Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests.

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/µL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free DNA Sequencing Assays.

BACKGROUND: Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing. METHODS: We characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables. RESULTS: Length bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used. CONCLUSIONS: Substantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling.

Tissue biomechanics regulate a wide range of cellular functions, but the influences on epidermal homeostasis and repair remain unclear. Here, we examined the role of extracellular matrix stiffness on human keratinocyte behavior using elastomeric substrates with defined mechanical properties. Increased matrix stiffness beyond normal physiologic levels promoted keratinocyte proliferation but did not alter the ability to self-renew or terminally differentiate. Activation of epidermal growth factor (EGF) signaling mediated the proliferative response to matrix stiffness and depended on focal adhesion assembly and cytoskeletal tension. Comparison of normal skin with keloid scar tissue further revealed an upregulation of EGF signaling within the epidermis of stiffened scar tissue. We conclude that matrix stiffness regulates keratinocyte proliferation independently of changes in cell fate and is mediated by EGF signaling. These findings provide mechanistic insights into how keratinocytes sense and respond to their mechanical environment, and suggest that matrix biomechanics may play a role in the pathogenesis keloid scar formation.

Minor collagens of the skin with not so minor functions.

The structure and function of the skin relies on the complex expression pattern and organisation of extracellular matrix macromolecules, of which collagens are a principal component. The fibrillar collagens, types I and III, constitute over 90% of the collagen content within the skin and are the major determinants of the strength and stiffness of the tissue. However, the minor collagens also play a crucial regulatory role in a variety of processes, including cell anchorage, matrix assembly, and growth factor signalling. In this article, we review the expression patterns, key functions and involvement in disease pathogenesis of the minor collagens found in the skin. While it is clear that the minor collagens are important mediators of normal tissue function, homeostasis and repair, further insight into the molecular level structure and activity of these proteins is required for translation into clinical therapies.

Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces.

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings.

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/µL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes.

The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.

Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft and the mechanism of wound healing.

Epidermal grafting for wound healing involves the transfer of the epidermis from a healthy location to cover a wound. The structural difference of the epidermal graft in comparison to the split-thickness skin graft and full-thickness skin graft contributes to the mechanism of effect. While skin grafting is an epidermal transfer, little is known about the precise mechanism of wound healing by epidermal graft. This paper aims to explore the evolution of the epidermal graft harvesting system over the last five decades, the structural advantages of epidermal graft for wound healing and the current hypotheses on the mechanism of wound healing by epidermal graft. Three mechanisms are proposed: keratinocyte activation, growth factor secretion and reepithelialisation from the wound edge. We evaluate and explain how these processes work and integrate to promote wound healing based on the current in vivo and in vitro evidence. We also review the ongoing clinical trials evaluating the efficacy of epidermal graft for wound healing. The epidermal graft is a promising alternative to the more invasive conventional surgical techniques as it is simple, less expensive and reduces the surgical burden for patients in need of wound coverage.

Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells.

Human epidermal stem cells express high levels of ß1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1(+) cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1(+) cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1(+) cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of ß1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and ß1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment.

Adipogenic Differentiation of hMSCs is Mediated by Recruitment of IGF-1r Onto the Primary Cilium Associated With Cilia Elongation.

