Diwei Ho

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

Paraspeckles are mammalian subnuclear bodies built on a long noncoding RNA and are enriched in RNA binding proteins with prion-like domains; two of these proteins, RBM14 and FUS, use these domains to hold paraspeckles together.

The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.

Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration

There is a need to develop economical, efficient and widely available therapeutic approaches to enhance the rate of skin wound healing. The optimal outcome of wound healing is restoration to the pre-wound quality of health. In this study we investigate the cellular response to biological stimuli using functionalized nanofibers from the self-assembling peptide, RADA16. We demonstrate that adding different functional motifs to the RADA16 base peptide can influence the rate of proliferation and migration of keratinocytes and dermal fibroblasts. Relative to unmodified RADA16; the Collagen I motif significantly promotes cell migration, and reduces proliferation.

Dose-Dependent Therapeutic Distinction between Active and Passive Targeting Revealed Using Transferrin-Coated PGMA Nanoparticles.

The paradigm of using nanoparticle-based formulations for drug delivery relies on their enhanced passive accumulation in the tumor interstitium. Nanoparticles with active targeting capabilities attempt to further enhance specific delivery of drugs to the tumors via interaction with overexpressed cellular receptors. Consequently, it is widely accepted that drug delivery using actively targeted nanoparticles maximizes the therapeutic benefit and minimizes the off-target effects. However, the process of nanoparticle mediated active targeting initially relies on their passive accumulation in tumors. In this article, it is demonstrated that these two tumor-targeted drug delivery mechanisms are interrelated and dosage dependent. It is reported that at lower doses, actively targeted nanoparticles have distinctly higher efficacy in tumor inhibition than their passively targeted counterparts. However, the enhanced permeability and retention effect of the tumor tissue becomes the dominant factor influencing the efficacy of both passively and actively targeted nanoparticles when they are administered at higher doses. Importantly, it is demonstrated that dosage is a pivotal parameter that needs to be taken into account in the assessment of nanoparticle mediated targeted drug delivery.

Multimodal and multifunctional stealth polymer nanospheres for sustained drug delivery

We report the preparation of fluorescent and magnetic PMMA nanospheres, and a corresponding PEGylated ‘stealth’ analogue prepared using a block copolymer. The nanospheres contain encapsulated magnetite nanoparticles and fluorescent BODIPY dyes, including a new such dye with pH-sensitive fluorescent emission. The new dye could potentially be used as an indicator of the immediate physiological environment. The nanospheres were non-toxic at up to 500 μg ml−1 in PC12 cells. Lomerizine, a lipophilic calcium channel blocker, was also encapsulated in the nanospheres and displayed sustained, pH-dependent release characteristics. The nanospheres may be of use to release lomerizine and other water-insoluble drugs at central nervous system injury sites.

An Unexpected Transient Breakdown of the Blood Brain Barrier Triggers Passage of Large Intravenously Administered Nanoparticles.

The highly restrictive blood-brain barrier (BBB) plays a critically important role in maintaining brain homeostasis and is pivotal for proper neuronal function. The BBB is currently considered the main limiting factor restricting the passage of large (up to 200 nm) intravenously administered nanoparticles to the brain. Breakdown of the barrier occurs as a consequence of cerebrovascular diseases and traumatic brain injury. In this article, we report that remote injuries in the CNS are also associated with BBB dysfunction. In particular, we show that a focal partial transection of the optic nerve triggers a previously unknown transient opening of the mammalian BBB that occurs in the visual centres. Importantly, we demonstrate that this transient BBB breakdown results in a dramatic change in the biodistribution of intravenously administered large polymeric nanoparticles which were previously deemed as BBB-impermeable.

