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Abstract: Several proteins are covalently bound to the cell wall glucan (glucan-associated proteins (GAPs)) in Candida albicans and different drugs may cause their modulation. Proteomic analysis is a suitable approach to study differential GAP patterns between control and drug-treated cells. Since antimycotics induce variation in GAP content, we investigated the effect of a sublethal dose of micafungin and observed a clear increase in Bgl2p, an enzyme with glucanosyltransferase activity, with respect to a general decrease in cell wall protein content. Immunoelectron microscopy using mouse antiserum confirmed this increase of Bgl2p on the outer cell wall but also revealed a dramatic increase in the immature Bgl2p isoform in the cytoplasm of drug-treated cells. Since this increased expression of Bgl2p is clearly dependent upon micafungin treatment, this enzyme appears to be one of the survival strategies of C. albicans and thus could be considered the molecular basis of antifungal resistance and also as a potential valuable candidate for future vaccine development. [Copyright &y& Elsevier]

Although very attractive for noninvasive specimen collection, saliva has not yet been considered a relevant bodily fluid for the diagnosis and prognosis of diseases. The functional roles of specific salivary peptides and proteins have also not yet been studied in detail. Recent proteomic analysis of human whole saliva has shown that salivary biomarkers could contribute to the detection of local and systemic diseases, provided the standardization of proper sampling procedures exists. Recently, interesting and novel functions for different families of specific secretory peptides and proteins have been demonstrated, which could be a basis for the design of peptidomimetics with relevant biotechnological applications. In this review, we focus on the most recent advances in analysing salivary proteins and their potential application in biotechnology. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the second most common cause of cancer death, leading to almost 1 million deaths per year. Despite constant progress in surgical and therapeutic protocols, the 5-year survival rate of advanced CRC patients remains extremely poor. Colorectal Cancer Stem Cells (CRC-CSCs) are endowed with unique stemness-related properties responsible for resistance, relapse and metastasis. The development of novel therapeutics able to tackle CSCs while avoiding undesired toxicity is a major need for cancer treatment. Natural products are a large reservoir of unexplored compounds with possible anticancer bioactivity, sustainability, and safety. The family of meroterpenoids derived from sponges share interesting bioactive properties. Bioassay-guided fractionation of a meroterpenoids extract led to the isolation of three compounds, all cytotoxic against several cancer cell lines: Metachromins U, V and W. In this study, we evaluated the anticancer potential of the most active one, Metachromins V (MV), on patient-derived CRC-CSCs. MV strongly impairs CSCs-viability regardless their mutational background and the cytotoxic effect is maintained on therapy-resistant metastatic CSCs. MV affects cell cycle progression, inducing a block in G2 phase in all the cell lines tested and more pronouncedly in CRC-CSCs. Moreover, MV triggers an important reorganization of the cytoskeleton and a strong reduction of Rho GTPases expression, impairing CRC-CSCs motility and invasion ability. By Proteomic analysis identified a potential molecular target of MV: CCAR1, that regulates apoptosis under chemotherapy treatments and affect β-catenin pathway. Further studies will be needed to confirm and validate these data in in vivo experimental models • The development of novel therapeutics able to tackle CSCs is a major need for cancer treatment. • The family of meroterpenoids derived from sponges share interesting bioactive properties. • Metachromins V has anticancer potential on patient-derived CRC-CSCs. • A potential molecular target of Methacromins V has been identified: CCAR1, that regulates apoptosis under chemotherapy. [ABSTRACT FROM AUTHOR]

By-products of the fish industry can be a source of valuable molecules, with different technological applications. Fish bones contain both organic and inorganic phases (collagen/other proteins and calcium phosphate minerals respectively), with several uses and among them in cosmetics and medicine. Using the common extraction protocols, only one of the two phases can be extracted, while the other one is degraded. In the present work a method of extraction which allows the obtainment of both proteins and calcium phosphates was developed. Bones from two different species (salmon and sea bream) were tested. Saline solutions based on (NH 4)HCO 3 /(NH 4) 2 HPO 4 or (NH 4)HCO 3 were employed for the protein extraction. Analysis of the organic phase showed that the main extracted proteins were collagen in its different forms (especially α-1); some differences were observed between salmon and sea bream and between the methods of extraction. As for the mineral phase, after calcination at 700 °C, single phase hydroxyapatite was obtained with (NH 4)HCO 3 solution, while the use of (NH 4)HCO 3 /(NH 4) 2 HPO 4 led to a biphasic material of β-tricalcium phosphate and β-calcium pyrophosphate. Biocompatibility tests performed on both organic and inorganic extracts (with fibroblast and osteoblast-like cellular lines) showed their non-toxicity and, hence, their potential suitability for cosmetic or biomedical applications. This work shows that it is possible to obtain a more complete valorisation of the fish bones, with the simultaneous extraction of organic and inorganic valuable compounds. The principles of this process could be applied also to bones of other species, with a positive impact on the environment for the reduction of the wastes and a better use of all available resources. [Display omitted] • Innovative extraction process was developed for valorisation of fish bones. • Both organic (proteins) and inorganic (calcium phosphates) phases were extracted. • The process did not use toxic solvent but a saline aqueous solution. • The main component of the protein fraction was collagen, in its different forms. • Both proteins and calcium phosphates showed high biocompatibility. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a serious health and social problem that will have a substantial impact in the coming years on the world health and economy. Thus, the increasing demand for innovative antibiotics, has prompted many researchers in the medical, microbiological, and biochemical fields to exploit the properties of antimicrobial peptides (AMPs). When properly used, designed, and conveyed, AMPs can really represent a valid alternative to conventional drugs especially in situations that are particularly difficult to treat such as chronic infections found in Cystic Fibrosis (CF) patients. In this review we focused on the applications of AMPs in the specific field of CF, illustrating different types of peptides from natural, naturally modified, synthetic as well as the different strategies used to overcome the barriers, and the physiological conditions in which AMPs must operate. [ABSTRACT FROM AUTHOR]

