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.

Elemental Mapping in a Preclinical Animal Model Reveals White Matter Copper Elevation in the Acute Phase of Central Nervous System Trauma.

Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.

Transitional dynamics from mercury to cyanide-based processing in artisanal and small-scale gold mining: Social, economic, geochemical, and environmental considerations.

Artisanal and small-scale gold mining (ASGM) is the leading global source of anthropogenic mercury (Hg) release to the environment. Top-down mercury reduction efforts have had limited results, but a bottom-up embrace of cyanide (CN) processing could eventually displace mercury amalgamation for gold recovery. However, ASGM transitions to cyanidation nearly always include an overlap phase, with mercury amalgamation then cyanidation being used sequentially. This paper uses a transdisciplinary approach that combines natural and social sciences to develop a holistic picture of why mercury and cyanide converge in gold processing and potential impacts that may be worse than either practice in isolation. We show that socio-economic factors drive the comingling of mercury and cyanide practices in ASGM as much or more so than technical factors. The resultant Hg-CN complexes have been implicated in increasing the mobility of mercury, compared to elemental mercury used in Hg-only processing. To support future inquiry, we identify key knowledge gaps including the role of Hg-CN complexes in mercury oxidation, transport, and fate, and possible links to mercury methylation. The global extent and increase of mercury and cyanide processing in ASGM underscores the importance of further research. The immediacy of the problem also demands interim policy responses while research advances, though ultimately, the well-documented struggles of mercury reduction efforts in ASGM temper optimism about policy responses to the mercury-cyanide transition.

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.

Perceptions and realities of mercury contamination in a Peruvian artisanal and small-scale gold mining (ASGM) community.

Artisanal and small-scale gold mining (ASGM) is the leading global source of mercury pollution. Efforts to reduce or eliminate mercury use in ASGM have produced limited results, in part because they do not engage the complex socio-technical nature of mercury issues in ASGM. The paper takes a multidisciplinary approach to understand the mercury issue with a socio-technical lens, pairing sampling of mercury in soils with surveys of miners' and residents' perceptions of mercury pollution and its dispersion. The research was conducted in Secocha, an ASGM boomtown in southern Peru. Mercury levels in soils exceeded relevant standards in both industrial zones (average of 72.6 mg/kg, versus the Peruvian standard of 24 mg/kg) and residential/urban zones (average of 9.5 mg/kg, versus the Peruvian standard of 6.6 mg/kg). Mercury levels were highest where processing and gold buying activity were concentrated. Surveys revealed that miners and residents correctly assumed mercury pollution to be highest in those areas. However, respondents seemed to underestimate the extent of mercury pollution in other parts of town, and many believed that only those who handle mercury directly were affected by it. Respondents also placed low priority on reducing mercury pollution. Miners' and residents' partial knowledge about mercury contamination and the low priority accorded to the issue suggest that mercury reduction efforts would likely be met with indifference and potentially resistance.

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.

Millimeter waves alter DNA secondary structures and modulate the transcriptome in human fibroblasts.

As millimetre wave (MMW) frequencies of the electromagnetic spectrum are increasingly adopted in modern technologies such as mobile communications and networking, characterising the biological effects is critical in determining safe exposure levels. We study the exposure of primary human dermal fibroblasts to MMWs, finding MMWs trigger genomic and transcriptomic alterations. In particular, repeated 60 GHz, 2.6 mW cm-2, 46.8 J cm-2 d-1 MMW doses induce a unique physiological response after 2 and 4 days exposure. We show that high dose MMWs induce simultaneous non-thermal alterations to the transcriptome and DNA structural dynamics, including formation of G-quadruplex and i-motif secondary structures, but not DNA damage.

Stability and context of intercalated motifs (i-motifs) for biological applications.

DNA is naturally dynamic and can self-assemble into alternative secondary structures including the intercalated motif (i-motif), a four-stranded structure formed in cytosine-rich DNA sequences. Until recently, i-motifs were thought to be unstable in physiological cellular environments. Studies demonstrating their existence in the human genome and role in gene regulation are now shining light on their biological relevance. Herein, we review the effects of epigenetic modifications on i-motif structure and stability, and biological factors that affect i-motif formation within cells. Furthermore, we highlight recent progress in targeting i-motifs with structure-specific ligands for biotechnology and therapeutic purposes.

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.

Enhancing Engineering Ethics: Role Ethics and Corporate Social Responsibility.

