Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Ferroptosis precedes apoptosis to facilitate specific death signalling by fatty acids.

Cell death is physiologically induced by specific mediators. However, our power to trigger the process in selected cells is quite limited. The protandric shrimp Hippolyte inermis offers a possible answer. Here, we analyse a de novo transcriptome of shrimp post-larvae fed on diatoms. The sex ratio of diatom-fed shrimps versus shrimps fed on control diets was dramatically altered, demonstrating the disruption of the androgenic gland, and their transcriptome revealed key modifications in gene expression. A wide transcriptomic analysis, validated by real-time qPCR, revealed that ferroptosis represents the primary factor to re-shape the body of this invertebrate, followed by further apoptotic events, and our findings open biotechnological perspectives for controlling the destiny of selected tissues. Ferroptosis was detected here for the first time in a crustacean. In addition, this is the first demonstration of a noticeable effect prompted by an ingested food, deeply impacting the gene networks of a young metazoan, definitely determining its future physiology and sexual differentiation.

Pro-Apoptotic Activity of the Marine Sponge Dactylospongia elegans Metabolites Pelorol and 5-epi-Ilimaquinone on Human 501Mel Melanoma Cells.

The natural environment represents an important source of drugs that originates from the terrestrial and, in minority, marine organisms. Indeed, the marine environment represents a largely untapped source in the process of drug discovery. Among all marine organisms, sponges with algae represent the richest source of compounds showing anticancer activity. In this study, the two secondary metabolites pelorol (PEL) and 5-epi-ilimaquinone (EPI), purified from Dactylospongia elegans were investigated for their anti-melanoma activity. PEL and EPI induced cell growth repression of 501Mel melanoma cells in a concentration- and time-dependent manner. A cell cycle block in the G1 phase by PEL and EPI was also observed. Furthermore, PEL and EPI induced significant accumulation of DNA histone fragments in the cytoplasmic fraction, indicating a pro-apoptotic effect of both compounds. At the molecular level, PEL and EPI induced apoptosis through the increase in pro-apoptotic BAX expression, confirmed by the decrease in its silencing miR-214-3p and the decrease in the anti-apoptotic BCL-2, MCL1, and BIRC-5 mRNA expression, attested by the increase in their silencing miRNAs, i.e., miR-193a-3p and miR-16-5p. In conclusion, our data indicate that PEL and EPI exert cytotoxicity activity against 501Mel melanoma cells promoting apoptotic signaling and inducing changes in miRNA expression and their downstream effectors. For these reasons could represent promising lead compounds in the anti-melanoma drug research.

Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation.

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized ß-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of ß-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.

Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma.

BACKGROUND: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. METHODS: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and -5p transfection in cutaneous melanoma cell lines are investigated. RESULTS: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. CONCLUSIONS: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

Diclofenac-Derived Hybrids for Treatment of Actinic Keratosis and Squamous Cell Carcinoma.

In this work, hybrid compounds 1-4 obtained by conjugation of the non-steroidal anti-inflammatory drug diclofenac, with natural molecules endowed with antioxidant and antiproliferative activity were prepared. The antiproliferative activity of these hybrids was evaluated on immortalized human keratinocyte (HaCaT) cells stimulated with epidermal growth factor (EGF), an actinic keratosis (AK) model, and on human squamous cell carcinoma (SCC) cells (A431). Hybrid 1 presented the best activity in both cell models. Self-assembling surfactant nanomicelles have been chosen as the carrier to drive the hybrid 1 into the skin; the in vitro permeation through and penetration into pig ear skin have been evaluated. Among the nanostructured formulations tested, Nano3Hybrid20 showed a higher tendency of the hybrid 1 to be retained in the skin rather than permeating it, with a desirable topical and non-systemic action. On these bases, hybrid 1 may represent an attractive lead scaffold for the development of new treatments for AK and SCC.

Anticancer Activity of Euplotin C, Isolated from the Marine Ciliate Euplotes crassus, Against Human Melanoma Cells.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma.

Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

Cytotoxic Activity of Oleocanthal Isolated from Virgin Olive Oil on Human Melanoma Cells.

Oleocanthal is one of the phenolic compounds of extra virgin olive oil with important anti-inflammatory properties. Although its potential anticancer activity has been reported, only limited evidence has been provided in cutaneous malignant melanoma. The present study is aimed at investigating the selective in vitro antiproliferative activity of oleocanthal against human malignant melanoma cells. Since oleocanthal is not commercially available, it was obtained as a pure standard by direct extraction and purification from extra virgin olive oil. Cell viability experiments carried out by WST-1 assay demonstrated that oleocanthal had a remarkable and selective activity for human melanoma cells versus normal dermal fibroblasts with IC50s in the low micromolar range of concentrations. Such an effect was paralleled by a significant inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression. These findings may suggest that extra virgin olive oil phenolic extract enriched in oleocanthal deserves further investigation in skin cancer.

AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Nutraceutical Features of the Phycobiliprotein C-Phycocyanin: Evidence from Arthrospira platensis (Spirulina).

Arthrospira platensis, commonly known as Spirulina, is a photosynthetic filamentous cyanobacterium (blue-green microalga) that has been utilized as a food source since ancient times. More recently, it has gained significant popularity as a dietary supplement due to its rich content of micro- and macro-nutrients. Of particular interest is a water soluble phycobiliprotein derived from Spirulina known as phycocyanin C (C-PC), which stands out as the most abundant protein in this cyanobacterium. C-PC is a fluorescent protein, with its chromophore represented by the tetrapyrrole molecule phycocyanobilin B (PCB-B). While C-PC is commonly employed in food for its coloring properties, it also serves as the molecular basis for numerous nutraceutical features associated with Spirulina. Indeed, the comprehensive C-PC, and to some extent, the isolated PCB-B, has been linked to various health-promoting effects. These benefits encompass conditions triggered by oxidative stress, inflammation, and other pathological conditions. The present review focuses on the bio-pharmacological properties of these molecules, positioning them as promising agents for potential new applications in the expanding nutraceutical market.

Bioactive Molecules from Marine Diatoms and Their Value for the Nutraceutical Industry.

The search for novel sources of nutrients is among the basic goals for achievement of sustainable progress. In this context, microalgae are relevant organisms, being rich in high-value compounds and able to grow in open ponds or photobioreactors, thus enabling profitable exploitation of aquatic resources. Microalgae, a huge taxon containing photosynthetic microorganisms living in freshwater, as well as in brackish and marine waters, typically unicellular and eukaryotic, include green algae (Chlorophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae) and diatoms (Bacillariophyceae). In recent decades, diatoms have been considered the most sustainable sources of nutrients for humans with respect to other microalgae. This review focuses on studies exploring their bio-pharmacological activities when relevant for human disease prevention and/or treatment. In addition, we considered diatoms and their extracts (or purified compounds) when relevant for specific nutraceutical applications.

Omega-3 PUFAs as a Dietary Supplement in Senile Systemic Amyloidosis.

Eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6), two omega-3 poly-unsaturated fatty acids (PUFAs), are the main components in oil derived from fish and other marine organisms. EPA and DHA are commercially available as dietary supplements and are considered to be very safe and contribute to guaranteeing human health. Studies report that PUFAs have a role in contrasting neurodegenerative processes related to amyloidogenic proteins, such as ß-amyloid for AD, α-synuclein in PD, and transthyretin (TTR) in TTR amyloidosis. In this context, we investigated if EPA and DHA can interact directly with TTR, binding inside the thyroxin-binding pockets (T4BP) that contribute to the tetramer stabilization. The data obtained showed that EPA and DHA can contribute to stabilizing the TTR tetramer through interactions with T4BP.

Tanshinone IIA and Cryptotanshinone Counteract Inflammation by Regulating Gene and miRNA Expression in Human SGBS Adipocytes.

