John A. Scott

Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.

Microalgae cultivation in a novel top-lit gas-lift open bioreactor.

This work investigated a top-lit open microalgae bioreactor that uses a gas-lift system to enable deeper production depths, thereby significantly reducing the footprint. Growth of Scenedesmus sp. in a one-meter deep system by sparged with 6% CO2-enhanced air was evaluated. The results gave comparable volumetric biomass productivity (0.06 g(dw) L(-1) day(-1)), but around three-times higher areal productivity (60.0 g(dw)m(-)(2) day(-)(1)) than reported for traditional raceways. The lipid content of the Scenedesmus sp. was increased by 27% with an enhanced level of CO2 in the sparging gas.

Bioprospecting for acidophilic lipid-rich green microalgae isolated from abandoned mine site water bodies

With fossil fuel sources in limited supply, microalgae show tremendous promise as a carbon neutral source of biofuel. Current microalgae biofuel strategies typically rely on growing high-lipid producing laboratory strains of microalgae in open raceways or closed system photobioreactors. Unfortunately, these microalgae species are found to be sensitive to environmental stresses or competition by regional strains. Contamination by invasive species can diminish productivity of commercial algal processes. A potential improvement to current strategies is to identify high-lipid producing microalgae, which thrive in selected culture conditions that reduce the risk of contamination, such as low pH. Here we report the identification of a novel high-lipid producing microalgae which can tolerate low pH growth conditions. Lig 290 is a Scenedesmus spp. isolated from a low pH waterbody (pH = 4.5) in proximity to an abandoned lignite mine in Northern Ontario, Canada. Compared to a laboratory strain of Scendesmus dimorphus, Lig 290 demonstrated robust growth rates, a strong growth profile, and high lipid production. As a consequence, Lig 290 may have potential application as a robust microalgal species for use in biofuel production.

Characterizing nerve growth factor–p75NTR interactions and small molecule inhibition using surface plasmon resonance spectroscopy

Nerve growth factor (NGF) is critical for the proliferation, differentiation, and survival of neurons through its binding to the p75(NTR) and TrkA receptors. Dysregulation of NGF has been implicated in several pathologies, including neurodegeneration (i.e., Parkinsons and Alzheimers diseases) and both inflammatory and neuropathic pain states. Therefore, small molecule inhibitors that block NGF-receptor interactions have significant therapeutic potential. Small molecule antagonists ALE-0540, PD90780, Ro 08-2750, and PQC 083 have all been reported to inhibit NGF from binding the TrkA receptor. Interestingly, the characterization of the ability of these molecules to block NGF-p75(NTR) interactions has not been performed. In addition, the inhibitory action of these molecules has never been evaluated using surface plasmon resonance (SPR) spectroscopy, which has been proven to be highly useful in drug discovery applications. In the current study, we used SPR biosensors to characterize the binding of NGF to the p75(NTR) receptor in addition to characterizing the inhibitory potential of the known NGF antagonists. The results of this study provide the first evaluation of the ability of these compounds to block NGF binding to p75(NTR) receptor. In addition, only PD90780 was effective at inhibiting the interaction of NGF with p75(NTR), suggesting receptor selectivity between known NGF inhibitors.

Using surface plasmon resonance spectroscopy to characterize the inhibition of NGF-p75NTR and proNGF-p75NTR interactions by small molecule inhibitors

Nerve growth factor (NGF), a member of the neurotrophin family, acts to influence the survival and differentiation of neurons in both the central and peripheral nervous systems via its binding to the p75(NTR) and TrkA receptors. Its precursor, proNGF, has been shown to be the dominant form of NGF in the central nervous system, suggesting a biological function beyond its role as a precursor. Like NGF, proNGF is known to bind the p75(NTR) receptor. The dysregulation of both NGF and proNGF have been implicated in several pathologies, including neurodegenerative diseases linked to p75(NTR)-mediated apoptotic signaling. Therefore, the identification of small molecule inhibitors capable of inhibiting both NGF and proNGF-p75(NTR) interactions may be of therapeutic interest. In the present study, we examine the inhibitory action of known small molecule-based inhibitors PD90780, ALE-0540, Ro 08-2750, and PQC 083, as well as novel derivatives of these compounds, using surface plasmon resonance (SPR) spectroscopy.

A Surface Plasmon Resonance Spectroscopy Method for Characterizing Small-Molecule Binding to Nerve Growth Factor

Small-molecule inhibitors have been previously investigated to identify possible therapeutics for the treatment of chronic pain. In the present study, known nerve growth factor (NGF) inhibitors identified by 125I-NGF binding were characterized using affinity and binding evaluations by surface plasmon resonance (SPR) spectroscopy. A novel strategy for characterizing NGF inhibitors was used to determine the binding affinity (KD) and saturation ability of each compound with immobilized NGF. Seventy-four percent of compounds screened demonstrated a positive binding event to NGF. A KD less than 10 μM and a percent saturation greater than 50% were used as thresholds to identify inhibitors that would warrant further investigation. This study details for the first time a methodology that can be used to directly characterize the binding event between small-molecule inhibitors and NGF.

Bioprospecting freshwater microalgae for antibacterial activity from water bodies associated with abandoned mine sites

Abstract: Bacterial resistance to antibiotics necessitates the search for new sources of microorganisms able to produce these needed molecules, which are typically secondary metabolites produced as a protective mechanism. Microalgae can produce a wide range of secondary metabolites as a response to environmental stress but have been the subject of little research as potential sources of antibiotics. As a step towards assessing their potential, we isolated 40 freshwater green microalgae from water bodies with a wide range of metal concentrations and pH values that were near abandoned mine sites in northern Canada. Microalgae from this region and these types of water bodies had not been previously investigated for antibacterial properties. Forty methanolic microalgal extracts were obtained, analysed and tested against Gram-positive and Gram-negative bacteria, and 37.5% inhibited the human pathogen Staphylococcus aureus (Bacilli). This is a higher ‘hit-rate’ than in previously published results, and furthermore, the minimum inhibitory concentrations against S. aureus were notably much lower than any other reported work. This is the first time such environments have been assessed, and whilst no clear association was observed between the metals and pH analysed and antibacterial activity, the findings indicate that microalgae from anthropogenically stressed environments are a potential source of antibacterial compounds. That is, sites that are typically regarded as having no value and often very negatively perceived are potential sources of valuable bioactive compounds. Therefore, future studies are necessary to determine what environmental thresholds are associated with the antibacterial activity of the freshwater green microalgae thriving in these environments.

Nerve growth factor inhibitor with novel‐binding domain demonstrates nanomolar efficacy in both cell‐based and cell‐free assay systems

Nerve growth factor (NGF), a member of the neurotrophin family, is known to regulate the development and survival of a select population of neurons through the binding and activation of the TrkA receptor. Elevated levels of NGF have been associated with painful pathologies such as diabetic neuropathy and fibromyalgia. However, completely inhibiting the NGF signal could hold significant side effects, such as those observed in a genetic condition called congenital insensitivity to pain and anhidrosis (CIPA). Previous methods of screening for NGF‐inhibitors used labeling techniques which have the potential to alter molecular interactions. SPR spectroscopy and NGF‐dependent cellular assays were utilized to identify a novel NGF‐inhibitor, BVNP‐0197 (IC50 = 90 nmol/L), the first NGF‐inhibitor described with a high nanomolar NGF inhibition efficiency. The present study utilizes molecular modeling flexible docking to identify a novel binding domain in the loop II/IV cleft of NGF.