Fighting Microbial Infections from Escherichia coli O157:H7: The Combined Use of Three Essential Oils of the Cymbopogon Genus and a Derivative of Esculentin-1a Peptide.

The absence of effective therapy against Escherichia coli O157:H7 infections has led to the need to develop new antimicrobial agents. As the use of synergistic combinations of natural antimicrobial compounds is growing as a new weapon in the fight against multidrug-resistant bacteria, here, we have tested new synergistic combinations of natural agents. Notably, we investigated a possible synergistic effect of combinations of essential oils and natural peptides to counteract the formation of biofilm. We chose three essential oils (i.e., Cymbopogon citratus, C. flexuosus and C. martinii) and one peptide already studied in our previous works. We determined the fractional inhibitory concentration (FIC) by analyzing the combination of the peptide derived from esculentin-1a, Esc(1-21), with the three essential oils. We also studied the effects of combinations by time-kill curves, scanning electron microscopy on biofilm and Sytox Green on cell membrane permeability. Finally, we analyzed the expression of different genes implicated in motility, biofilm formation and stress responses. The results showed a different pattern of gene expression in bacteria treated with the mixtures compared to those treated with the peptide or the single C. citratus essential oil. In conclusion, we demonstrated that the three essential oils used in combination with the peptide showed synergy against the E. coli O157:H7, proving attractive as an alternative strategy against E. coli pathogen infections.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

Phytochemical Analysis and In Vitro Antileukemic Activity of Alkaloid-Enriched Extracts from Vinca sardoa (Stearn) Pignatti.

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1-15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations.

Gold nanorods derivatized with CTAB and hydroquinone or ascorbic acid: spectroscopic investigation of anisotropic nanoparticles of different shapes and sizes.

Gold nanorods stabilized by binary ligand mixtures of cetyltrimethylammonium bromide (CTAB, primary ligand) and ascorbic acid or hydroquinone were investigated by complementary synchrotron radiation-induced spectroscopies and microscopies, with the aim to find evidence of the influence of the secondary ligand molecular and chemical structure on the nanorod shapes and size ratios. Indeed, as it is well known that the CTAB interaction with Ag(i) ions at the NR surface plays a key role in directing the anisotropic growth of nanorods, the possibility to finely control the NR shape and dimension by opportunely selecting the secondary ligands opens new perspectives in the design and synthesis of anisotropic nanoparticles.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

"In vivo" and "in vitro" antimicrobial activity of Origanum vulgare essential oil and its two phenolic compounds on clinical isolates of Candida spp.

A limited therapeutic arsenal is currently available against Candida infections that show high resistance to antifungal agents. For this reason, there is a great need to prioritize testing therapeutic agents for the treatment of candidiasis. The use of essential oils and their phytoconstituents has been emphasized as a new therapeutic approach. The cell surface hydrophobicity (CSH), polysaccharide content, antimicrobial activity of essential oil from Origanum vulgare L. (OVEO), and its two phenolic compounds carvacrol and thymol were evaluated in four different Candida spp. (Candida albicans and emerging non-albicans Candida (NAC) species, such as C. glabrata, C. tropicalis, and C. krusei). The results showed the differences between Candida species; for example, C. tropicalis revealed higher resistance than other strains to different natural molecule treatments. The ultrastructural variabilities in the biomembranes and cell walls of these Candida spp. might explain the different biological effects observed after OVEO, carvacrol and thymol treatments. Therefore, to study the biological effects of these natural compounds on Candida strains, the samples were observed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Moreover, the release of cellular materials and their "in vivo" antimicrobial activity on infected G. mellonella larvae were evaluated. The novelty of this study is the demonstration that exists a close correlation between both structural architecture of cell walls and biomembranes' organization with cell fungal responses to essential oils treatments. Overall, these results suggest practical limits to the predictability.

Peptide-Mediated Targeted Delivery of Aloe-Emodin as Anticancer Drug.

Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs' cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.

Ultrastructural Damages to H1N1 Influenza Virus Caused by Vapor Essential Oils.

