNA methyltransferases are time consuming. So, we need to multiplex operations and expensive instrumentation. To overcome the limitations of conventional methods, new methods such as microfluidic platforms and lateral flow tests have been developed to evaluate DNA methylation. The microfluidic tests are based on optical and electrical biosensing. These tests able us to can analyze DNA methylation with high efficiency and sensitivity without the need for expensive equipment and skilled people. Lateral flow strip tests are another type of rapid, simple, and sensitive test with advanced technology used to assess DNA methylation. Lateral flow strip tests are based on optical biosensors. This review attempts to evaluate new methods for assessing DNA extraction, DNA methylation and DNA methyltransferase activity as well as recent developments in microfluidic technology application for bisulfite treatment and restriction enzyme (bisulfite free), and lateral flow relying on their application in the field of recognition of DNA methylation in blood and body fluids. Also, the advantages and disadvantages of each test are reviewed. Finally, future prospects for the development of the microfluidics biodevices for the detection of DNA methylation is briefly discussed.

Multiplex bioassaying of cancer proteins and biomacromolecules: Nanotechnological, structural and technical perspectives.

Since the specific proteins (carbohydrate antigens, ligands and interleukins) get raised up in body tissue or fluids in cancer cases, early detection of them will provide an effective treatment and survival rate. Sensitive and accurate determination of multiple cancer proteins can be engaged in chorus by simultaneous/multiplex detection in the biomedical fields. Bioassaying technology is one of the non-invasive, high-sensitive, and economical methods. Currently, extensive application of nanomaterial (biocompatible polymers, metallic and metal oxide) in bioassays resulted in ultra-high sensitive and selective diagnosis. This review article focuses on types of multiplex bioassays for delicate and specific determination of cancer proteins for diagnostic aims. It also covers two modes of multiplex bioassays as multi labeled bioassays and spatially-separated test zones (multi-electrode mode). In this review, the nanotechnological, structural, and technical perspectives in the multiplex analysis of cancer proteins were discussed. Finally, the use of different types of nanomaterials, polysaccharides, biopolymers and their advantages in signal amplification are discussed.

Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics.

Antibody-based therapeutic classes are evolving from monoclonal antibodies to antibody derivatives with complex structures to achieve advanced therapeutic effect. These antibody derivatives may contain multiple functional domains and are often vulnerable to in vivo biotransformation. Understanding the pharmacokinetics of these antibody derivatives requires a sophisticated bioanalytical approach to carefully characterize the whole drug and each functional domain with respect to quantity, functionality enabled by biotransformation, and corresponding immune responses. Ligand binding assays and liquid chromatography-mass spectrometry assays are predominantly used in bioanalytical support of monoclonal antibodies and are continuously used for antibody derivatives such as antibody drug conjugate and bispecific antibodies. However, they become increasingly cumbersome in coping with increased complexity of drug modality and associated biotransformation. In this mini-review, we examined the current pharmacokinetic assays in the literature for antibody drug conjugate and bispecific antibodies, and presented our view of promising bioanalytical technologies to address the distinct bioanalytical needs of complex modalities.

Transferability study of the BINACLE (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity.

The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the "binding and cleavage" (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market. Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products.

Development of a Highly Specific Anti-drug Antibody Assay in Support of a Nanoparticle-based Therapeutic.