Inflammation of the adipose tissue contributes to the onset and progression of several chronic obesity-related diseases. The two most important lipophilic diterpenoid compounds found in the root of Salvia milthorrhiza Bunge (also called Danshen), tanshinone IIA (TIIA) and cryptotanshinone (CRY), have many favorable pharmacological effects. However, their roles in obesity-associated adipocyte inflammation and related sub-networks have not been fully elucidated. In the present study, we investigated the gene, miRNAs and protein expression profile of prototypical obesity-associated dysfunction markers in inflamed human adipocytes treated with TIIA and CRY. The results showed that TIIA and CRY prevented tumor necrosis factor (TNF)-α induced inflammatory response in adipocytes, by counter-regulating the pattern of secreted cytokines/chemokines associated with adipocyte inflammation (CCL2/MCP-1, CXCL10/IP-10, CCL5/RANTES, CXCL1/GRO-α, IL-6, IL-8, MIF and PAI-1/Serpin E1) via the modulation of gene expression (as demonstrated for CCL2/MCP-1, CXCL10/IP-10, CCL5/RANTES, CXCL1/GRO-α, and IL-8), as well as related miRNA expression (miR-126-3p, miR-223-3p, miR-124-3p, miR-155-5p, and miR-132-3p), and by attenuating monocyte recruitment. This is the first demonstration of a beneficial effect by TIIA and CRY on adipocyte dysfunction associated with obesity development and complications, offering a new outlook for the prevention and/or treatment of metabolic diseases.

The phenolic compounds tyrosol and hydroxytyrosol counteract liver fibrogenesis via the transcriptional modulation of NADPH oxidases and oxidative stress-related miRNAs.

Liver fibrosis is the result of a chronic pathological condition caused by the activation of hepatic stellate cells (HSCs), which induces the excessive deposition of extracellular matrix. Fibrogenesis is sustained by an exaggerated production of reactive oxidative species (ROS) by NADPH oxidases (NOXs), which are overactivated in hepatic inflammation. In this study, we investigated the antifibrotic properties of two phenolic compounds of natural origin, tyrosol (Tyr) and hydroxytyrosol (HTyr), known for their antioxidant and anti-inflammatory effects. We assessed Tyr and HTyr antifibrotic and antioxidant activity both in vitro, by a co-culture of LX2, HepG2 and THP1-derived Mϕ macrophages, set up to simulate the hepatic microenvironment, and in vivo, in a mouse model of liver fibrosis obtained by carbon tetrachloride treatment. We evaluated the mRNA and protein expression of profibrotic and oxidative markers (α-SMA, COL1A1, NOX1/4) by qPCR and/or immunocytochemistry or immunohistochemistry. The expression of selected miRNAs in mouse livers were measured by qPCR. Tyr and HTyr reduces fibrogenesis in vitro and in vivo, by downregulating all fibrotic markers. Notably, they also modulated oxidative stress by restoring the physiological levels of NOX1 and NOX4. In vivo, this effect was accompanied by a transcriptional regulation of inflammatory genes and of 2 miRNAs involved in the control of oxidative stress damage (miR-181-5p and miR-29b-3p). In conclusion, Tyr and HTyr exert antifibrotic and anti-inflammatory effects in preclinical in vitro and in vivo models of liver fibrosis, by modulating hepatic oxidative stress, representing promising candidates for further development.

Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands.

The design of dualsteric/bitopic agents as single chemical entities able to simultaneously interact with both the orthosteric and an allosteric binding site represents a novel approach in medicinal chemistry. Biased dualsteric/bitopic agents could enhance certain signaling pathways while diminishing the others that cause unwanted side effects. We have designed, synthesized, and functionally characterized the first CB2R heterobivalent bitopic ligands. In contrast to the parent orthosteric compound, our bitopic ligands selectively target CB2R versus CB1R and show a functional selectivity for the cAMP signaling pathway versus ßarrestin2 recruitment. Moreover, the most promising bitopic ligand FD-22a displayed anti-inflammatory activity in a human microglial cell inflammatory model and antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Finally, computational studies clarified the binding mode of these compounds inside the CB2R, further confirming their bitopic nature.

