HelixComplex snail mucus as a potential technology against O3 induced skin damage

Mucus form H. aspersa muller has been reported to have several therapeutic proprieties, such as antimicrobial activity, skin protection and wound repair. In this study, we have analyzed H. aspersa mucus (Helixcomplex) bio-adhesive efficacy and its defensive properties against the ozone (O3) (0.5 ppm for 2 hours) exposure in human keratinocytes and reconstructed human epidermis models. Cytotoxicity, tissue morphology and cytokine levels were determined. We confirmed HelixComplex regenerative and bio-adhesive properties, the latter possibly via the characteristic mucopolysaccharide composition. In addition, HelixComplex was able to protect from O3 exposure by preventing oxidative damage and the consequent pro-inflammatory response in both 2D and 3D models. Based on this study, it is possible to suggest HelixComplex as a potentially new protective technology against pollution induced skin damage

Circadian Clock and OxInflammation: Functional Crosstalk in Cutaneous Homeostasis

Circadian rhythms are biological oscillations that occur with an approximately 24 h period and optimize cellular homeostasis and responses to environmental stimuli. A growing collection of data suggests that chronic circadian disruption caused by novel lifestyle risk factors such as shift work, travel across time zones, or irregular sleep-wake cycles has long-term consequences for human health. Among the multiplicity of physiological systems hypothesized to have a role in the onset of pathologies in case of circadian disruption, there are redox-sensitive defensive pathways and inflammatory machinery. Due to its location and barrier physiological role, the skin is a prototypical tissue to study the influence of environmental insults induced OxInflammation disturbance and circadian system alteration. To better investigate the link among outdoor stressors, OxInflammation, and circadian system, we tested the differential responses of keratinocytes clock synchronized or desynchronized, in an in vitro inflammatory model exposed to O3. Being both NRF2 and NF-κB two key redox-sensitive transcription factors involved in cellular redox homeostasis and inflammation, we analyzed their activation and expression in challenged keratinocytes by O3. Our results suggest that a synchronized circadian clock not only facilitates the protective role of NRF2 in terms of a faster and more efficient defensive response against environmental insults but also moderates the cellular damage resulting from a condition of chronic inflammation. Our results bring new insights on the role of circadian clock in regulating the redox-inflammatory crosstalk influenced by O3 and possibly can be extrapolated to other pollutants able to affect the oxinflammatory cellular processes

Biophysical characterization of antimicrobial peptides activity: from in vitro to ex vivo techniques.

Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune defense system of many living organisms varying from prokaryotes to eukaryotes, including humans. Due to their broad-spectrum activity and low level of induced resistance, these short aminoacid sequences represent a novel class of potential antimicrobial agents. Besides the development of anti-bacterial drugs, AMPs constitute ideal molecular models for the design of molecules with wide-ranging nanomedical applications, such as anti-tumorigenic agents and pharmacological tools to cure channelopaties. Several techniques are currently used to shed light on the mechanisms of action of AMPs, ranging from the characterization of the interaction between peptides and biomimetic membranes and/or intracellular targets, to the study of AMPs effects on pathogens, living cells and tissues. Comprehensive and multiscale studies are crucial to design new AMPs and to identify molecules that can boost their activity. In this minireview we summarize the most recent achievements in AMP-characterization, with a special emphasis on the integration of biophysical approaches, which can synergistically help to bridge the gap between in vitro and ex vivo investigations

Biophysical characterization of antimicrobial peptides activity: From in vitro to ex vivo techniques

Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune defense system of many living organisms varying from prokaryotes to eukaryotes, including humans. Due to their broad-spectrum activity and low level of induced resistance, these short aminoacid sequences represent a novel class of potential antimicrobial agents. Besides the development of anti-bacterial drugs, AMPs constitute ideal molecular models for the design of molecules with wide-ranging nanomedical applications, such as anti-tumorigenic agents and pharmacological tools to cure channelopaties. Several techniques are currently used to shed light on the mechanisms of action of AMPs, ranging from the characterization of the interaction between peptides and biomimetic membranes and/or intracellular targets, to the study of AMPs effects on pathogens, living cells and tissues. Comprehensive and multiscale studies are crucial to design new AMPs and to identify molecules that can boost their activity. In this minireview we summarize the most recent achievements in AMP-characterization, with a special emphasis on the integration of biophysical approaches, which can synergistically help to bridge the gap between in vitro and ex vivo investigations

A pressure-polishing set-up to fabricate patch pipettes that seal on virtually any membrane, yielding low access resistance and efficient intracellular perfusion.