Engineering ethics calls the attention of engineers to professional codes of ethical responsibility and personal values, but the practice of ethics in corporate settings can be more complex than either of these. Corporations too have cultures that often include corporate social responsibility (CSR) practices and policies, but few discussions of engineering ethics make any explicit reference to CSR. This article proposes critical attention to CSR and role ethics as an opportunity to help prepare engineers to think through the ethics of their professional practice. After a brief overview of the evolution of social responsibility within engineering ethics in the United States, this article shares empirical research with practicing engineers in the mining and energy industries to explore how their formal ethics training did and did not prepare them to grapple with the ethical dimensions of their professional practice. It then illustrates the ways in which these dilemmas and the strategies employed for navigating them are framed within CSR policies and practices and resonate more strongly with role ethics rather than ethical theory as currently taught in most US engineering programs. The article concludes that engineering ethics teaching and learning would benefit from explicitly incorporating critical discussions of role ethics and CSR.

Modulating gene expression in breast cancer via DNA secondary structure and the CRISPR toolbox.

Breast cancer is the most commonly diagnosed malignancy in women, and while the survival prognosis of patients with early-stage, non-metastatic disease is ∼75%, recurrence poses a significant risk and advanced and/or metastatic breast cancer is incurable. A distinctive feature of advanced breast cancer is an unstable genome and altered gene expression patterns that result in disease heterogeneity. Transcription factors represent a unique therapeutic opportunity in breast cancer, since they are known regulators of gene expression, including gene expression involved in differentiation and cell death, which are themselves often mutated or dysregulated in cancer. While transcription factors have traditionally been viewed as 'undruggable', progress has been made in the development of small-molecule therapeutics to target relevant protein-protein, protein-DNA and enzymatic active sites, with varying levels of success. However, non-traditional approaches such as epigenetic editing, transcriptional control via CRISPR/dCas9 systems, and gene regulation through non-canonical nucleic acid secondary structures represent new directions yet to be fully explored. Here, we discuss these new approaches and current limitations in light of new therapeutic opportunities for breast cancers.

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.

High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A π-stacking interactions for small-molecule recognition.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

Protein corona formation moderates the release kinetics of ion channel antagonists from transferrin-functionalized polymeric nanoparticles.

Transferrin (Tf)-functionalized p(HEMA-ran-GMA) nanoparticles were designed to incorporate and release a water-soluble combination of three ion channel antagonists, namely zonampanel monohydrate (YM872), oxidized adenosine triphosphate (oxATP) and lomerizine hydrochloride (LOM) identified as a promising therapy for secondary degeneration that follows neurotrauma. Coupled with a mean hydrodynamic size of 285 nm and near-neutral surface charge of -5.98 mV, the hydrophilic nature of the functionalized polymeric nanoparticles was pivotal in effectively encapsulating the highly water soluble YM872 and oxATP, as well as lipophilic LOM dissolved in water-based medium, by a back-filling method. Maximum loading efficiencies of 11.8 ± 1.05% (w/w), 13.9 ± 1.50% (w/w) and 22.7 ± 4.00% (w/w) LOM, YM872 and oxATP respectively were reported. To obtain an estimate of drug exposure in vivo, drug release kinetics assessment by HPLC was conducted in representative physiological milieu containing 55% (v/v) human serum at 37 °C. In comparison to serum-free conditions, it was demonstrated that the inevitable adsorption of serum proteins on the Tf-functionalized nanoparticle surface as a protein corona impeded the rate of release of LOM and YM872 at both pH 5 and 7.4 over a period of 1 hour. While the release of oxATP from the nanoparticles was detectable for up to 30 minutes under serum-free conditions at pH 7.4, the presence of serum proteins and a slightly acidic environment impaired the detection of the drug, possibly due to its molecular instability. Nevertheless, under representative physiological conditions, all three drugs were released in combination from Tf-functionalized p(HEMA-ran-GMA) nanoparticles and detected for up to 20 minutes. Taken together, the study provided enhanced insight into potential physiological outcomes in the presence of serum proteins, and suggests that p(HEMA-ran-GMA)-based therapeutic nanoparticles may be promising drug delivery vehicles for CNS therapy.

