Reduced WNT4 expression in normal skin fibroblasts leads to 'Dupuytren-like' changes in the transcriptome.

Background: Dupuytren's disease (DD) is a fibro-proliferative disorder of unknown aetiology. Previous studies have implicated multiple WNT signalling genes/proteins in Dupuytren pathology, including WNT4. However, it is not yet clear whether WNT signalling dysregulation plays an important role in the initiation of the disease or progression. The aim of this study was to determine if loss of WNT4 expression triggered 'Dupuytren-like' changes in the transcriptome of healthy skin fibroblasts. Methods: Fibroblasts were isolated from the wrists of healthy adult males and from the wrists and disease cord tissue from males in a family positive for Dupuytren's disease. Normal skin fibroblasts from healthy controls were treated with WNT4 siRNA and scrambled controls. RNASeq was used to analyse the transcriptomes of disease and non-disease fibroblasts from patients with Dupuytren's as well as in siRNA treated and non-treated control fibroblasts. Results: Analysis of the transcriptomes from DD patient and normal skin fibroblasts showed significant differences, including in WNT4 expression. Downregulation of WNT4 in normal skin fibroblasts using siRNA led to 'DD-like' changes in the transcriptome. Conclusion: In people susceptible to DD WNT4 is downregulated even in non-fibrotic fibroblasts. Knockdown of WNT4 in normal fibroblasts led to changes that made cells 'DD-like'. This study shows that WNT4 is down regulated in 'non-disease' cells, and that downregulating WNT4 in normal skin fibroblasts leads to widespread 'DD like' changes in the transcriptome, suggesting WNT4 downregulation is a key driver of DD.

A Systematic Review to Compare Electrical, Magnetic, and Optogenetic Stimulation for Peripheral Nerve Repair.

The purpose of this systematic review was to assess the currently available evidence for the use of external stimulation to modulate neural activity and promote peripheral nerve regeneration. The most common external stimulations are electrical stimulation (ES), optogenetic stimulation (OS), and magnetic stimulation (MS). Understanding the comparative effectiveness of these stimulation methods is pivotal in advancing therapeutic interventions for peripheral nerve injuries. This systematic review focused on these three external stimulation modalities as potential strategies to enhance peripheral nerve repair (PNR). We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses framework to systematically evaluate and compare the efficiency of ES, OS, and MS in PNR. The review included studies published between 2018 and 2023 using ES, OS, or MS for PNR focused on enhancing recovery of peripheral nerve injuries in rodent models identified through PubMed and Google Scholar. The search strategies and inclusion criteria identified 19 studies (13 ES, 4 OS, and 2 MS) for detailed analysis, focusing on critical parameters such as functional recovery, histological outcomes, and electrophysiological data. Although ES demonstrated a consistent improvement in all the analyses, high-frequency repetitive MS (HFr-MS) emerged as a promising modality. HFr-MS demonstrated accelerated PNR, as histological and electrophysiological evidence indicated. In contrast, OS exhibited superior functional recovery outcomes. Notable limitations include constrained MS and OS data sets and the challenge of comparing relative improvements because of methodological diversity in evaluation techniques. Our findings underscore the potential of HFr-MS and OS in PNR while emphasizing the critical need for standardized testing protocols to facilitate meaningful cross-study comparisons. External stimulations have the potential to improve functional recovery in patients with nerve injury.

Transcriptomic Analysis Reveals the Heterogeneous Role of Conducting Films Upon Electrical Stimulation.

Central nervous system (CNS) injuries and neurodegenerative diseases have markedly poor prognoses and can result in permanent dysfunction due to the general inability of CNS neurons to regenerate. Differentiation of transplanted stem cells has emerged as a therapeutic avenue to regenerate tissue architecture in damaged areas. Electrical stimulation is a promising approach for directing the differentiation outcomes and pattern of outgrowth of transplanted stem cells, however traditional inorganic bio-electrodes can induce adverse effects such as inflammation. This study demonstrates the implementation of two organic thin films, a polymer/reduced graphene oxide nanocomposite (P(rGO)) and PEDOT:PSS, that have favorable properties for implementation as conductive materials for electrical stimulation, as well as an inorganic indium tin oxide (ITO) conductive film. Transcriptomic analysis reveals that electrical stimulation improves neuronal differentiation of SH-SY5Y cells on all three films, with the greatest effect for P(rGO). Unique material- and electrical stimuli-mediated effects are observed, associated with differentiation, cell-substrate adhesion, and translation. The work demonstrates that P(rGO) and PEDOT:PSS are highly promising organic materials for the development of biocompatible, conductive scaffolds that will enhance electrically-aided stem cell therapeutics for CNS injuries and neurodegenerative diseases.