PEGylated biotherapeutics can elicit anti-PEG (polyethylene glycol) immune responses in patients treated with this category of drugs. While anti-PEG antibody assays for this class of biotherapeutics have become a common element of the clinical immunogenicity testing strategy, the overall antibody incidence induced by the nanoparticle (NP) delivery system (such as ACCURINS®) has not been fully studied to date. To support the immunogenicity assessment of one of Pfizer's NP-based therapeutics, consisting of gedatolisib (GEDA) encapsulated in ACCURINS® (GEDA-NP), we developed an anti-GEDA-NP antibody (ADA) assay on the MSD platform for the detection of GEDA-NP induced ADA in human serum. The focus of our strategy was on developing a clinically relevant ADA assay and systematically addressing assay interference through rigorous assay optimization. Our efforts led to a fit-for-purpose assay for the detection of anti-GEDA-NP ADA in serum samples obtained from breast cancer patients. Results from method qualification indicated robust assay performance, as highlighted by inter and intra-assay precision within 25% CV for all controls, and reproducible response profiles across multiple runs during the assessment of assay cut points with breast cancer samples. The assay sensitivity was between 4.3 ng/mL and 123 ng/mL for surrogate positive controls of IgG and IgM isotypes, respectively. Additionally, assay interference from nonspecific matrix proteins and circulating drug was addressed, which ensured accurate assessment of ADA incidence that can be attributed to GEDA-NP.

Agrochemicals with estrogenic endocrine disrupting properties: Lessons Learned?

Many agrochemicals have endocrine disrupting properties. A subset of these chemicals is characterized as "estrogenic". In this review, we describe several distinct ways that chemicals used in crop production can affect estrogen signaling. Using three agrochemicals as examples (DDT, endosulfan, and atrazine), we illustrate how screening tests such as the US EPA's EDSP Tier 1 assays can be used as a first-pass approach to evaluate agrochemicals for endocrine activity. We then apply the "Key Characteristics" approach to illustrate how chemicals like DDT can be evaluated, together with the World Health Organization's definition of an endocrine disruptor, to identify data gaps. We conclude by describing important issues that must be addressed in the evaluation and regulation of hormonally active agrochemicals including mixture effects, efforts to reduce vertebrate animal use, chemical prioritization, and improvements in hazard, exposure, and risk assessments.

High-Throughput Fluorescence Assays for Ion Channels and GPCRs.

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.

Thyroid disrupting chemicals and developmental neurotoxicity - New tools and approaches to evaluate hormone action.

It is well documented that thyroid hormone (TH) action is critical for normal brain development and is mediated by both nuclear and extranuclear pathways. Given this dependence, the impact of environmental endocrine disrupting chemicals that interfere with thyroid signaling is a major concern with direct implications for children's health. However, identifying thyroid disrupting chemicals in vivo is primarily reliant on serum thyroxine (T4) measurements within greater developmental and reproductive toxicity assessments. These studies do not examine known TH-dependent phenotypes in parallel, which complicates chemical evaluation. Additionally, there exist no recommendations regarding what degree of serum T4 dysfunction is adverse, and little consideration is given to quantifying TH action within the developing brain. This review summarizes current testing strategies in rodent models and discusses new approaches for evaluating the developmental neurotoxicity of thyroid disrupting chemicals. This includes assays to identify adverse cellular effects of the brain by both immunohistochemistry and gene expression, which would compliment serum T4 measures. While additional experiments are needed to test the full utility of these approaches, incorporation of these cellular and molecular assays could enhance chemical evaluation in the regulatory arena.

Microfluidics-Implemented Biochemical Assays: From the Perspective of Readout.

Over the past decades, microfluidics has emerged as an increasingly important tool to perform biochemical assays for diagnosis and healthcare. The precise fluid control and molecule manipulation within microfluidics greatly contribute to developing assays with simplicity and convenience. The advantages of microfluidics, including decreased consumption of reagents and samples, lower operating and analysis time, much lower cost, and higher integration and automation over traditional systems, offer a great platform to meet the needs of point-of-care applications. In this Review, versatile strategies are outlined and recent advances in microfluidics-implemented assays are discussed from the perspective of readout, because a convenient and straightforward readout is what a biochemical assay requires and the end user desires. Functions and properties arising from each readout are reviewed and the advantages and limitations of each readout are discussed together with current challenges and future perspectives.

Perspective on CETSA Literature: Toward More Quantitative Data Interpretation.

