Forced Intercalation (FIT)-Aptamers.

Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.

Morphogens enable interacting supracellular phases that generate organ architecture.

During vertebrate organogenesis, increases in morphological complexity are tightly coupled to morphogen expression. In this work, we studied how morphogens influence self-organizing processes at the collective or "supra"-cellular scale in avian skin. We made physical measurements across length scales, which revealed morphogen-enabled material property differences that were amplified at supracellular scales in comparison to cellular scales. At the supracellular scale, we found that fibroblast growth factor (FGF) promoted "solidification" of tissues, whereas bone morphogenetic protein (BMP) promoted fluidity and enhanced mechanical activity. Together, these effects created basement membrane-less compartments within mesenchymal tissue that were mechanically primed to drive avian skin tissue budding. Understanding this multiscale process requires the ability to distinguish between proximal effects of morphogens that occur at the cellular scale and their functional effects, which emerge at the supracellular scale.

A narrative review of lateral meniscus transplantation with the bridge in slot: technique and outcomes.

Objective: This narrative review aims to detail the indications, technique, and published outcomes of the bridge in slot technique for lateral meniscus allograft transplantation (LMAT) and to serve as a concise reference for orthopaedists looking to incorporate this method into their practice. Background: The menisci are crucial to normal knee function but are commonly injured; partial and subtotal meniscectomy are frequently performed to address meniscal pathology. Following these procedures, a substantial number of patients go on to develop degenerative joint changes accompanied by pain and disability. LMAT is an attractive option for young, active, lateral meniscal-deficient patients who seek pain relief and improved function but who are not yet prepared to undergo arthroplasty. In the properly indicated patient, the bridge in slot technique is a reliable and effective method for LMAT. Methods: Using a narrative style, this review outlines the indications and preoperative assessment for LMAT, the detailed technical steps for the bridge in slot technique, postoperative considerations, and trends in the surgical outcomes literature. The presented technique is consistent with the senior author's clinical experience and with published literature and the discussed outcomes are elicited from a focused review of recent peer-reviewed sources. Conclusions: The bridge in slot technique is a reliable and effective method for LMAT and is supported by the literature. This technique may confidently be used in patients with severe lateral meniscal pathology who are not yet candidates for arthroplasty.

Solving an MHC allele-specific bias in the reported immunopeptidome.

Identification of MHC class I-bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, increased detection of cancer germline antigen-derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view of the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.