Primary cilia are single non-motile organelles that provide a highly regulated compartment into which specific proteins are trafficked as a critical part of various signaling pathways. The absence of primary cilia has been shown to prevent differentiation of human mesenchymal stem cells (hMSCs). Changes in primary cilia length are crucial for regulating signaling events; however it is not known how alterations in cilia structure relate to differentiation. This study tested the hypothesis that changes in primary cilia structure are required for stem cell differentiation. hMSCs expressed primary cilia that were labeled with acetylated alpha tubulin and visualized by confocal microscopy. Chemically induced differentiation resulted in lineage specific changes in cilia length and prevalence which were independent of cell cycle. In particular, adipogenic differentiation resulted in cilia elongation associated with the presence of dexamethasone, while insulin had an inhibitory effect on cilia length. Over a 7-day time course, adipogenic differentiation media resulted in cilia elongation within 2 days followed by increased nuclear PPARγ levels; an early marker of adipogenesis. Cilia elongation was associated with increased trafficking of insulin-like growth factor-1 receptor ß (IGF-1Rß) into the cilium. This was reversed on inhibition of elongation by IFT-88 siRNA transfection, which also decreased nuclear PPARγ. This is the first study to show that adipogenic differentiation requires primary cilia elongation associated with the recruitment of IGF-1Rß onto the cilium. This study may lead to the development of cilia-targeted therapies for controlling adipogenic differentiation and associated conditions such as obesity.

"Paper Machine" for Molecular Diagnostics.

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.

Integrin-mediated adhesion and mechano-sensing in cutaneous wound healing.

Integrin receptors mediate the interactions between cells and the extracellular matrix. They not only provide anchorage and a physical linkage to the matrix but also participate in cell signaling and the regulation of diverse cellular functions. In the epidermis of the skin, integrins are essential for tissue structure and integrity, and, under normal homeostatic conditions, the ß1 subunit specifically controls the balance between proliferation and terminal differentiation. Integrin expression can also dynamically respond to changes in the cell's environment, and integrin-mediated adhesion is required for keratinocyte migration and re-epithelialization during wound repair. Importantly, integrins participate in keratinocyte mechanotransduction and could potentially regulate cell behavior within the altered mechanical microenvironment of a wound. While the complete functions of integrin receptors in cutaneous wound healing have yet to be determined, recent evidence suggests that cell-matrix interactions are perturbed in chronic and non-healing wounds. Integrins may therefore be a potential therapeutic target for improving wound repair and tissue regeneration.

Nucleocytoplasmic shuttling: a common theme in mechanotransduction.

Cells sense their mechanical and physical environment through diverse mechanisms, and these interactions specify a wide range of responses including growth, survival, migration and differentiation. Although much work has focused on dissecting the adhesive and structural components of the cell responsible for transducing external mechanical forces into biochemical signalling cascades, only recently have studies begun to examine how mechanical signals are transmitted to the nucleus and activate specific gene expression programmes. One necessary step in these processes is the transport of signalling molecules from the cytoplasm to the nucleus. The SRF (serum-response factor) and YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif) pathways are known mediators of this process in multiple cell types, including mesenchymal stem cells, keratinocytes, mammary epithelial cells and smooth muscle cells. In addition, recent evidence suggests a potential role for ß-catenin and Smad signalling in mechanotransduction, but further mechanistic studies are needed to prove this hypothesis. As a model system, the epidermis of the skin is one tissue in which nucleocytoplasmic shuttling mediates cellular mechanosensing and is essential for tissue development, homoeostasis and repair. We propose that nuclear translocation is a common element of mechanotransduction conserved across multiple cell types and tissues.

Biomechanical Activation of Keloid Fibroblasts Promotes Lysosomal Remodeling and Exocytosis.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. Although biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fiber formation, and focal adhesion assembly. Elevated actomyosin contractility in KDFs disrupted the normal mechanical regulation of extracellular matrix deposition and conferred resistance on myosin inhibitors. Transcriptional profiling identified mechanically regulated pathways in normal dermal fibroblasts and KDFs, including the actin cytoskeleton, Hippo signaling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on actomyosin contractility. However, KDFs displayed marked changes in lysosome organization and an increase in lysosomal exocytosis, which was mediated by actomyosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to extracellular matrix mechanics and promotes lysosomal exocytosis.

Extracellular-matrix tethering regulates stem-cell fate.

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

Biosensors for the detection of waterborne pathogens.

