Inflammation protein quantification by multiple reaction monitoring mass spectrometry in lipopolysaccharide-stimulated THP-1 cells

Rationale: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. Methods: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. Results: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. Conclusions: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis

Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease

Chronic respiratory infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients, and are characterized by the development of multidrug resistance (MDR) phenotype and biofilm formation, generally recalcitrant to treatment with conventional antibiotics. Hence, novel eective strategies are urgently needed. Antimicrobial peptides represent new promising therapeutic agents. Here, we analyze for the first time the ecacy of three versions of a cryptide identified in human apolipoprotein B (ApoB, residues 887-922) towards bacterial strains clinically isolated from CF patients. Antimicrobial and anti-biofilm properties of ApoB-derived cryptides have been analyzed by broth microdilution assays, crystal violet assays, confocal laser scanning microscopy and scanning electron microscopy. Cell proliferation assays have been performed to test cryptide eects on human host cells. ApoB-derived cryptides have been found to be endowed with significant antimicrobial and anti-biofilm properties towards Pseudomonas and Burkholderia strains clinically isolated from CF patients. Peptides have been also found to be able to act in combination with the antibiotic ciprofloxacin, and they are harmless when tested on human bronchial epithelial mesothelial cells. These findings open interesting perspectives to cryptide applicability in the treatment of chronic lung infections associated with CF disease

Edible Films Made of Dried Olive Leaf Extract and Chitosan: Characterization and Applications

Nowadays a possible strategy in food preservation consists of the use of active and functional packaging to improve safety and ensure a longer shelf life of food products. Many studies refer to chitosan-based films because of the already-known chitosan (CH) antibacterial and antifungal activity. In this work, we developed CH-based films containing Dried Olive Leaf Extract (DOLE) obtained by Naviglio extractor, with the aim to investigate the polyphenols yield and the antioxidant activity of this extract entrapped in CH-based-edible films. Olive tree cultivation produces a huge amount of byproducts that are usually simply burned. Phenolic compounds are already studied for their beneficial effects on human health. Some studies reported that phenols isolated from olive leaves have been shown to inhibit the growth of different strains of microorganisms. Thus, the antimicrobial effect of DOLE-containing films against bacterial strains (Salmonella enterica subsp. enterica serovar Typhimurium ATCC® 14028, Salmonella enteritidis RIVM 706, and Enterococcus faecalis ATCC® 29212) was tested in vitro. The DOLE component of the films is effective in inhibiting all the bacteria tested in a dose-dependent manner. Thus, it was demonstrated that these edible films can act as active bioplastics when used to wrap hamburgers in substitution for baking paper, which is normally used

Host defense peptides identified in human apolipoprotein B as natural food bio-preservatives: Evaluation of their biosafety and digestibility

The employment of chemical agents in the food industry is raising several concerns by consumers and is leading to an increasing interest in natural food preservatives. Among alternatives, host defense peptides (HDPs) have attracted great interest for their ability to preserve food samples from contamination without altering their quality, taste, and organoleptic properties. Recently, we evaluated the applicability of ApoB-derived peptides as novel food bio-preservatives and demonstrated their ability to prevent chicken meat sample contamination when immobilized on chitosan films. To perform a further step towards the applicability of these peptides in the food field, here we evaluated peptides biosafety and digestibility. To do this, we used a multidisciplinary approach including the evaluation of the peptides' toxicity and antimicrobial activity, the analysis of resistance phenotype development, an in silico prediction of the peptides' susceptibility to proteases and the evaluation of the peptides' stability in simulated gastric and intestinal fluids. ApoB-derived peptides were found to be nontoxic when tested on human gastric carcinoma cells SNU-1 and on human colon-rectal adenocarcinoma cells HT-29, and not to induce resistance phenotype in Salmonella strains. Bioinformatic analyses showed that the peptides are susceptible to several proteases, as also confirmed by experiments in simulated gastric and intestinal fluids. Altogether these findings open interesting perspectives to the future applicability of ApoB-derived peptides as novel food biopreservatives

Antimicrobial and antibiofilm activity of specialized metabolites isolated from Centaurea hyalolepis

