Creating wounds in cell monolayers using micro-jets.

Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a contactless method to create wounds with any imaginable 2D pattern in cell monolayers using the micro-jets of either media or an immiscible and biocompatible fluorocarbon (i.e., FC40). We also combine this with another method that allows automation and multiplexing using standard Petri dishes. A dish is filled with a thin film of media overlaid with FC40, and the two liquids are reshaped into an array of microchambers within minutes. Each chamber in such a grid is isolated from others by the fluid walls of FC40. Cells are now added, allowed to grow into a monolayer, and wounds are created using the microjets; then, healing is monitored by microscopy. As arrays of chambers can be made using media and Petri dishes familiar to biologists, and as dishes fit seamlessly into their incubators, microscopes, and workflows, we anticipate that this assay will find wide application in wound healing.

Nerve growth factor-mediated photoablation of nociceptors reduces pain behavior in mice.

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here, we describe novel technology to selectively photoablate TrkA-positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near-infrared illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic, and joint pain. To target peripheral nociceptors, we generated a SNAP-tagged NGF derivative NGF that binds to TrkA/p75 receptors but does not provoke signaling in TrkA-positive cells or elicit pain behaviors in mice. NGF was coupled to the photosensitizer IRDye700DX phthalocyanine (IR700) and injected subcutaneously. After near-infrared illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic, and neuropathic pain, mechanical hypersensitivity was abolished for 3 weeks after a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then reinnervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGF-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.

Lactate signalling regulates fungal ß-glucan masking and immune evasion.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of ß-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces ß-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to ß-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.