Biology subjects

McNulty, A.

Publications and source records attributed to McNulty, A..

3 recordsLinked to original sources

bioRxiv2020

Osteoarthritis: articular chondrocyte inflammatory signaling leads to enhanced gene expression and function of mechanotransduction channel Piezo1 as a pathogenic feedforwardmechanism

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies (1, 2). We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury (3, 4). We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1 (IL-1) upregulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+, at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically-induced deformation-microtrauma. As intracellular substrates of this novel OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1 we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. In ensemble, these signaling and genetic re-programming events represent a novel and detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA. Significance StatementOsteoarthritis affecting weight-bearing joints is a global health problem, causing loss of mobility and enormous healthcare costs. Disease-modifying approaches are lacking. Here, we report a new cellular mechanism of inflammatory signaling in chondrocytes, the cellular substrate of cartilage. We show how osteoarthritis-relevant levels of interleukin-1 reprogram articular chondrocytes so that they become more susceptible to mechanical trauma, which chondrocytes sense via Piezo1/2 mechanosensitive ion channels. We uncover that IL-1 enhances gene expression of Piezo1 in primary articular chondrocytes underlying Piezo1 gain-of-function. We elucidate the new signaling pathway, from membrane to nucleus, including transcription factors that enhance Piezo1-expression. We also define detrimental effects of gain-of-function of Piezo1, for mechanotransduction and at-rest, that suggest this new reprogramming mechanism to contribute to osteoarthritis pathogenesis.

molecular biology↗

bioRxiv2020

A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues

Mechanobiologic signals regulate cellular responses under physiologic and pathologic conditions. Using synthetic biology and tissue engineering, we developed a mechanically-responsive bioartificial tissue that responds to mechanical loading to produce a pre-programmed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically-sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. We engineered these cells into living tissues that respond to mechanical loading by producing the anti-inflammatory biologic drug, interleukin-1 receptor antagonist. Chondrocyte TRPV4 is activated by osmotic loading and not direct cellular deformation, suggesting tissue loading is transduced into an osmotic signal that activates TRPV4. Either osmotic or mechanical loading of tissues transduced with TRPV4-responsive circuits protected constructs from inflammatory degradation by interleukin-1. This synthetic mechanobiology approach was used to develop a mechanogenetic system to enable long-term, autonomously regulated drug delivery driven by physiologically-relevant loading.

synthetic biology↗

bioRxiv2019

Systematic assessment of commercially available low-input miRNA library preparation kits

High-throughput sequencing is increasingly favoured to assay the presence and abundance of micro RNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. However, although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human biological total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers sequence (UMI) tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of expected miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

bioinformatics↗