Tailoring the stress response of human skin cells by substantially limiting the nuclear localization of angiogenin.

Human angiogenin (hANG) is the most studied stress-induced ribonuclease (RNase). In physiological conditions it performs its main functions in nucleoli, promoting cell proliferation by rDNA transcription, whereas it is strongly limited by its inhibitor (RNH1) throughout the rest of the cell. In stressed cells hANG dissociates from RNH1 and thickens in the cytoplasm where it manages the translational arrest and the recruitment of stress granules, thanks to its propensity to cleave tRNAs and to induce the release of active halves. Since it exists a clear connection between hANG roles and its intracellular routing, starting from our recent findings on heterologous ANG (ANG) properties in human keratinocytes (HaCaT cells), here we designed a variant unable to translocate into the nucleus with the aim of thoroughly verifying its potentialities under stress. This variant, widely characterized for its structural features and biological attitudes, shows more pronounced aid properties than unmodified protein. The collected evidence thus fully prove that ANG stress-induced skills in assisting cellular homeostasis are strictly due to its cytosolic localization. This study opens an interesting scenario for future studies regarding both the strengthening of skin defences and in understanding the mechanism of action of these special enzymes potentially suitable for any cell type.

The Antimicrobial, Antibiofilm and Anti-Inflammatory Activities of P13#1, a Cathelicidin-like Achiral Peptoid.

Cationic antimicrobial peptides (CAMPs) are powerful molecules with antimicrobial, antibiofilm and endotoxin-scavenging activities. These properties make CAMPs very attractive drugs in the face of the rapid increase in multidrug-resistant (MDR) pathogens, but they are limited by their susceptibility to proteolytic degradation. An intriguing solution to this issue could be the development of functional mimics of CAMPs with structures that enable the evasion of proteases. Peptoids (N-substituted glycine oligomers) are an important class of peptidomimetics with interesting benefits: easy synthetic access, intrinsic proteolytic stability and promising bioactivities. Here, we report the characterization of P13#1, a 13-residue peptoid specifically designed to mimic cathelicidins, the best-known and most widespread family of CAMPs. P13#1 showed all the biological activities typically associated with cathelicidins: bactericidal activity over a wide spectrum of strains, including several ESKAPE pathogens; the ability to act in combination with different classes of conventional antibiotics; antibiofilm activity against preformed biofilms of Pseudomonas aeruginosa, comparable to that of human cathelicidin LL-37; limited toxicity; and an ability to inhibit LPS-induced proinflammatory effects which is comparable to that of "the last resource" antibiotic colistin. We further studied the interaction of P13#1 with SDS, LPSs and bacterial cells by using a fluorescent version of P13#1. Finally, in a subcutaneous infection mouse model, it showed antimicrobial and anti-inflammatory activities comparable to ampicillin and gentamicin without apparent toxicity. The collected data indicate that P13#1 is an excellent candidate for the formulation of new antimicrobial therapies.

Simultaneous Irradiation with UV-A, -B, and -C Lights Promotes Effective Decontamination of Planktonic and Sessile Bacteria: A Pilot Study.

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Cytotoxicity of an Innovative Pressurised Cyclic Solid-Liquid (PCSL) Extract from Artemisia annua.

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid-liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

Ribotoxin-like proteins from Boletus edulis: structural properties, cytotoxicity and in vitro digestibility.

Porcini are edible mushrooms widely used in cooking due to their extraordinary taste. Despite this, cases of food poisoning have been reported in the recent literature also for ingestion of porcini. Here, we report the isolation from Boletus edulis fruiting bodies of two novel ribotoxin-like proteins (RL-Ps), enzymes already studied in other organisms for their toxicity. These RL-Ps, named Edulitin 1 (16-kDa) and Edulitin 2 (14-kDa), show peculiar structural and enzymatic differences, which probably reflect their different bio-activities and a dose/time dependent toxicity (Edulitin 2) on normal and tumoral human cells. Particularly interesting is the resistance to proteolysis of Edulitin 2, for which it was observed that its toxicity was abolished only after heat treatment (90 °C) followed by proteolysis. As mushroom poisoning is a serious food safety issue, data here presented confirm the existence of toxins also in porcini and the importance of a proper cooking before their consumption.

The structural features of an ancient ribonuclease from Salmo salar reveal an intriguing case of auto-inhibition.

The superfamily of vertebrate ribonucleases, a large group of evolutionarily related proteins, continues to provide interesting structural and functional information. In particular, the crystal structure of SS-RNase-2 from Salmo salar (SS2), here presented, has revealed a novel auto-inhibition mechanism that enriches the number of inhibition strategies observed in some members of the family. Within an essentially unmodified RNase folding, the SS2 active site cleft is in part obstructed by the collapse of an extra pentapeptide inserted in the C-terminal region. This unexpected intrusion alters the organization of the catalytic triad by pushing one catalytic histidine off the pocket. Possible mechanisms to remove the active site obstruction have also been studied through the production of two mutants that provide useful information on the functionality of this intriguing version of the ribonuclease superfamily.

Dietary Micronutrient Management to Treat Mitochondrial Dysfunction in Diet-Induced Obese Mice.

Obesity and associated metabolic disturbances, which have been increasing worldwide in recent years, are the consequences of unhealthy diets and physical inactivity and are the main factors underlying non-communicable diseases (NCD). These diseases are now responsible for about three out of five deaths worldwide, and it has been shown that they depend on mitochondrial dysfunction, systemic inflammation and oxidative stress. It was also demonstrated that several nutritional components modulating these processes are able to influence metabolic homeostasis and, consequently, to prevent or delay the onset of NCD. An interesting combination of nutraceutical substances, named DMG-gold, has been shown to promote metabolic and physical wellness. The aim of this research was to investigate the metabolic, inflammatory and oxidative pathways modulated by DMG-gold in an animal model with diet-induced obesity. Our data indicate that DMG-gold decreases the metabolic efficiency and inflammatory state and acts as an antioxidant and detoxifying agent, modulating mitochondrial functions. Therefore, DMG-gold is a promising candidate in the prevention/treatment of NCD.

A Marine Isolate of Bacillus pumilus Secretes a Pumilacidin Active against Staphylococcus aureus.

Producing antimicrobials is a common adaptive behavior shared by many microorganisms, including marine bacteria. We report that SF214, a marine-isolated strain of Bacillus pumilus, produces at least two different molecules with antibacterial activity: a molecule smaller than 3 kDa active against Staphylococcus aureus and a molecule larger than 10 kDa active against Listeria monocytogenes. We focused our attention on the anti-Staphylococcus molecule and found that it was active at a wide range of pH conditions and that its secretion was dependent on the growth phase, medium, and temperature. A mass spectrometry analysis of the size-fractionated supernatant of SF214 identified the small anti-Staphylococcus molecule as a pumilacidin, a nonribosomally synthesized biosurfactant composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. The analysis of the SF214 genome revealed the presence of a gene cluster similar to the srfA-sfp locus encoding the multimodular, nonribosomal peptide synthases found in other surfactant-producing bacilli. However, the srfA-sfp cluster of SF214 differed from that present in other surfactant-producing strains of B. pumilus by the presence of an insertion element previously found only in strains of B. safensis.