Development and Evaluation of a Point-of-Care Test in a Low-Resource Setting with High Rates of Chlamydia trachomatis Urogenital Infections in Fiji.

Rapid and precise detection of Chlamydia trachomatis, the leading global cause of sexually transmitted infections (STI), at the point of care (POC) is required for treatment decisions to prevent transmission and sequelae, including pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and preterm birth. We developed a rapid POC test (POCT), termed LH-POCT, which uses loop-mediated amplification (LAMP) of nucleic acids. We performed a head-to-head comparison with the Cepheid Xpert CT/NG assay using clinician-collected, deidentified paired vaginal samples from a parent study that consecutively enrolled symptomatic and asymptomatic females over 18 years of age from the Ministry of Health and Medical Services Health Centers in Fiji. Samples were processed by the Xpert CT/NG assay and LH-POCT, blinded to the comparator. Discrepant samples were resolved by quantitative PCR. Deidentified clinical data and tests for Trichomonas vaginalis, Candida, and bacterial vaginosis (BV) were provided. There were a total of 353 samples from 327 females. C. trachomatis positivity was 16.7% (59/353), while the prevalence was 16.82% (55/327) after discrepant resolution. Seven discrepant samples resolved to four false negatives, two false positives, and one true positive for the LH-POCT. The sensitivity of the LH-POCT was 93.65% (95% confidence interval [CI], 84.53% to 98.24%), and specificity was 99.31% (95% CI, 97.53% to 99.92%). Discrepant samples clustered among women with vaginal discharge and/or BV. The prototype LH-POCT workflow has excellent performance, meeting many World Health Organization ASSURED criteria for POC tests, including a sample-to-result time of 35 min. Our LH-POCT holds promise for improving clinical practice to prevent and control C. trachomatis STIs in diverse health care settings globally.

Spatiomechanical Modulation of EphB4-Ephrin-B2 Signaling in Neural Stem Cell Differentiation.

Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling.

Single-cell western blotting.

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-µm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

Conjugation of proteins to polymer chains to create multivalent molecules.

The activation of cellular signaling cascades, critical for regulating cell function and fate, often involves changes in the organization of receptors in the cell membrane. Using synthetic multivalent ligands to control the nanoscale organization of cellular receptors into clusters is an attractive approach to elicit desired downstream cellular responses, since multivalent ligands can be significantly more potent than their corresponding monovalent ligands. Synthetic multivalent ligands can serve as both versatile biological tools and potent nanoscale therapeutics, for example in applications to harness them to control stem cell fate in vitro and in vivo. Here we describe the use of recombinant protein expression and bioconjugate chemistry to synthesize multivalent ligands that have the potential to regulate cell signaling in a variety of cell types.

Multivalent ligands control stem cell behaviour in vitro and in vivo.

There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

Engineered cell homing.

One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.

Methods for embryoid body formation: the microwell approach.

Embyroid body (EB) formation is a key step in many embryonic stem cell (ESC) differentiation protocols. The EB mimics the structure of the developing embryo, thereby providing a means of obtaining any cell lineage. Traditionally, the two methods of EB formation are suspension and hanging drop. The suspension method allows ESCs to self-aggregate into EBs in a nonadherent dish. The hanging drop method suspends ESCs on the lid of a dish and EBs form through aggregation at the bottom of the drops. Recently, alternative methods of EB formation have been developed that allow for highly accurate control of EB size and shape, resulting in reproducibly produced homogeneous EBs. This control is potentially useful for directed differentiation, as recent studies have shown that EB size may be a useful determinant of the resulting differentiated cell types. One particular approach to generate homogeneous EBs utilizes nonadhesive microwell structures. The methodology associated with this technique, along with the traditional approaches of suspension and hanging drop, is the focus of this chapter.

Chemical engineering of mesenchymal stem cells to induce a cell rolling response.

Covalently conjugated sialyl Lewis X (SLeX) on the mesenchymal stem cell (MSC) surface through a biotin-streptavidin bridge imparts leukocyte-like rolling characteristics without altering the cell phenotype and the multilineage differentiation potential. We demonstrate that the conjugation of SLeX on the MSC surface is stable, versatile, and induces a robust rolling response on P-selectin coated substrates. These results indicate the potential to increase the targeting efficiency of any cell type to specific tissue.