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents. [ABSTRACT FROM AUTHOR]

The use of inhalable nanoparticles (NPs) for cystic fibrosis (CF) has been advocated as a promising tool to improve the efficacy of antimicrobials taking advantage of their ability to penetrate airway mucus and pathogen biofilm and to release the drug in or in proximity to the enclosed bacteria. Here, inhalable calcium phosphate (CaP) NPs were functionalized with colistin (Col) which is one of the most active antimicrobials against Gram-negative bacteria. The adsorption kinetic and isotherm of Col on CaP-NPs were investigated and fitted according to different mathematical models and revealed an electrostatic interaction between positively charged amine groups of Col and negatively charged surface of CaP-NPs. The maximum Col payload was of about 50 mg g−1 of CaP-NPs. After functionalization, despite an increase of size (213 vs 95 nm), in citrate solution, CaP-NPs maintained a dimension and surface charge considered suitable for crossing mucus barrier. CaP-NPs do not interact with mucin and are able to permeate a layer of artificial mucus. In vitro tests on pulmonary cells demonstrated that CaP-NPs are not cytotoxic up to a concentration of 125 μg mL−1. The antimicrobial and antibiofilm activity of Col loaded CaP-NPs tested on Pseudomonas aeruginosa RP73, a clinical strain isolated from a CF patient, was similar to that of free Col demonstrating that the therapeutic effect of Col adsorbed on CaP-NPs was retained. This work represents the first attempt to use CaP-NPs as delivery system for the CF treatment. The encouraging results open the way to further studies. The adsorption of colistin (Col), an antimicrobial against Gram-negative bacteria, on calcium phosphate nanoparticles was evaluated. In vitro tests demonstrated that the samples are not-cytotoxic towards pulmonary cells and the activity of Col was retained. Results open the way for a new formulation to treat cystic fibrosis Pseudomonas aeruginosa infections [Display omitted] • Calcium phosphate nanoparticles were successfully functionalized with colistin. • The interaction of nanoparticles and colistin is of electrostatic nature. • Nanoparticles are not mucoadhesive and able to permeate a layer of artificial mucus. • Pulmonary cells well tolerate an acute administration of nanoparticles. • Colistin adsorbed on nanoparticles maintains antimicrobial and antibiofilm activity. [ABSTRACT FROM AUTHOR]

Abstract: A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC50 of 2.2μM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, β-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage. [Copyright &y& Elsevier]

Abstract: A series of N-substituted-3-[(2′-hydroxy-4′-prenyloxy)-phenyl]-5-phenyl-4,5-dihydro-(1H)-pyrazolines were synthesized and tested on human monoamine oxidase-A and -B isoforms. Structure–activity relationships and molecular modelling showed that some substitutions, such as benzyloxy or chlorine atom, improve the best interaction with active site of hMAO-B. [ABSTRACT FROM AUTHOR]

Abstract: Hepcidin 25 (hep-25) is a peptide primarily produced by human liver with a central role in iron homeostasis. Its isoform, hepcidin 20 (hep-20), has an unknown function and lacks the first five aminoacids of the amino-terminal portion. This sequence is crucial for iron regulation by hep-25 and contains a molecular motif able to bind metals. Aim of this study, was to evaluate the antibacterial properties of both peptides in vitro, against a wide range of bacterial clinical isolates and in different experimental conditions. Although both peptides were found to be bactericidal against a variety of clinical isolates with different antibiotic resistance profiles, hep-20 was active at lower concentrations than hep-25, in most of the cases. Killing kinetics, carried on in sodium-phosphate buffer at pH 7.4, demonstrated that bactericidal activity occurred not earlier than 30–90min of incubation. Bactericidal activity of hep-25 was slightly enhanced in the presence of copper, while the same metal did not affect the activity of hep-20. Interestingly, bactericidal activity of both hepcidins was highly enhanced at acidic pH. Acidic pH (pH 5.0 and 6.6) not only reduced the microbicidal concentrations of hepcidins, but also shortened the killing times of both peptides, as compared to pH 7.4. Combining hep-20 and hep-25 at pH 5.0 a bactericidal effect could be obtained at very low concentrations of both peptides. These results render hepcidins interesting for the design of new drugs for the treatment of infections occurring in body districts with physiologic acidic pH. [ABSTRACT FROM AUTHOR]