Use of saliva-based qPCR diagnostics for the accurate, rapid, and inexpensive detection of strep throat.

OBJECTIVES: Outpatient health care facilities are essential for quickly diagnosing common infectious diseases such as bacterial and viral pharyngitis. The only form of pharyngitis requiring antibiotics is strep throat (ST); however, antibiotic prescription rates are much higher than ST prevalence, suggesting antibiotics are being inappropriately prescribed. Current rapid ST diagnostics may be contributing to this problem due to the low sensitivity and variable specificity of these tests. It is best practice to verify a negative ST diagnosis with a group A Streptococcus (GAS) culture, but many clinics do not perform this test due to the additional cost and 24-72 h required to obtain results. This indicates there is great need for more accurate rapid diagnostic tools in outpatient facilities. We hypothesized that next generation qPCR technology could be adapted to detect GAS DNA from saliva samples (instead of the traditional throat swab) by creating a simple, fast, and inexpensive protocol. METHODS: Saliva specimens collected from patients at James Madison University Health Center were used to test the effectiveness of our Chelex 100-based rapid DNA extraction method, followed by a fast protocol developed for the Open qPCR machine to accurately detect ST. RESULTS: Our final saliva processing and qPCR protocol required no specialized training to perform and was able to detect ST with 100 % sensitivity and 100 % specificity (n=102) in 22-26 min, costing only $1.12 per sample. CONCLUSIONS: Saliva can be rapidly analyzed via qPCR for the accurate and inexpensive detection of ST.

Tissue-Like 3D Standard and Protocols for Microscope Quality Management.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

Individual cells generate their own self-reinforcing contact guidance cues through local matrix fiber remodeling.

Directed cell migration arises from cells following a microenvironmental gradient (e.g. of a chemokine) or polarizing feature (e.g. a linear structure). However cells not only follow, but in many cases, also generate directionality cues by modifying their microenvironment. This bi-directional relationship is seen in the alignment of extracellular matrix (ECM) fibers ahead of invading cell masses. The forces generated by many migrating cells cause fiber alignment, which in turn promotes further migration in the direction of fiber alignment via contact guidance and durotaxis. While this positive-feedback relationship has been widely described for cells invading en masse, single cells are also able to align ECM fibers, as well as respond to contact guidance and durotaxis cues, and should therefore exhibit the same relationship. In this study, we directly tested this hypothesis by studying the migration persistence of individual HT-1080 fibrosarcoma cells migrating in photocrosslinked collagen matrices with limited remodeling potential. Our results demonstrate that this positive-feedback relationship is indeed a fundamental aspect of cell migration in fibrillar environments. We observed that the cells' inability to align and condense fibers resulted in a decrease in persistence relative to cells in native collagen matrices and even relative to isotropic (glass) substrates. Further experiments involving 2D collagen and electrospun polymer scaffolds suggest that substrates composed of rigid, randomly oriented fibers reduce cells' ability to follow another directionality cue by forcing them to meander to follow the available adhesive area (i.e. fibers). Finally, our results demonstrate that the bi-directional relationship between cell remodeling and migration is not a "dimensionality" effect, but a fundamental effect of fibrous substrate structure.

A Review of the Scientific Rigor, Reproducibility, and Transparency Studies Conducted by the ABRF Research Groups.

Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and they have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications.

Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

Contact guidance-cell polarization by anisotropic substrate features-is integral to numerous physiological processes; however the complexities of its regulation are only beginning to be discovered. In particular, cells polarize to anisotropic features under non-muscle myosin II (MII) inhibition, despite MII ordinarily being essential for polarized cell migration. Here, we investigate the ability of cells to sense and respond to fiber alignment in the absence of MII activity. We find that contact guidance is determined at the level of individual protrusions, which are individually guided by local fiber orientation, independent of MII. Protrusion stability and persistence are functions of adhesion lifetime, which depends on fiber orientation. Under MII inhibition, adhesion lifetime no longer depends on fiber orientation; however the ability of protrusions to form closely spaced adhesions sequentially without having to skip over gaps in adhesive area, biases protrusion formation along fibers. The co-alignment of multiple protrusions polarizes the entire cell; if the fibers are not aligned, contact guidance of individual protrusions still occurs, but does not produce overall cell polarization. These results describe how aligned features polarize a cell independently of MII and demonstrate how cellular contact guidance is built on the local alignment of adhesions and individual protrusions.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Matrix microarchitecture and myosin II determine adhesion in 3D matrices.

