Edel Durack

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration

BackgroundCell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration.ResultsThe objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation.ConclusionsWe conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Salt: A Review of its Role in Food Science and Public Health

Salt has been used in food preparation since pre-history and is still routinely added in modern food manufacturing processes. Salt may be added at the table or during cooking; however a large amount of dietary salt originates from processed foods (∼60-70% of adult daily dietary salt). Excess salt intake is strongly linked to increases in blood pressure (hypertension) and subsequently pre-disposes individuals to the onset of cardiovascular disease (CVD), a leading cause of death in developed countries. Excess dietary salt intake has also been linked to the development of kidney disease, aggravation of asthma conditions and to the onset of osteoporosis. Results from large numbers of human studies have led to worldwide initiatives being put in place in order to reduce current levels of salt consumption. Hence there is an urgent need to reduce excess dietary salt intake from processed foods in particular. Current daily consumption figures can be as high as ∼10-12g NaCl per day while an intake of 4g salt per day is sufficient. Therefore a target reduction to an intake of 6g per day is the goal for a number of public health agencies worldwide. An incremental reduction in salt content in foods appears to be the best strategy to reduce population blood pressure values. However any reduction in salt content in processed foods must not impact on product safety or sensory quality. In this review various public health aspects of salt intake are considered. Additionally, the technological implications of salt reduction in foods are discussed and the possibilities for replacement of salt by addition of salt replacers, herbs, spices and other food ingredients to maintain product quality are highlighted.

RACK1 promotes neurite outgrowth by scaffolding AGAP2 to FAK.

RACK1 binds proteins in a constitutive or transient manner and supports signal transmission by engaging in diverse and distinct signalling pathways. The emerging theme is that RACK1 functions as a signalling switch, recruiting proteins to form distinct molecular complexes. In focal adhesions, RACK1 is required for the regulation of FAK activity and for integrating a wide array of cellular signalling events including the integration of growth factor and adhesion signalling pathways. FAK is required for cell adhesion and migration and has a well-established role in neurite outgrowth and in the developing nervous system. However, the mechanism by which FAK activity is regulated in neurons remains unknown. Using neuronal cell lines, we determined that differentiation of these cells promotes an interaction between the scaffolding protein RACK1 and FAK. Disruption of the RACK1/FAK interaction leads to decreased neurite outgrowth suggesting a role for the interaction in neurite extension. We hypothesised that RACK1 recruits proteins to FAK, to regulate FAK activity in neuronal cells. To address this, we immunoprecipitated RACK1 from rat hippocampus and searched for interacting proteins by mass spectrometry. We identified AGAP2 as a novel RACK1-interacting protein. Having confirmed the RACK1-AGAP2 interaction biochemically, we show RACK1-AGAP2 to localise together in the growth cone of differentiated cells, and confirm that these proteins are in complex with FAK. This complex is disrupted when RACK1 expression is suppressed using siRNA or when mutants of RACK1 that do not interact with FAK are expressed in cells. Similarly, suppression of AGAP2 using siRNA leads to increased phosphorylation of FAK and increased cell adhesion resulting in decreased neurite outgrowth. Our results suggest that RACK1 scaffolds AGAP2 to FAK to regulate FAK activity and cell adhesion during the differentiation process.

Role of Biorelevant Dissolution Media in the Selection of Optimal Salt Forms of Oral Drugs: Maximizing the Gastrointestinal Solubility and in Vitro Activity of the Antimicrobial Molecule, Clofazimine