The discovery of plant-derived compounds that are able to combat antibiotic-resistant pathogens is an urgent demand. Over years, Centaurea hyalolepis attracted considerable attention because of its beneficial medical properties. Phytochemical analyses revealed that Centaurea plant species contain several metabolites, such as sesquiterpene lactones (STLs), essential oils, flavonoids, alkaloids, and lignans.The organic extract of C. hyalolepis plant, collected in Palestine, showed significant antimicrobial properties towards a panel of Gram-negative and Gram-positive bacterial strains when the Minimal Inhibitory Concentration (MIC) values were evaluated by broth microdilution assays. A bio-guided fractionation of the active extract via multiple steps of column and thin layer chromatography allowed us to obtain three main compounds. The isolated metabolites were identified as the STLs cnicin, 11β,13-dihydrosalonitenolide and salonitenolide by spectroscopic and spectrometric analyses. Cnicin conferred the strongest antimicrobial activity among the identified compounds. Moreover, the evaluation of its antibiofilm activity by biomass assays through crystal violet staining revealed almost 30% inhibition of biofilm formation in the case of A. baumannii ATCC 17878 strain. Furthermore, the quantification of carbohydrates and proteins present in the extracellular polymeric substance (EPS) revealed the ability of cnicin to significantly perturb biofilm structure. Based on these promising results, further investigations might open interesting perspectives to its applicability in biomedical field to counteract multidrug resistant infections

Host defence peptides identified in human apolipoprotein B as promising antifungal agents

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents

Novel Antimicrobial Strategies to Prevent Biofilm Infections in Catheters after Radical Cystectomy: A Pilot Study

Catheter-associated infections in bladder cancer patients, following radical cystectomy or ureterocutaneostomy, are very frequent, and the development of antibiotic resistance poses great challenges for treating biofilm-based infections. Here, we characterized bacterial communities from catheters of patients who had undergone radical cystectomy for muscle-invasive bladder cancer. We evaluated the efficacy of conventional antibiotics, alone or combined with the human ApoB-derived antimicrobial peptide r(P)ApoBLAla, to treat ureteral catheter-colonizing bacterial communities on clinically isolated bacteria. Microbial communities adhering to indwelling catheters were collected during the patients' regular catheter change schedules (28 days) and extracted within 48 h. Living bacteria were characterized using selective media and biochemical assays. Biofilm growth and novel antimicrobial strategies were analyzed using confocal laser scanning microscopy. Statistical analyses confirmed the relevance of the biofilm reduction induced by conventional antibiotics (fosfomycin, ceftriaxone, ciprofloxacin, gentamicin, and tetracycline) and a well-characterized human antimicrobial peptide r(P)ApoBLAla (1:20 ratio, respectively). Catheters showed polymicrobial communities, with Enterobactericiae and Proteus isolates predominating. In all samples, we recorded a meaningful reduction in biofilms, in both biomass and thickness, upon treatment with the antimicrobial peptide r(P)ApoBLAla in combination with low concentrations of conventional antibiotics. The results suggest that combinations of conventional antibiotics and human antimicrobial peptides might synergistically counteract biofilm growth on ureteral catheters, suggesting novel avenues for preventing catheter-associated infections in patients who have undergone radical cystectomy and ureterocutaneostomy

Insights into the interaction of the N-terminal amyloidogenic polypeptide of ApoA-I with model cellular membranes

BACKGROUND: About twenty variants of apolipoprotein A-I (ApoA-I) are associated to hereditary systemic amyloidoses. Although the molecular bases of this disease are still largely unknown, it has been hypothesized that ApoA-I proteolysis is a key event in pathogenesis, since it triggers the release of an N-terminal fragment (80-100 residue long) that misfolds to form amyloid deposits in peripheral organs and tissues. It is also known that cell membrane lipids play a key role in the fibrillogenic pathway. In the case of ApoA-I related amyloidosis caused by L174S mutation, the 93-residue N-terminal fragment of ApoA-I ([1-93]ApoA-I) was found to be the major constituent of ex vivo fibrils. METHODS: With the main goal to investigate the interaction of either [1-93]ApoA-I and ApoA-I with biomimetic membranes, we set-up an experimental system based on the Raman Tweezers methodology. We tested GUVs composed by two types of zwitterionic lipids with a different fluidity degree, i.e. dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC). RESULTS: We found that [1-93]ApoA-I induces conformational disorder in an ordered lipid bilayer. When interacting with fluid phases, instead, the fragment was found to be able to penetrate the membrane bilayer inducing an alignment of lipid chains. CONCLUSIONS: The interaction features of [1-93]ApoA-I with biomimetic membranes strongly depend on the lipid phase. Full-length ApoA-I was found to have similar effects, even if significantly less pronounced. GENERAL SIGNIFICANCE: Our observations shed light on still largely unknown molecular bases of ApoA-I fibrillogenic domain interaction with membranes

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