Influenza viruses are transmitted from human to human via airborne droplets and can be transferred through contaminated environmental surfaces. Some works have demonstrated the efficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined the liquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viral cytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs and their vapor phase by the head-space gas chromatography-mass spectrometry technique, observing that the major component found in each liquid EO is the same one of the corresponding vapor phases, with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrity of virus particles was checked by negative staining transmission electron microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes. We speculated that the most abundant components of the vapor EOs might directly interfere with influenza virus envelope structures or mask viral structures important for early steps of viral infection.

Terpinen-4-ol, the Main Bioactive Component of Tea Tree Oil, as an Innovative Antimicrobial Agent against Legionella pneumophila.

Legionella pneumophila (Lp), responsible for a severe pneumonia called Legionnaires' disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti-Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06-0.125% v/v and MBC range of 0.25-0.5% v/v. The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v/v against Lp. SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.

Design, Synthesis, and In Vitro, In Silico and In Cellulo Evaluation of New Pyrimidine and Pyridine Amide and Carbamate Derivatives as Multi-Functional Cholinesterase Inhibitors.

Alzheimer disease is an age-linked neurodegenerative disorder representing one of the greatest medical care challenges of our century. Several drugs are useful in ameliorating the symptoms, even if none could stop or reverse disease progression. The standard approach is represented by the cholinesterase inhibitors (ChEIs) that restore the levels of acetylcholine (ACh) by inhibiting the acetylcholinesterase (AChE). Still, their limited efficacy has prompted researchers to develop new ChEIs that could also reduce the oxidative stress by exhibiting antioxidant properties and by chelating the main metals involved in the disease. Recently, we developed some derivatives constituted by a 2-amino-pyrimidine or a 2-amino-pyridine moiety connected to various aromatic groups by a flexible amino-alkyl linker as new dual inhibitors of AChE and butyrylcholinesterase (BChE). Following our previous studies, in this work we explored the role of the flexible linker by replacing the amino group with an amide or a carbamic group. The most potent compounds showed higher selectivity against BChE in respect to AChE, proving also to possess a weak anti-aggregating activity toward Aß42 and tau and to be able to chelate Cu2+ and Fe3+ ions. Molecular docking and molecular dynamic studies proposed possible binding modes with the enzymes. It is noteworthy that these compounds were predicted as BBB-permeable and showed low cytotoxicity on the human brain cell line.

High Activity of N-Acetylcysteine in Combination with Beta-Lactams against Carbapenem-Resistant Klebsiella pneumoniae and Acinetobacter baumannii.

AIM: The aim of the study was to evaluate the in vitro activity of N-acetylcysteine (NAC), alone or in combination with beta-lactams, against carbapenem-resistant Klebsiella pneumoniae (CR-Kp) and Acinetobacter baumannii (CR-Ab). METHODS: The antibacterial activity of each compound was tested by broth microdilution and the synergism was evaluated by the checkerboard method. Killing studies of NAC alone and in combination with beta-lactams were performed. Bacterial morphological changes were investigated with scanning electron microscopy (SEM). RESULTS: Overall, 30 strains were included (15 CR-Kp and 15 CR-Ab). The NAC Minimal Inhibitory Concentrations (MIC)50/90 were 5/5 and 2.5/5 mg/mL for CR-Kp and CR-Ab, respectively. For both microorganisms, NAC, in addition to beta-lactams (meropenem for CR-Kp, meropenem and ampicillin/sulbactam for CR-Ab, respectively), was able to enhance their activity. The killing studies showed a rapid and concentration-dependent activity of NAC alone; the addition of NAC to meropenem or ampicillin/sulbactam at subinhibitory concentrations induced a fast and lasting bactericidal activity that persisted over time. The SEM analyses showed evident morphological alterations of the bacterial cells following incubation with NAC, alone and in combination with meropenem. CONCLUSIONS: NAC demonstrated a high in vitro activity against CR-Kp and CR-Ab and was able to enhance beta-lactams' susceptibility in the tested strains. The preliminary data on the SEM analyses confirmed the in vitro results.