Clofazimine is an antimycobacterial agent that is routinely used for the treatment of leprosy. Clofazimine has also been shown to have high clinical potential for the treatment of many Gram-positive pathogens, including those that exhibit high levels of antibiotic resistance in the medical community. The use of clofazimine against these pathogens has largely been limited by the inherently poor water solubility of the drug substance. In this work, the possibility of repurposing and reformulating clofazimine to maximize its clinical potential is investigated. To achieve this, the potential of novel salt forms of clofazimine as supersaturating drug-delivery vehicles to enhance the aqueous solubility and gastrointestinal solubility of the drug substance was explored. The solution properties of seven novel salt forms, identified during an initial screening process, were examined in water and in a gastrointestinal-like media and were compared and contrasted with those of the free base, clofazimine, and the commercial formulation of the drug, Lamprene. The stability of the most promising solid forms was tested, and their bioactivity against Staphylococcus aureus was also compared with that of the clofazimine free base and Lamprene. Salts forms which showed superior stability as well as solubility and activity to the commercial drug formulation were fully characterized using a combination of spectroscopic techniques, including X-ray diffraction, solid-state NMR, and Fourier transform infrared spectroscopy.

Delivery of a Hydrophobic Drug into the Lower Gastrointestinal System via an Endogenous Enzyme-Mediated Carrier Mechanism: an in vitro study.

&NA; Clofazimine (CFZ) is a hydrophobic antibiotic agent which exhibits poor solubility. This poor solubility was overcome herein by the formulation of CFZ with the digestive enzyme pepsin. It is shown that pepsin can actively bind 11 CFZ molecules in the proteins native gastric environment, forming a CFZ‐pepsin complex. A dynamic dissolution system, representing both the gastric and intestinal system, was used to analyze this CFZ‐pepsin complex, revealing that only CFZ which binds to pepsin in the gastric environment remains in solution in the intestinal environment. The CFZ‐pepsin complex displays adequate solution stability for the delivery of CFZ into the lower intestinal system. In vitro bioactivity assays against Clostridium difficile demonstrated the effectiveness of this CFZ‐pepsin complex for the treatment of infectious diseases in the lower intestinal system. Graphical abstract Figure. No caption available.

Co-reductive fabrication of carbon nanodots with high quantum yield for bioimaging of bacteria

A simple and straightforward synthetic approach for carbon nanodots (C-dots) is proposed. The strategy is based on a one-step hydrothermal chemical reduction with thiourea and urea, leading to high quantum yield C-dots. The obtained C-dots are well-dispersed with a uniform size and a graphite-like structure. A synergistic reduction mechanism was investigated using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The findings show that using both thiourea and urea during the one-pot synthesis enhances the luminescence of the generated C-dots. Moreover, the prepared C-dots have a high distribution of functional groups on their surface. In this work, C-dots proved to be a suitable nanomaterial for imaging of bacteria and exhibit potential for application in bioimaging thanks to their low cytotoxicity.

Image-Based Tracking of Anticancer Drug-Loaded Nanoengineered Polyelectrolyte Capsules in Cellular Environments Using a Fast Benchtop Mid-Infrared (MIR) Microscope

Drug delivery monitoring and tracking in the human body are two of the biggest challenges in targeted therapy to be addressed by nanomedicine. The ability of imaging drugs and micro-/nanoengineered drug carriers and of visualizing their interactions at the cellular interface in a label-free manner is crucial in providing the ability of tracking their cellular pathways and will help understand their biological impact, allowing thus to improve the therapeutic efficacy. We present a fast, label-free technique to achieve high-resolution imaging at the mid-infrared (MIR) spectrum that provides chemical information. Using our custom-made benchtop infrared microscope using a high-repetition-rate pulsed laser (80 MHz, 40 ps), we were able to acquire images with subwavelength resolution (0.8 × λ) at very high speeds. As a proof-of-concept, we embarked on the investigation of nanoengineered polyelectrolyte capsules (NPCs) containing the anticancer drug, docetaxel. These NPCs were synthesized using a layer-by-layer approach built upon a calcium carbonate (CaCO3) core, which was then removed away with ethylenediaminetetraacetic acid. The obtained MIR images show that NPCs are attached to the cell membrane, which is a good step toward an efficient drug delivery. This has been confirmed by both three-dimensional confocal fluorescence and stimulated emission depletion microscopy. Coupled with additional instrumentation and data processing advancements, this setup is capable of video-rate imaging speeds and will be significantly complementing current super-resolution microscopy techniques while providing an unperturbed view into living cells.