Waterborne bacterial, viral and parasitic pathogens are a global health concern and their rapid and specific detection in contaminated potable water is of utmost importance. Biosensors using a variety of biorecognition molecules and transduction methodologies have been reported, and have the potential to enable highly sensitive detection of the analyte of interest in a short time with high specificity. However, there are several obstacles to the detection of waterborne pathogens-they tend to be present at very low concentrations in the environment and environmental samples contain numerous inhibitors of enzymatic reactions and interfering organisms and particulates. Here we present a review of the current state of biosensor technology with regard to the improvements needed over standard detection methods and the challenges presented by real environmental samples. Further, we identify future areas of focus necessary to realize novel detection devices capable of supplanting the gold standards of today.

Micro-total analysis system for virus detection: microfluidic pre-concentration coupled to liposome-based detection.

An integrated microfluidic biosensor is presented that combines sample pre-concentration and liposome-based signal amplification for the detection of enteric viruses present in environmental water samples. This microfluidic approach overcomes the challenges of long assay times of cell culture-based methods and the need to extensively process water samples to eliminate inhibitors for PCR-based methods. Here, viruses are detected using an immunoassay sandwich approach with the reporting antibodies tagged to liposomes. Described is the development of the integrated device for the detection of environmentally relevant viruses using feline calicivirus (FCV) as a model organism for human norovirus. In situ fabricated nanoporous membranes in glass microchannels were used in conjunction with electric fields to achieve pre-concentration of virus-liposome complexes and therefore enhance the antibody-virus binding efficiency. The concentrated complexes were eluted to a detection region downstream where captured liposomes were lysed to release fluorescent dye molecules that were then quantified using image processing. This system was compared to an optimized electrochemical liposome-based microfluidic biosensor without pre-concentration. The limit of detection of FCV of the integrated device was at 1.6 × 10(5) PFU/mL, an order of magnitude lower than that obtained using the microfluidic biosensor without pre-concentration. This significant improvement is a key step toward the goal of using this integrated device as an early screening system for viruses in environmental water samples.

Fabrication of Human Skin Equivalents Using Decellularized Extracellular Matrix.

There is a growing demand for in vitro models of human tissues that recapitulate the complex structures and functions found in vivo, and the biomaterials that support these physiologically relevant models are essential underpinning technologies. Here, we present an optimized protocol for generating human skin equivalents (HSEs) using a dermal matrix isolated from decellularized porcine skin. The decellularized extracellular matrix (dECM) contains a complex mixture of fibrillar collagens and matrisomal proteins that mimic native skin and can be produced in large quantities. The procedure for decellularization, digestion, and solubilization of the dECM is described in detail. In addition, we provide instructions for how to construct a three-dimensional HSE model using the dECM as the dermal support matrix for human keratinocytes and dermal fibroblasts. Recent studies from our laboratory have shown that HSEs generated using porcine dECM display improved epidermal differentiation and stratification compared to existing protocols using type I collagen gels. Thus, dECM-based biomaterials are a useful tool for replicating human skin physiology in vitro and developing advanced human skin models for therapeutic discovery and testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of decellularized extracellular matrix from porcine skin Basic Protocol 2: Generation of human skin equivalents.

Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function.

Adhesive cues from the extracellular matrix (ECM) specify the size and shape of the nucleus via mechanical forces transmitted through the cytoskeleton. However, the effects of these biophysical stimuli on internal nuclear architecture and cellular responses remain poorly understood. This study investigates the direct impact of ECM adhesion on nucleolar remodeling in human keratinocytes using micropatterned substrates. Limited adhesion on small micropatterns promotes fusion of nucleoli, alongside a reduction in nuclear volume and condensation of heterochromatin. These changes in nucleolar architecture are mediated by altered chromatin biomechanics and depend on integration of the nucleus with the actin cytoskeleton. Functionally, nucleolar remodeling regulates ribogenesis and protein synthesis in keratinocytes and is associated with specific transcriptional changes in ribogenesis genes. Together, these findings demonstrate that cell shape and nuclear morphology control nucleolar structure and function and implicate the nucleolus as a key mechano-sensing element within the cell.