Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2-independent of its chiral state-significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.

Aggregation of the natively unfolded protein αsynuclein (α-Syn) has been widely correlated to the neuronal death associated with Parkinson's disease. Mutations and protein overaccumulation can promote the aggregation of α-Syn into oligomers and fibrils. Recent work has suggested that α-Syn oligomers can permeabilize the neuronal membrane, promoting calcium influx and cell death. However, the mechanism of this permeabilization is still uncertain and has yet to be characterized in live cells. This work uses scanning ion conductance microscopy (SICM) to image, in real time and without using chemical probes, the topographies of live SH-SY5Y neuroblastoma cells after exposure to α-Syn oligomers. Substantial morphological changes were observed, with micrometer-scale hills and troughs observed at lower α-Syn concentrations (1.00 μM) and large, transient pores observed at higher α-Syn concentrations (6.0 μM). These findings suggest that α-Syn oligomers may permeabilize the neuronal membrane by destabilizing the lipid bilayer and opening transient pores.

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Nanosensors built with pre-pulled glass nanopipettes, including bare or chemically modified nanopipettes and fully or partially filled solid nanoelectrodes, have found applications in chemical and biological sensing via resistive-pulse, current rectification, and electrochemical sensing. These nanosensors are easily fabricated and provide advantages through their needle-like geometry with nanometer-sized tips, making them highly sensitive and suitable for local measurements in extremely small samples. The variety in the geometry and layout have extended sensing capabilities. In this review, we will outline the fundamentals in fabrication, modification, and characterization of those pre-pulled glass nanopipette based nanosensors and highlight the most recent progress in their development and applications in real-time monitoring of biological processes, chemical ion sensing, and single entity analysis.

Parkinson's disease (PD) is recognized as the second most common neurodegenerative disorder and has affected approximately one million people in the United States alone. A large body of evidence has suggested that deposition of aggregated alpha-synuclein (α-Syn), a brain protein abundant near presynaptic termini, in intracellular protein inclusions (Lewy bodies) results in neuronal cell damage and ultimately contributes to the progression of PD. However, the exact mechanism is still unclear. One hypothesis is that α-Syn aggregates disrupt the cell membrane's integrity, eventually leading to cell death. We used scanning ion conductance microscopy (SICM) to monitor the morphological changes of SH-SY5Y neuroblastoma cells and observed dramatic disruption of the cell membrane after adding α-Syn aggregates to the culturing media. This work demonstrates that SICM can be applied as a new approach to studying the cytotoxicity of α-Syn aggregates.