New Pyrimidine and Pyridine Derivatives as Multitarget Cholinesterase Inhibitors: Design, Synthesis, and In Vitro and In Cellulo Evaluation.

A new series of pyrimidine and pyridine diamines was designed as dual binding site inhibitors of cholinesterases (ChEs), characterized by two small aromatic moieties separated by a diaminoalkyl flexible linker. Many compounds are mixed or uncompetitive acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE) nanomolar inhibitors, with compound 9 being the most active on Electrophorus electricus AChE (EeAChE) (Ki = 0.312 µM) and compound 22 on equine BChE (eqBChE) (Ki = 0.099 µM). Molecular docking and molecular dynamic studies confirmed the interaction mode of our compounds with the enzymatic active site. UV-vis spectroscopic studies showed that these compounds can form complexes with Cu2+ and Fe3+ and that compounds 18, 20, and 30 have antioxidant properties. Interestingly, some compounds were also able to reduce Aß42 and tau aggregation, with compound 28 being the most potent (22.3 and 17.0% inhibition at 100 µM on Aß42 and tau, respectively). Moreover, the most active compounds showed low cytotoxicity on a human brain cell line and they were predicted as BBB-permeable.

Oxidative Stress and Male Fertility: Role of Antioxidants and Inositols.

Infertility is defined as a couple's inability to conceive after at least one year of regular unprotected intercourse. This condition has become a global health problem affecting approximately 187 million couples worldwide and about half of the cases are attributable to male factors. Oxidative stress is a common reason for several conditions associated with male infertility. High levels of reactive oxygen species (ROS) impair sperm quality by decreasing motility and increasing the oxidation of DNA, of protein and of lipids. Multi-antioxidant supplementation is considered effective for male fertility parameters due to the synergistic effects of antioxidants. Most of them act by decreasing ROS concentration, thus improving sperm quality. In addition, other natural molecules, myo-inositol (MI) and d-chiro-inositol (DCI), ameliorate sperm quality. In sperm cells, MI is involved in many transduction mechanisms that regulate cytoplasmic calcium levels, capacitation and mitochondrial function. On the other hand, DCI is involved in the downregulation of steroidogenic enzyme aromatase, which produces testosterone. In this review, we analyze the processes involving oxidative stress in male fertility and the mechanisms of action of different molecules.

Phytochemical Analysis and Biological Activities of the Ethanolic Extract of Daphne sericea Vahl Flowering Aerial Parts Collected in Central Italy.

In this paper, the first phytochemical analysis of the ethanolic extract of Daphne sericea Vahl flowering aerial parts collected in Italy and its biological activities were reported. Eleven compounds were identified i.e., α-linolenic acid (1), tri-linoleoyl-sn-glycerol (2), pheophorbide a ethyl ester (3), pilloin (4), sinensetin (5), yuanhuanin (6), rutamontine (7), syringin (8), p-coumaric acid (9), p-anisic acid (10) and caffeic acid (11). To the best of our knowledge, compounds (1-4, 7-8 and 10) were isolated from D. sericea for the first time during this work, whereas sinensetin (5) represents a newly identified component of the entire Thymelaeaceae family. The extract was found to possess radical scavenging against both DPPH• and 2,2'-azino-bis(3-thylbenzothiazoline-6-sulfonic acid (ABTS•+) radicals, with at least a 40-fold higher potency against the latter. Moreover, chelating abilities against both ferrous and ferric ions have been highlighted, thus suggesting a possible indirect antioxidant power of the extract. Although the precise bioactive compounds remain to be discovered, the polyphenolic constituents, including phenolic acids, tannins and flavonoids, seem to contribute to the antioxidant power of the phytocomplex. In addition, the extract produced cytotoxic effects in MDA-MB-231 and U87-MG cancer cell lines, especially at the concentration of 625 µg/mL and after 48-72 h. Further studies are required to clarify the contribution of the identified compounds in the bioactivities of the extract and to support possible future applications.

Antifungal Carvacrol Loaded Chitosan Nanoparticles.