High-resolution visualisation of antisense oligonucleotide release from polymers in cells.

Antisense oligonucleotides (ASOs) are a well-established therapeutic modality based on RNA interference, but low cellular uptake, limited ability to direct ASO trafficking, and a range of intracellular barriers to successful activity compromise both gene silencing outcomes and clinical translations. Herein, we demonstrate that polymers can increase ASO internalisation via intracellular trafficking pathways that are distinct from lipid-based delivery reagents. For the first time, we spatially define internalisation and dissociation stages in the polymer-mediated cytosolic delivery of ASOs using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which enables visualisation of ASO localisation at the organelle level. We find that polymer-ASO complexes are imported into cells, from which free ASO enters the cytosol following complex dissociation. This information enables a better understanding of the intracellular trafficking pathways of nucleic acid therapeutics and may be exploited for therapeutic delivery to enhance the effectiveness of nucleic acid therapeutics in the future.

Local therapy with combination TLR agonists stimulates systemic anti-tumor immunity and sensitizes tumors to immune checkpoint blockade.

Toll-like receptor (TLR) agonists are being developed as anti-cancer therapeutics due to their potent immunostimulatory properties. However, clinical trials testing TLR agonists as monotherapy have often failed to demonstrate significant improvement over standard of care. We hypothesized that the anti-cancer efficacy of TLR agonist immunotherapy could be improved by combinatorial approaches. To prevent increased toxicity, often seen with systemic combination therapies, we developed a hydrogel to deliver TLR agonist combinations at low doses, locally, during cancer debulking surgery. Using tumor models of WEHI 164 and bilateral M3-9-M sarcoma and CT26 colon carcinoma, we assessed the efficacy of pairwise combinations of poly(I:C), R848, and CpG in controlling local and distant tumor growth. We show that combination of the TLR3 agonist poly(I:C) and TLR7/8 agonist R848 drives anti-tumor immunity against local and distant tumors. In addition, combination of local poly(I:C) and R848 sensitized tumors to systemic immune checkpoint blockade, improving tumor control. Mechanistically, we demonstrate that local therapy with poly(I:C) and R848 recruits inflammatory monocytes to the tumor draining lymph nodes early in the anti-tumor response. Finally, we provide proof of concept for intraoperative delivery of poly(I:C) and R848 together via a surgically applicable biodegradable hydrogel.

Preferential formation of Z-RNA over intercalated motifs in long noncoding RNA.

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.

Docetaxel Inhibits Epithelial-Mesenchymal Transition in Human Mammary Cells.

Epithelial-mesenchymal transition (EMT) is a reversible and dynamic biological process in which epithelial cells acquire mesenchymal characteristics including enhanced stemness and migratory ability. EMT can facilitate cancer metastasis and is a known driver of cellular resistance to common chemotherapeutic drugs, such as docetaxel. Current chemotherapeutic practices such as docetaxel treatment can promote EMT and increase the chance of tumor recurrence and resistance, calling for new approaches in cancer treatment. Here we show that prolonged docetaxel treatment at a sub-IC50 concentration inhibits EMT in immortalized human mammary epithelial (HMLE) cells. Using immunofluorescence, flow cytometry, and bulk transcriptomic sequencing to assess EMT progression, we analyzed a range of cellular markers of EMT in docetaxel-treated cells and observed an upregulation of epithelial markers and downregulation of mesenchymal markers in the presence of docetaxel. This finding suggests that docetaxel may have clinical applications not only as a cytotoxic drug but also as an inhibitor of EMT-driven metastasis and multidrug resistance depending on the concentration of its use.

G4-DNA formation and chromatin remodelling are interdependent in human cells.

DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.

Separating Convective from Diffusive Mass Transport Mechanisms in Ionic Liquids by Redox Pro-fluorescence Microscopy.

The study of electrochemical reactivity requires analytical techniques capable of probing the diffusion of reactants and products to and from electrified interfaces. Information on diffusion coefficients is often obtained indirectly by modeling current transients and cyclic voltammetry data, but such measurements lack spatial resolution and are accurate only if mass transport by convection is negligible. Detecting and accounting for adventitious convection in viscous and wet solvents, such as ionic liquids, is technically challenging. We have developed a direct, spatiotemporally resolved optical tracking of diffusion fronts which can detect and resolve convective disturbances to linear diffusion. By tracking the movement of an electrode-generated fluorophore, we demonstrate that parasitic gas evolving reactions lead to 10-fold overestimates of macroscopic diffusion coefficients. A hypothesis is put forward linking large barriers to inner-sphere redox reactions, such as hydrogen gas evolution, to the formation of cation-rich overscreening and crowding double layer structures in imidazolium-based ionic liquids.

