Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis

Background and Aims: Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. Results: Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. Conclusions: We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma

Stem Cell Markers in Neuroblastoma—An Emerging Role for LGR5

The prognostic value of cancer stem cell markers in various cancer subtypes is a well documented research area. Our findings show that the stem cell marker Lgr5 is associated with an aggressive phenotype in neuroblastoma. Here, we discuss these findings within the context of recent studies in several cancers such as lung, colorectal and intestinal cancer, glioblastoma and ewing's sarcoma. Neuroblastoma continues to be an elusive disease, due to its heterogeneous presentation ranging from spontaneous regression to aggressive metastatic disease and intertwined genetic variability. Currently, the most significant prognostic marker of high risk disease and poor prognosis is amplification of the MYCN oncogene, which is found in approximately 25% of cases. With this in mind, there is still much to learn about the driving mechanisms of this aggressive pediatric tumor. Neuroblastoma development is thought to be the result of aberrant differentiation of the cell of origin, embryonic neural crest cells which then migrate and invade during the developmental stage. Aberrant cells are those which would, under normal conditions form the mature tissues of the sympathetic ganglia and adrenal medulla. Tumors are known to develop indiscriminately along the radius of the sympathetic ganglia, although it is well established that the adrenal glands are fundamentally the most common primary site

Keeping up with the COVID's—Could siRNA-based antivirals be a part of the answer?

Coronavirus disease 2019 (COVID-19) is a highly contagious viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This deadly infection has resulted in more than 5.2 million deaths worldwide. The global rollout of COVID-19 vaccines has without doubt saved countless lives by reducing the severity of symptoms for patients. However, as the virus continues to evolve, there is a risk that the vaccines and antiviral designed to target the infection will no longer be therapeutically viable. Furthermore, there remain fears over both the short and long-term side effects of repeat exposure to currently available vaccines. In this review, we discuss the pros and cons of the vaccine rollout and promote the idea of a COVID medicinal toolbox made up of different antiviral treatment modalities, and present some of the latest therapeutic strategies that are being explored in this respect to try to combat the COVID-19 virus and other COVID viruses that are predicted to follow. Lastly, we review current literature on the use of siRNA therapeutics as a way to remain adaptable and in tune with the ever-evolving mutation rate of the COVID-19 virus.Peer reviewe

Development and characterisation of star-shaped nanoparticles to deliver therapeutic siRNA to medulloblastoma

Brain cancer kills more children in Australia than any other disease. Medulloblastoma (MB) accounts for approximately 20% of all childhood brain tumours. Chemoresistance, relapse and treatment related toxicity are common for this disease. There is an urgent need to develop new effective and less toxic treatments. Gene therapies which use short-interfering RNA (siRNA) to silence the expression of a target gene have great potential for the treatment of a host of human diseases including cancer. However, a major hurdle for the clinical translation of siRNA drugs is the need for a delivery vehicle to allow siRNA to internalise into cells. Nanoparticles may offer a solution to this problem and can be used as delivery vehicles for siRNA. No studies have examined the potential of star nanoparticles for the treatment of MB. The aims of this study were to: 1) define the biological conditions for di-block copolymer nanoparticles (star nanoparticles) to deliver siRNA to MB cells in vitro to silence the expression of a gene (Polo-Like Kinase 1, PLK1) which is highly expressed in MB cells and plays a major role in promoting tumour growth; 2) determine whether star nanoparticles could deliver siRNA to solid tumours in mice; and 3) examine whether Star nanoparticle-siRNA (star-siRNA) could penetrate a blood-brain barrier (BBB). Results from this thesis demonstrate that star-siRNA form monodisperse nanoparticles with a size of 19 nm. Star-siRNA is internalised into MB cells in vitro and can silence PLK1 expression leading to mitotic arrest, DNA damage and apoptosis. Star-siRNA was non-toxic to mice and siRNA could be effectively delivered to subcutaneous MB tumours to silence PLK1 expression which induced apoptosis. Finally, using in vitro and in vivo models we showed that star-siRNA could penetrate the BBB. Importantly, star nanoparticles delivered siRNA to the brains of mice with growing orthotopic MB tumours. Collectively, results presented in this thesis demonstrate for the first time the potential of star nanoparticles to deliver siRNA to induce apoptosis in MB cells in vitro and in vivo. Star-siRNA nanodrugs may be a novel therapeutic strategy to inhibit MB growth and increase patient survival

Tailored fluorosurfactants through controlled/living radical polymerization for highly stable microfluidic droplet generation

Droplet-based microfluidics represents a disruptive technology in the field of chemistry and biology through the generation and manipulation of sub-microlitre droplets. To avoid droplet coalescence, fluoropolymer-based surfactants are commonly used to reduce the interfacial tension between two immiscible phases to stabilize droplet interfaces. However, the conventional preparation of fluorosurfactants involves multiple steps of conjugation reactions between fluorinated and hydrophilic segments to form multiple-block copolymers. In addition, synthesis of customized surfactants with tailored properties is challenging due to the complex synthesis process. Here, we report a highly efficient synthetic method that utilizes living radical polymerization (LRP) to produce fluorosurfactants with tailored functionalities. Compared to the commercialized surfactant, our surfactants outperform in thermal cycling for polymerase chain reaction (PCR) testing, and exhibit exceptional biocompatibility for cell and yeast culturing in a double-emulsion system. This breakthrough synthetic approach has the potential to revolutionize the field of droplet-based microfluidics by enabling the development of novel designs that generate droplets with superior stability and functionality for a wide range of applications.</p

