Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

A High-Throughput Automated Confocal Microscopy Platform for Quantitative Phenotyping of Nanoparticle Uptake and Transport in Spheroids.

There is a high demand for advanced, image-based, automated high-content screening (HCS) approaches to facilitate phenotypic screening in 3D cell culture models. A major challenge lies in retaining the resolution of fine cellular detail but at the same time imaging multicellular structures at a large scale. In this study, a confocal microscopy-based HCS platform in optical multiwell plates that enables the quantitative morphological profiling of populations of nonuniform spheroids obtained from HT-29 human colorectal cancer cells is described. This platform is then utilized to demonstrate a quantitative dissection of the penetration of synthetic nanoparticles (NP) in multicellular 3D spheroids at multiple levels of scale. A pilot RNA interference-based screening validates this methodology and identifies a subset of RAB GTPases that regulate NP trafficking in these spheroids. This technology is suitable for high-content phenotyping in 3D cell-based screening, providing a framework for nanomedicine drug development as applied to translational oncology.

Multitasking Rab Proteins in Autophagy and Membrane Trafficking: A Focus on Rab33b.

Autophagy (particularly macroautophagy) is a bulk degradation process used by eukaryotic cells in order to maintain adequate energy levels and cellular homeostasis through the delivery of long-lived proteins and organelles to the lysosome, resulting in their degradation. It is becoming increasingly clear that many of the molecular requirements to fulfil autophagy intersect with those of conventional and unconventional membrane trafficking pathways. Of particular interest is the dependence of these processes on multiple members of the Rab family of small GTP binding proteins. Rab33b is a protein that localises to the Golgi apparatus and has suggested functions in both membrane trafficking and autophagic processes. Interestingly, mutations in the RAB33B gene have been reported to cause the severe skeletal disorder, Smith-McCort Dysplasia; however, the molecular basis for Rab33b in this disorder remains to be determined. In this review, we focus on the current knowledge of the participation of Rab33b and its interacting partners in membrane trafficking and macroautophagy, and speculate on how its function, and dysfunction, may contribute to human disease.

Heritable Skeletal Disorders Arising from Defects in Processing and Transport of Type I Procollagen from the ER: Perspectives on Possible Therapeutic Approaches.

Rare bone disorders are a heterogeneous group of diseases, initially associated with mutations in type I procollagen (PC) genes. Recent developments from dissection at the molecular and cellular level have expanded the list of disease-causing proteins, revealing that disruption of the machinery that handles protein secretion can lead to failure in PC secretion and in several cases result in skeletal dysplasia. In parallel, cell-based in vitro studies of PC trafficking pathways offer clues to the identification of new disease candidate genes. Together, this raises the prospect of heritable bone disorders as a paradigm for biosynthetic protein traffic-related diseases, and an avenue through which therapeutic strategies can be explored.Here, we focus on human syndromes linked to defects in type I PC secretion with respect to the landscape of biosynthetic and protein transport steps within the early secretory pathway. We provide a perspective on possible therapeutic interventions for associated heritable craniofacial and skeletal disorders, considering different orders of complexity, from the cellular level by manipulation of proteostasis pathways to higher levels involving cell-based therapies for bone repair and regeneration.

Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport.

The Sar1 GTPase coordinates the assembly of coat protein complex-II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER-to-Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII-mediated transport in mammalian cells, we used small interfering RNA (siRNA)-mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER-to-Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen-I is inhibited. These data provide proof-of-principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII.

The UCD nanosafety workshop (03 December 2018): towards developing a consensus on safe handling of nanomaterials within the Irish university labs and beyond - a report.

Careful handling of the nanomaterials (NMs) in research labs is crucial to ensure a safe working environment. As the largest university in Ireland, University College Dublin (UCD) has invested significant resources to update researchers working with NMs. Due to sizes often <100 nm, the NMs including nanoparticles, harbor unprecedented materialistic properties, for example, enhanced reactivity, conductivity, fluorescence, etc. which albeit conferring the NMs an edge over bulk materials regarding the applied aspects; depending on the dose, also render them to be toxic. Thus, a set of regulatory guidelines have emerged regarding safe handling of the NMs within occupational set-ups. Unfortunately, the current regulations based on the toxic chemicals and carcinogens are often confusing, lack clarity, and difficult to apply for the NMs. As a research-intensive university, a diverse range of research activities occur within the UCD labs, and it is difficult, at times impossible, for the UCD Safety, Insurance, Operational Risk & Compliance (SIRC) office to develop a set of common guidelines and cater throughout all its labs conducting research with the NMs. Hence, a necessity for dialog and exchange of ideas was felt across the UCD which encouraged the researchers including early stage researchers (e.g. PhDs, Postdocs) from multiple schools to participate in a workshop held on the 03 December 2018. The workshop tried to follow a pragmatic approach, where apart from discussing both the in vitro and in vivo scenarios, practical cases simulating situations faced frequently in the labs were discussed. This report summarizes the findings made during the workshop by this emerging critical mass in UCD.

Calcium sensing receptor activation by calcimimetic R-568 in human amniotic fluid mesenchymal stem cells: correlation with osteogenic differentiation.

Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are ß-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.