When performing whole-cell configuration recordings, it is important to minimize series resistance to reduce the time constant of charging the cell membrane capacitance and to reduce error in membrane potential control. To this end, an existing method was improved by widening the patch pipette shank through the calibrated combination of heat and air pressure. The heat was produced by passing current through a filament that was shaped appropriately to ensure a homogeneous heating of the pipette shank. Pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor connected to a contrast-intensified CCD camera and coupled to a modified bright-field stereomicroscope. By appropriately regulating the timing of air pressure and the application of heating, the pipette shank and, independently, the tip opening diameter were widened as desired. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed the sealing of a wide variety of cell types isolated from different amphibian, reptilian, fish, and mammalian tissues as well as a variety of artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure-polished pipettes was approximately one-fourth the size of the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed for fast intracellular perfusion as shown by fluorescence imaging, also via pulled quartz or plastic tubes, which could be inserted very close to the pipette tip

Divalent cations regulate pore formation of synthetic, naturally occurring alamethicin and selected analogs.

This paper studies the stability properties of the concepts of local compactness introduced by the authors in 1998. We show that all of these concepts are stable for contractive, expansive images and for products

Selective neuroinhibitory effects of taurine in slices of rat main olfactory bulb

Taurine is abundant in the main olfactory bulb, exceeding glutamate and GABA in concentration. In whole-cell patch-clamp recordings in rat olfactory bulb slices, taurine inhibited principal neurons, mitral and tufted cells. In these cells, taurine decreased the input resistance and caused a shift of the membrane potential toward the chloride equilibrium potential. The taurine actions were sustained under the blockade of transmitter release and were reversible and dose-dependent. At a concentration of 5 mM, typically used in this study, taurine showed 90% of its maximal effect. GABAA antagonists, bicuculline and picrotoxin, blocked the taurine actions, whereas the glycine receptor antagonist strychnine and GABAB antagonists, CGP 55845A and CGP 35348, were ineffective. These findings are consistent with taurine directly activating GABAA receptors and inducing chloride conductance. Taurine had no effect on periglomerular and granule interneurons. The subunit composition of GABAA receptors in these cells, differing from those in mitral and tufted cells, may account for taurine insensitivity of the interneurons. Taurine suppressed olfactory nerve-evoked monosynaptic responses of mitral and tufted cells while chloride conductance was blocked. This action was mimicked by the GABAB agonist baclofen and abolished by CGP 55845A; CGP 35348, which primarily blocks postsynaptic GABAB receptors, was ineffective. The taurine effect most likely was due to GABAB receptor-mediated inhibition of presynaptic glutamate release. Neither taurine nor baclofen affected responses of periglomerular cells. The lack of a baclofen effect implies that functional GABAB receptors are absent from olfactory nerve terminals that contact periglomerular cells. These results indicate that taurine decreases the excitability of mitral and tufted cells and their responses to olfactory nerve stimulation without influencing periglomerular and granule cells. Selective effects of taurine in the olfactory bulb may represent a physiologic mechanism that is involved in the inhibitory shaping of the activation pattern of principal neurons

Pore forming properties of cecropin-melittin hybrid peptide in a natural membrane

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH(2); CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in approximately 50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was >or=1 G Omega, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15

A Pressure-Polish Setup to Fabricate Patch Pipettes Yielding Low Access Resistance and Efficient Intracellular Perfusion

When performing whole-cell configuration recordings, it is important to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. To this goal, it was improved an existing method widening the patch pipette shank, through the calibrated combination of heat and air pressure. The heat was produced by passing current in a filament shaped appropriately to ensure an homogeneous heating of the pipette shank. The pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on a LCD monitor, connected to a contrast-intensified CCD camera, coupled to a modified bright-field stereomicroscope. By appropriately regulating timing of air pressure and heating application, the pipette shank was widened as desired, independently by the tip opening diameter. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed to seal on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure polished pipettes resulted ~4-fold smaller than the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed fast intracellular perfusion, as shown by fluorescence imaging, also via pulled quartz or plastic tubes, that could be inserted very close to the pipette tip