Topical application of an irreversible small molecule inhibitor of lysyl oxidases ameliorates skin scarring and fibrosis.

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.

Intracellular Communication between Synthetic Macromolecules.

Non-viral delivery is an important strategy for selective and efficient gene therapy, immunization, and RNA interference, which overcomes problems of genotoxicity and inherent immunogenicity associated with viral vectors. Liposomes and polymers are compelling candidates as carriers for intracellular, non-viral delivery, but maximal efficiencies of around 1% have been reported for the most advanced non-viral carriers. Here, we develop a library of dendronized bottlebrush polymers with controlled defects, displaying a level of precision surpassed only by biological molecules like DNA, RNA, and proteins. We test concurrent and competitive delivery of DNA and show for the first time that, while intracellular communication is thought to be an exclusively biomolecular phenomenon, such communication between synthetic macromolecular complexes can also take place. Our findings challenge the assumption that delivery agents behave as bystanders that enable transfection by passive intracellular release of genetic cargo and improve upon coarse strategies in intracellular carrier design lacking control over polymer sequence, architecture, and composition, leading to a hit-or-miss outcome. Understanding the communication that takes place between macromolecules will help improve the design of non-viral delivery agents and facilitate translation of genome engineering, vaccines, and nucleic acid-based therapies.

The role of G-Quadruplex DNA in Paraspeckle formation in cancer.

Paraspeckles are RNA-protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture.

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.

A dendronised polymer architecture breaks the conventional inverse relationship between porosity and mechanical properties of hydrogels.

We present a series of synthetic polymer hydrogels which break the traditional correlation between pore size and mechanical properties. The hydrogels are prepared from a dendronised polymer architecture based on a methacrylate copolymer to which poly(amido amine) dendrons are attached. Our approach will be useful in tailoring hydrogels for tissue engineering, controlled drug release, and flexible electronics.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

DNA G-Quadruplex and i-Motif Structure Formation Is Interdependent in Human Cells.

Guanine- and cytosine-rich nucleic acid sequences have the potential to form secondary structures such as G-quadruplexes and i-motifs, respectively. We show that stabilization of G-quadruplexes using small molecules destabilizes the i-motifs, and vice versa, indicating these gene regulatory controllers are interdependent in human cells. This has important implications as these structures are predominately considered as isolated structural targets for therapy, but their interdependency highlights the interplay of both structures as an important gene regulatory switch.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer.

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

Surface Diffusion of Dendronized Polymers Correlates with Their Transfection Potential.

Successful intracellular delivery of therapeutics requires interactions at several liquid-solid interfaces, including cell surface, endosomal membranes, and-depending on the therapeutic-the nuclear membrane. Understanding the dynamics of polymer kinetics at the liquid-solid interface is fundamental for the design of polymers for such biomedical delivery applications. However, the effect of polymer architecture and charge density on polymer kinetics is not readily investigated using routine techniques, and the role of such parameters in the context of gene delivery remains unknown. We adopted a synthetic strategy which enabled the systematic manipulation of charge density, flexibility, and molecular weight using a dendronized linear polymeric architecture. High-speed atomic force microscopy (HS-AFM) was used as a label-free method to directly observe the polymers' dynamic properties, such as velocity, displacement, and diffusion, in physiologically relevant conditions. Importantly, we found that the physical parameters measured by HS-AFM relate to the transfection potential of the individual polymers and may be a valuable tool in screening structural polymer variants.

A dendronized polymer variant that facilitates safe delivery of a calcium channel antagonist to the heart.

Therapeutic approaches for myocardial ischemia-reperfusion injury (MI) have been ineffective due to limited bioavailability and poor specificity. We have previously shown that a peptide that targets the α-interaction domain of the cardiac L-type calcium channel (AID-peptide) attenuates MI when tethered to transactivator of transcription sequence (TAT) or spherical nanoparticles. However some reservations remain regarding use of these delivery platforms due to the relationship with human immunodeficiency virus, off-target effects and toxicity. Here we investigate the use of linear dendronized polymers (denpols) to deliver AID-peptide as a potential MI therapy using in vitro, ex vivo and in vivo models. Optimized denpol-complexed AID-peptide facilitated in vitro cardiac uptake of AID-peptide, and reduced MI. Maximal in vivo cardiac uptake was achieved within the 2 h therapeutic time window for acute myocardial infarction. Importantly, optimized denpol-complexed AID-peptide was not toxic. This platform may represent an alternative therapeutic approach for the prevention of MI.