Nanomaterial integrated 3D printing for biomedical applications

3D printing technology, otherwise known as additive manufacturing, has provided a promising tool for manufacturing customized biomaterials for tissue engineering and regenerative medicine applications. A vast variety of biomaterials including metals, ceramics, polymers, and composites are currently being used as base materials in 3D printing. In recent years, nanomaterials have been incorporated into 3D printing polymers to fabricate innovative, versatile, multifunctional hybrid materials that can be used in many different applications within the biomedical field. This review focuses on recent advances in novel hybrid biomaterials composed of nanomaterials and 3D printing technologies for biomedical applications. Various nanomaterials including metal-based nanomaterials, metal-organic frameworks, upconversion nanoparticles, and lipid-based nanoparticles used for 3D printing are presented, with a summary of the mechanisms, functional properties, advantages, disadvantages, and applications in biomedical 3D printing. To finish, this review offers a perspective and discusses the challenges facing the further development of nanomaterials in biomedical 3D printing.</p

Alzheimer's Progenitor Amyloid-β Targets and Dissolves Microbial Amyloids and Impairs Biofilm Function

Alzheimer's disease (AD) is a leading form of dementia where the presence of extra-neuronal plaques of Amyloid-.. (A..) is a pathological hallmark. However, A.. peptide is also observed in the intestinal tissues of AD patients and animal models. In this study, it is reported that A.. monomers can target and disintegrate microbial amyloids of FapC and CsgA formed by opportunistic gut pathogens, Pseudomonas aeruginosa and Escherichia coli, explaining a potential role of A.. in the gut-brain axis. Employing a zebrafish-based transparent in vivo system and whole-mount live-imaging, A.. is observed to diffuse into the vasculature and subsequently localize with FapC or CsgA fibrils that were injected into the tail muscles of the fish. FapC aggregates, produced after A.. treatment (Fa..), present selective toxicity to SH-SY5Y neuronal cells while the intestinal Caco-2 cells are shown to phagocytose Fa.. in a non-toxic cellular process. After remodeling by A.., microbial fibrils lose their native function of cell adhesion with intestinal Caco-2 cells and A.. dissolves and detachs the microbial fibrils already attached to the cell membrane. Taken together, this study strongly indicates an anti-biofilm role for A.. monomers that can help aid in the future development of selective anti-Alzheimer's and anti-infective medicine.Peer reviewe

Nanoengineering Liquid Metal Core–Shell Nanostructures

Nanoengineering the composition and morphology of functional nanoparticles endows them to perform multiple tasks and functions. An intriguing strategy for creating multifunctional nanomaterials involves the construction of core–shell nanostructures, which have enabled promising applications in biomedicine, energy, sensing, and catalysis. Here, a straightforward nanoengineering approach is presented utilizing liquid metal nanoparticles and galvanic replacement to create diverse core–shell nanostructures. Controlled nanostructures including liquid metal core-gold nanoparticle shell (LM@Au), gold nanoparticle core-gallium oxide shell (Au@Ga oxide), and hollow Ga oxide nanoparticles are successfully fabricated. Remarkably, these investigations reveal that LM@Au exhibits exceptional photothermal performance, achieving an impressive conversion efficiency of 65.9%, which is five times that of gold nanoparticles. By leveraging the high photothermal conversion efficiency and excellent biocompatibility of LM@Au, its promising application in hyperthermia cancer therapy is demonstrated. This simple yet powerful nanoengineering strategy opens new avenues for the controlled synthesis of complex core–shell nanostructures, advancing various fields beyond biomedicine

Nanoengineering liquid metal core–shell nanostructures

Nanoengineering the composition and morphology of functional nanoparticles endows them to perform multiple tasks and functions. An intriguing strategy for creating multifunctional nanomaterials involves the construction of core–shell nanostructures, which have enabled promising applications in biomedicine, energy, sensing, and catalysis. Here, a straightforward nanoengineering approach is presented utilizing liquid metal nanoparticles and galvanic replacement to create diverse core–shell nanostructures. Controlled nanostructures including liquid metal core-gold nanoparticle shell (LM@Au), gold nanoparticle core-gallium oxide shell (Au@Ga oxide), and hollow Ga oxide nanoparticles are successfully fabricated. Remarkably, these investigations reveal that LM@Au exhibits exceptional photothermal performance, achieving an impressive conversion efficiency of 65.9%, which is five times that of gold nanoparticles. By leveraging the high photothermal conversion efficiency and excellent biocompatibility of LM@Au, its promising application in hyperthermia cancer therapy is demonstrated. This simple yet powerful nanoengineering strategy opens new avenues for the controlled synthesis of complex core–shell nanostructures, advancing various fields beyond biomedicine.</p