The cellular thermal shift assay (CETSA) was introduced in 2013 to investigate drug-target engagement inside live cells and tissues. As with all thermal shift assays, the response measured by CETSA is not simply governed by ligand affinity to the investigated target protein, but the thermodynamics and kinetics of ligand binding and protein unfolding also contribute to the observed protein stabilization. This limitation is commonly neglected in current applications of the method to validate the target of small-molecule probes. Instead, there is an eagerness to make direct comparisons of CETSA measurements with functional and phenotypic readouts from cells at 37 °C. Here, we present a perspective of the early CETSA literature and put the accumulated data into a quantitative context. The analysis includes annotation of ~270 peer-reviewed papers, the majority of which do not consider the underlying biophysical basis of CETSA. We also detail what future technology developments are needed to enable CETSA-based optimization of structure-activity relationships and more appropriate comparisons of these data with functional or phenotypic responses. Finally, we describe ongoing developments in assay formats that allow for CETSA measurements at single-cell resolution, with the aspiration to allow differentiation in cellular target engagement between cells in co-cultures and more complex models, such as organoids and potentially even tissue.

Recent development of portable imaging platforms for cell-based assays.

Cell-based assays including both cell identification assays and cell functional assays are broadly used in life science research and for medical applications. Traditionally, cell-based assays are performed in professional laboratories. Although such an approach is reliable, it significantly limits the accessibility of cell-based assays by many researchers without specialized cell imaging facilities and does not permit on-site use at most clinics. Over recent years, growing efforts have been made to overcome this limitation by developing various portable cell imaging platforms. Herein, we provide a timely review on the recent development of portable imaging systems and their biomedical applications. We describe the respective applications of three main classes of portable imaging technologies including miniaturized microscope, lens-free imaging device and smartphone-based microscope for cell-based assays. We conclude by discussing our views on the opportunities, problems and future directions of this emerging field.

Alternatives to current flow cytometry data analysis for clinical and research studies.

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment.

Hydroxyvitamin D assays: An historical perspective from DEQAS.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

Lessons Learned: Transfer of the High-Definition Circulating Tumor Cell Assay Platform to Development as a Commercialized Clinical Assay Platform.

Planning and transfer of a new technology platform developed in an academic setting to a start-up company for medical diagnostic product development may appear daunting and costly in terms of complexity, time, and resources. In this review we outline the key steps taken and lessons learned when a technology platform developed in an academic setting was transferred to a start-up company for medical diagnostic product development in the interest of elucidating development toolkits for academic groups and small start-up companies starting on the path to commercialization and regulatory approval.

Toxicogenomics in Environmental Science.

This chapter reviews the current knowledge and recent progress in the field of environmental, aquatic ecotoxicogenomics with a focus on transcriptomic methods. In ecotoxicogenomics the omics technologies are applied for the detection and assessment of adverse effects in the environment, and thus are to be distinguished from omics used in human toxicology [Snape et al., Aquat Toxicol 67:143-154, 2004]. Transcriptomic methods in ecotoxicology are applied to gain a mechanistic understanding of toxic effects on organisms or populations, and thus aim to bridge the gap between cause and effect. A worthwhile effect-based interpretation of stressor induced changes on the transcriptome is based on the principle of phenotypic-anchoring [Paules, Environ Health Perspect 111:A338-A339, 2003]. Thereby, changes on the transcriptomic level can only be identified as effects if they are clearly linked to a specific stressor-induced effect on the macroscopic level. By integrating those macroscopic and transcriptomic effects, conclusions on the effect-inducing type of the stressor can be drawn. Stressor-specific effects on the transcriptomic level can be identified as stressor-specific induced pathways, transcriptomic patterns, or stressors-specific genetic biomarkers. In this chapter, examples of the combined application of macroscopic and transcriptional effects for the identification of environmental stressors, such as aquatic pollutants, are given and discussed. By means of these examples, challenges on the way to a standardized application of transcriptomics in ecotoxicology are discussed. This is also done against the background of the application of transcriptomic methods in environmental regulation such as the EU regulation Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Workshop Report: Crystal City VI-Bioanalytical Method Validation for Biomarkers.

With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration's (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.