BACKGROUND: Reports of adhesions in cells growing in 3D vary widely-from nonexistent to very large and elongated-and are often in apparent conflict, due largely to our minimal understanding of the underlying mechanisms that determine 3D cell phenotype. We address this problem directly by systematically identifying mechanisms that determine adhesion in 3D matrices and, from our observations, develop principles widely applicable across 2D and 3D substrates. RESULTS: We demonstrate that nonmuscle myosin II activity guides adhesion phenotype in 3D as it does in 2D; however, in contrast to 2D, decreasing bulk matrix stiffness does not necessarily inhibit the formation of elongated adhesions. Even in soft 3D matrices, cells can form large adhesions in areas with appropriate local matrix fiber alignment. We further show that fiber orientation, apart from influencing local stiffness, modulates the available adhesive area and thereby determines adhesion size. CONCLUSIONS: Thus adhesion in 3D is determined by both myosin activity and the immediate microenvironment of each adhesion, as defined by the local matrix architecture. Important parameters include not only the resistance of the fiber to pulling (i.e., stiffness) but also the orientation and diameter of the fiber itself. These principles not only clarify conflicts in the literature and point to adhesion modulating factors other than stiffness, but also have important implications for tissue engineering and studies of tumor cell invasion.

Fluorescence fluctuation approaches to the study of adhesion and signaling.

Cell-matrix adhesions are large, multimolecular complexes through which cells sense and respond to their environment. They also mediate migration by serving as traction points and signaling centers and allow the cell to modify the surroucnding tissue. Due to their fundamental role in cell behavior, adhesions are germane to nearly all major human health pathologies. However, adhesions are extremely complex and dynamic structures that include over 100 known interacting proteins and operate over multiple space (nm-µm) and time (ms-min) regimes. Fluorescence fluctuation techniques are well suited for studying adhesions. These methods are sensitive over a large spatiotemporal range and provide a wealth of information including molecular transport dynamics, interactions, and stoichiometry from a single time series. Earlier chapters in this volume have provided the theoretical background, instrumentation, and analysis algorithms for these techniques. In this chapter, we discuss their implementation in living cells to study adhesions in migrating cells. Although each technique and application has its own unique instrumentation and analysis requirements, we provide general guidelines for sample preparation, selection of imaging instrumentation, and optimization of data acquisition and analysis parameters. Finally, we review several recent studies that implement these techniques in the study of adhesions.

Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells.

Elevated levels of tissue crosslinking are associated with numerous diseases (cancer stroma, organ fibrosis), and also eliminate the otherwise remarkable clinical successes of tissue-derived scaffolds, instead eliciting a foreign body reaction. Nevertheless, it is not well understood how the initial physical and biochemical properties of cellular microenvironments, stem cell niches, or of 3D tissue scaffolds guide the assembly and remodeling of new extracellular matrix (ECM) that is ultimately sensed by cells. Here, we incorporated FRET-based mechanical strain sensors, either into cell-derived ECM scaffolds or into the fibronectin (Fn) matrix assembled by reseeded fibroblasts, and demonstrated the following. Cell-generated tensile forces change the conformation of Fn in both 3D scaffolds and new matrix over time. The time course by which new matrix fibers are stretched by reseeded cells is accelerated by scaffold crosslinking. Importantly, stretching Fn fibers increases their elastic modulus (rigidity) and alters their biochemical display. Regulated by Fn fiber unfolding, more soluble Fn binds to the native than to the crosslinked scaffolds. Additionally, matrix assembly of fibroblasts is decreased by scaffold crosslinking. Taken together, scaffold crosslinking has a multifactorial impact on the microenvironment that reseeded cells assemble and respond to, with far-reaching implications for tissue engineering and disease physiology.

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites.

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Optimization strategies for electrospun silk fibroin tissue engineering scaffolds.

As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well-established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSCs) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSCs showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds.

Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response.

While the mechanical properties of a substrate or engineered scaffold can govern numerous aspects of cell behavior, cells quickly start to assemble their own matrix and will ultimately respond to their self-made extracellular matrix (ECM) microenvironments. Using fluorescence resonance energy transfer (FRET), we detected major changes in the conformation of a constituent ECM protein, fibronectin (Fn), as cells fabricated a thick three-dimensional (3D) matrix over the course of three days. These data provide the first evidence that matrix maturation occurs and that aging is associated with increased stretching of fibronectin fibrils, which leads to at least partial unfolding of the secondary structure of individual protein modules. A comparison of the conformations of Fn in these 3D matrices with those constructed by cells on rigid and flexible polyacrylamide surfaces suggests that cells in maturing matrices experience a microenviroment of gradually increasing rigidity. In addition, further matrix stiffening is caused by active Fn fiber alignment parallel to the contractile axis of the elongated fibroblasts, a cell-driven effect previously described for other fibrillar matrices. The fibroblasts, therefore, not only cause matrix unfolding, but reciprocally respond to the altered Fn matrix properties by up-regulating their own rigidity response. Consequently, our data demonstrate for the first time that a matured and aged matrix has distinctly different physical and biochemical properties compared to a newly assembled matrix. This might allow cells to specifically recognise the age of a matrix.

Force-induced unfolding of fibronectin in the extracellular matrix of living cells.

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.