From Life in the Sea to the Clinic: The Marine Drugs Approved and under Clinical Trial.

In the last decades Blue Growth policy in european and non-european countries produced a great impulse in applied marine sciences, comprehending the research of new bioactive molecules in marine organisms. These organisms are a great source of natural compounds with unique features resulting from the huge variability of marine habitats and species living in them. Most of the marine compounds in use and in clinical trials are drugs for cancer therapy and many of them are conjugated to antibody to form antibody-drug conjugates (ADCs). Severe pain, viral infections, hypertriglyceridemia, obesity, Alzheimer's and other CNS diseases are further target conditions for these pharmaceuticals. This review summarizes the state-of-the-art marine drugs focusing on the most successful results in the fast expanding field of marine pharmacology.

Pharmacological Activities of Extracts and Compounds Isolated from Mediterranean Sponge Sources.

Marine pharmacology is an exciting and growing discipline that blends blue biotechnology and natural compound pharmacology together. Several sea-derived compounds that are approved on the pharmaceutical market were discovered in sponges, marine organisms that are particularly rich in bioactive metabolites. This paper was specifically aimed at reviewing the pharmacological activities of extracts or purified compounds from marine sponges that were collected in the Mediterranean Sea, one of the most biodiverse marine habitats, filling the gap in the literature about the research of natural products from this geographical area. Findings regarding different Mediterranean sponge species were individuated, reporting consistent evidence of efficacy mainly against cancer, infections, inflammatory, and neurological disorders. The sustainable exploitation of Mediterranean sponges as pharmaceutical sources is strongly encouraged to discover new compounds.

Apolipoprotein E Polymorphism and Oxidative Stress in Human Peripheral Blood Cells: Can Physical Activity Reactivate the Proteasome System through Epigenetic Mechanisms?

Alzheimer's disease (AD) is characterized by proteasome activity impairment, oxidative stress, and epigenetic changes, resulting in ß-amyloid (Aß) production/degradation imbalance. Apolipoprotein E (ApoE) is implicated in Aß clearance, and particularly, the ApoE ε4 isoform predisposes to AD development. Regular physical activity is known to reduce AD progression. However, the impact of ApoE polymorphism and physical exercise on Aß production and proteasome system activity has never been investigated in human peripheral blood cells, particularly in erythrocytes, an emerging peripheral model used to study biochemical alteration. Therefore, the influence of ApoE polymorphism on the antioxidant defences, amyloid accumulation, and proteasome activity was here evaluated in human peripheral blood cells depending on physical activity, to assess putative peripheral biomarkers for AD and candidate targets that could be modulated by lifestyle. Healthy subjects were enrolled and classified based on the ApoE polymorphism (by the restriction fragment length polymorphism technique) and physical activity level (Borg scale) and grouped into ApoE ε4/non-ε4 carriers and active/non-active subjects. The plasma antioxidant capability (AOC), the erythrocyte Aß production/accumulation, and the nuclear factor erythroid 2-related factor 2 (Nrf2) mediated proteasome functionality were evaluated in all groups by the chromatographic and immunoenzymatic assay, respectively. Moreover, epigenetic mechanisms were investigated considering the expression of histone deacetylase 6, employing a competitive ELISA, and the modulation of two key miRNAs (miR-153-3p and miR-195-5p), through the miRNeasy Serum/Plasma Mini Kit. ApoE ε4 subjects showed a reduction in plasma AOC and an increase in the Nrf2 blocker, miR-153-3p, contributing to an enhancement of the erythrocyte concentration of Aß. Physical exercise increased plasma AOC and reduced the amount of Aß and its precursor, involving a reduced miR-153-3p expression and a miR-195-5p enhancement. Our data highlight the impact of the ApoE genotype on the amyloidogenic pathway and the proteasome system, suggesting the positive impact of physical exercise, also through epigenetic mechanisms.