Epigenetic modification of cytosines fine tunes the stability of i-motif DNA.

i-Motifs are widely used in nanotechnology, play a part in gene regulation and have been detected in human nuclei. As these structures are composed of cytosine, they are potential sites for epigenetic modification. In addition to 5-methyl- and 5-hydroxymethylcytosine modifications, recent evidence has suggested biological roles for 5-formylcytosine and 5-carboxylcytosine. Herein the human telomeric i-motif sequence was used to examine how these four epigenetic modifications alter the thermal and pH stability of i-motifs. Changes in melting temperature and transitional pH depended on both the type of modification and its position within the i-motif forming sequence. The cytosines most sensitive to modification were next to the first and third loops within the structure. Using previously described i-motif forming sequences, we screened the MCF-7 and MCF-10A methylomes to map 5-methylcytosine and found the majority of sequences were differentially methylated in MCF7 (cancerous) and MCF10A (non-cancerous) cell lines. Furthermore, i-motif forming sequences stable at neutral pH were significantly more likely to be epigenetically modified than traditional acidic i-motif forming sequences. This work has implications not only in the epigenetic regulation of DNA, but also allows discreet tunability of i-motif stability for nanotechnological applications.

Lung CD4+ resident memory T cells remodel epithelial responses to accelerate neutrophil recruitment during pneumonia.

Previous pneumococcal experience establishes lung-resident IL-17A-producing CD4+ memory TRM cells that accelerate neutrophil recruitment against heterotypic pneumococci. Herein, we unravel a novel crosstalk between CD4+ TRM cells and lung epithelial cells underlying this protective immunity. Depletion of CD4+ cells in pneumococcus-experienced mice diminished CXCL5 (but not CXCL1 or CXCL2) and downstream neutrophil accumulation in the lungs. Epithelial cells from experienced lungs exhibited elevated mRNA for CXCL5 but not other epithelial products such as GM-CSF or CCL20, suggesting a skewing by CD4+ TRM cells. Genome-wide expression analyses revealed a significant remodeling of the epithelial transcriptome of infected lungs due to infection history, ~80% of which was CD4+ cell-dependent. The CD4+ TRM cell product IL-17A stabilized CXCL5 but not GM-CSF or CCL20 mRNA in cultured lung epithelial cells, implicating posttranscriptional regulation as a mechanism for altered epithelial responses. These results suggest that epithelial cells in experienced lungs are effectively different, owing to their communication with TRM cells. Our study highlights the role of tissue-resident adaptive immune cells in fine-tuning epithelial functions to hasten innate immune responses and optimize defense in experienced lungs, a concept that may apply broadly to mucosal immunology.

TGF-ß-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.

Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-ß-induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca2+ influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.

Tumour suppression by targeted intravenous non-viral CRISPRa using dendritic polymers.

Aberrant gene expression is a hallmark of cancer. Although transcription is traditionally considered 'undruggable', the development of CRISPR-associated protein 9 (Cas9) systems offers enormous potential to rectify cancer-associated transcriptional abnormalities in malignant cells. However delivery of this technology presents a critical challenge to overcome in order to realize clinical translation for cancer therapy. In this article we demonstrate for the first time, a fully synthetic strategy to enable CRISPR-mediated activation (CRISPRa) of tumour suppressor genes in vivo using a targeted intravenous approach. We show this via highly efficient transcriptional activation of two model tumour suppressor genes, Mammary Serine Protease Inhibitor (MASPIN, SERPINB5) and cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed 6 (CCN6, WISP3), in a mouse model of breast cancer. In particular, we demonstrate that targeted intravenous delivery of can be achieved using a novel nanoscale dendritic macromolecular delivery agent, with negligible toxicity and long lasting therapeutic effects, outlining a targeted effective formulation with potential to treat aggressive malignancies.

Elucidating the Inability of Functionalized Nanoparticles to Cross the Blood-Brain Barrier and Target Specific Cells in Vivo.

The adsorption of serum proteins on the surface of nanoparticles (NPs) delivered into a biological environment has been known to alter NP surface properties and consequently their targeting efficiency. In this paper, we use random copolymer (p(HEMA- ran-GMA))-based NPs synthesized using 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA). We show that serum proteins bind to the NP and that functionalization with antibodies and peptides designed to facilitate NP passage across the blood-brain barrier (BBB) to bind specific cell types is ineffective. In particular, we use systematic in vitro and in vivo analyses to demonstrate that p(HEMA- ran-GMA) NPs functionalized with HIV-1 trans-activating transcriptor peptide (known to cross the BBB) and α neural/glial antigen 2 (NG2) (known for targeting oligodendrocyte precursor cells (OPCs)), individually and in combination, do not specifically target OPCs and are unable to cross the BBB, likely due to the serum protein binding to the NPs.