The increased prevalence and incidence of fungal infections, of which Candida albicans represents one of the most life-threatening organisms, is prompting the scientific community to develop novel antifungal molecules. Many essential oils components are attracting attention for their interesting antifungal activities. Given the chemical and physical characteristics of these compounds, the use of appropriate nanodelivery systems is becoming increasingly widespread. In this study, chitosan nanoparticles were prepared using an ionic gelation procedure and loaded with the phenolic monoterpene carvacrol. After a bioassay guided optimization, the best nanoparticle formulation was structurally characterized by means of different spectroscopic (UV, FTIR and DLS) and microscopy techniques (SEM) and described for their functional features (encapsulation efficiency, loading capacity and release kinetics). The antifungal activity of this formulation was assayed with different Candida spp., both in planktonic and biofilm forms. From these studies, it emerged that the carvacrol loaded nanoparticles were particularly active against planktonic forms and that the antibiofilm activity was highly dependent on the species tested, with the C. tropicalis and C. krusei strains resulting as the most susceptible.

Drug Delivery Systems of Natural Products in Oncology.

In recent decades, increasing interest in the use of natural products in anticancer therapy field has been observed, mainly due to unsolved drug-resistance problems. The antitumoral effect of natural compounds involving different signaling pathways and cellular mechanisms has been largely demonstrated in in vitro and in vivo studies. The encapsulation of natural products into different delivery systems may lead to a significant enhancement of their anticancer efficacy by increasing in vivo stability and bioavailability, reducing side adverse effects and improving target-specific activity. This review will focus on research studies related to nanostructured systems containing natural compounds for new drug delivery tools in anticancer therapies.

Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis.

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

New deferiprone derivatives as multi-functional cholinesterase inhibitors: design, synthesis and in vitro evaluation.

In order to obtain multi-functional molecules for Alzheimer's disease, a series of deferiprone derivatives has been synthesized and evaluated in vitro with the hypothesis that they can restore the cholinergic tone and attenuate the dyshomeostasis of the metals mainly involved in the pathology. These compounds were designed as dual binding site AChE inhibitors: they possess an arylalkylamine moiety connected via an alkyl chain to a 3-hydroxy-4-pyridone fragment, to allow the simultaneous interaction with catalytic active site (CAS) and peripheral anionic site (PAS) of the enzyme. Deferiprone moiety and 2-aminopyridine, 2-aminopyrimidine or 2,4-diaminopyrimidine groups have been incorporated into these compounds, in order to obtain molecules potentially able to chelate bio-metals colocalized in Aß plaques and involved in the generation of radical species. Synthesized compounds were tested by enzymatic inhibition studies towards EeAChE and eqBChE using Ellman's method. The most potent EeAChE inhibitor is compound 5a, with a Ki of 788 ± 51 nM, while the most potent eqBChE inhibitors are compounds 12 and 19, with Ki values of 182 ± 18 nM and 258 ± 25 nM respectively. Selected compounds, among the most potent cholinesterases inhibitors, were able to form complex with iron and in some cases with copper and zinc. Moreover, these compounds were characterized by low toxicity on U-87 MG Cell Line from human brain (glioblastoma astrocytoma).

Antioxidant, Antifungal, Antibiofilm, and Cytotoxic Activities of Mentha spp. Essential Oils.

Since ancient times, plants have been used to preserve food, or for their health properties. Essential oils are complex mixtures of volatile compounds that are obtained from botanical material, specifically from aromatic plants. Lamiaceae is one of the most important families in the production of essential oils, as it has both antioxidant and antimicrobial properties. The essential oils of Mentha (the Lamiaceae family) have been extensively studied for their biological actions. In this review, we report the antioxidant, antifungal, antibiofilm, and cytotoxic properties of Mentha spp. essential oils. The first objective is to provide comprehensive information about the use of essential oils in the treatment of fungal infections, or as antioxidants and integrative anticancer therapy. The second is to explore the evidence supporting its effectiveness in treating diseases without causing any serious adverse reactions.

Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938.

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.