Functional Reactive Polymer Electrospun Matrix

Synthetic multifunctional electrospun composites are a new class of hybrid materials with many potential applications. However, the lack of an efficient, reactive large-area substrate has been one of the major limitations in the development of these materials as advanced functional platforms. Herein, we demonstrate the utility of electrospun poly(glycidyl methacrylate) films as a highly versatile platform for the development of functional nanostructured materials anchored to a surface. The utility of this platform as a reactive substrate is demonstrated by grafting poly(N-isopropylacrylamide) to incorporate stimuli-responsive properties. Additionally, we demonstrate that functional nanocomposites can be fabricated using this platform with properties for sensing, fluorescence imaging, and magneto-responsiveness.

Nanoparticle-mediated internalisation and release of a calcium channel blocker

Lomerizine is a calcium channel blocker that selectively blocks L- and T-type Ca2+ channels, but it is effectively insoluble under physiological conditions. Herein we show that lomerizine can be released from a nanoparticle-based carrier by intracellular protonation in PC12 cells, suppressing Ca2+ influx in these cells in response to glutamate.

Achieving assessor accuracy on the International Standards for Neurological Classification of Spinal Cord Injury

Study design:A retrospective audit of assessor accuracy using the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) in three multicentre randomised controlled trials (SCIPA: Spinal Cord Injury and Physical Activity) spanning 2010–2014 with standards revised in 2011.Objectives:To investigate assessor accuracy of neurological classification after spinal cord injury.Setting:Australia and New Zealand.Methods:ISNCSCI examinations were undertaken by trained clinicians prior to randomisation. Data were recorded manually and ISNCSCI worksheets circulated to panels, consensus reached and worksheets corrected. An audit team used a 2014 computerised ISNCSCI algorithm to check manual worksheets. A second audit team assessed whether the 2014 computerised algorithm accurately reflected pre- and post-2011 ISNCSCI standards.Results:Of the 208 ISNCSCI worksheets, 24 were excluded. Of the remaining 184 worksheets, 47 (25.5%) were consistent with the 2014 computerised algorithm and 137 (74.5%) contained one or more errors. Errors were in motor (30.1%) or sensory (12.4%) levels, zone of partial preservation (24.0%), motor/sensory scoring (21.5%), ASIA Impairment Scale (AIS, 8.3%) and complete/incomplete classification (0.8%). Other difficulties included classification when anal contraction/sensation was omitted, incorrect neurological levels and violation of the ‘motor follows sensory rule in non-testable myotomes’ (7.4%). Panel errors comprised corrections that were incorrect or missed or incorrect changes to correct worksheets.Conclusion:Given inaccuracies in the manual ISNCSCI worksheets in this long-term clinical trial setting, continued training and a computerised algorithm are essential to ensure accurate scoring, scaling and classification of the ISNCSCI and confidence in clinical trials.

Maternal-placental-fetal biodistribution of multimodal polymeric nanoparticles in a pregnant rat model in mid and late gestation

Multimodal polymeric nanoparticles have many exciting diagnostic and therapeutic applications, yet their uptake and passage by the placenta, and applications in the treatment of pregnancy complications have not been thoroughly investigated. In this work, the maternal-fetal-placental biodistribution of anionic and cationic multimodal poly(glycidyl methacrylate) (PGMA) nanoparticles in pregnant rats at mid (ED10) and late (ED20) gestation was examined. Fluorescently-labelled and superparamagnetic PGMA nanoparticles functionalized with/without poly(ethyleneimine) (PEI) were administered to pregnant rats at a clinically-relevant dose and biodistribution and tissue uptake assessed. Quantitative measurement of fluorescence intensity or magnetic resonance relaxometry in tissue homogenates lacked the sensitivity to quantify tissue uptake. Confocal microscopy, however, identified uptake by maternal organs and the decidua (ectoplacental cone) and trophoblast giant cells of conceptuses at ED10. At ED20, preferential accumulation of cationic vs. anionic nanoparticles was observed in the placenta, with PGMA-PEI nanoparticles localised mainly within the chorionic plate. These findings highlight the significant impact of surface charge and gestational age in the biodistribution of nanoparticles in pregnancy, and demonstrate the importance of using highly sensitive measurement techniques to evaluate nanomaterial biodistribution and maternal-fetal exposure.