Microbiological safety of dry-cured fish from the raw material to the end of processing.

The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Companilactobacillus alimentarius: An extensive characterization of strains isolated from spontaneous fermented sausages.

Companilactobacillus alimentarius is a facultatively heterofermentative lactic acid bacterium (LAB) that is a significant constituent within the microbiota of various traditional fermented foods exerting several functions in fermentative or ripening processes. This species has been isolated from Spanish fermented sausages, where its frequency of isolation was comparable to those of Latilactobacillus sakei and Latilactobacillus curvatus. Despite to its presence in several niches, ecological information on this species is still scarce and only few publications report information about its safety features (i.e. antibiotic resistance). Since studies on C. alimentarius concern the analysis of a few individual traits regarding this species, a more extensive work on a larger number of isolates from the same matrix have been performed to allow a clearer interpretation of their phenotypic and technological characteristics. Specifically, 14 strains of C. alimentarius isolated from Mediterranean spontaneously fermented sausages, have been screened for their safety and technological characteristics (such as antibiotic resistance, biogenic amine production, inhibiting potential, growth at different temperatures and NaCl concentrations) and with phenotype microarrays with the aim to elucidate their potential role and contribution to sausage fermentation and ripening. In general, a wide variability was observed in relation to the parameters considered. Several of the tested strains were able to produce histamine, tyramine and putrescine while the antibiotic resistance greatly varied according to the strains, with the exception of vancomycin. In addition, C. alimentarius strains showed a relevant potential to grow in conditions of salt and temperature mimicking those found in fermented foods. In particular, the growth at 10 °C and in the presence of salt can explain the presence of C. alimentarius in sausages and its adaptation to fermented meat environment in which low temperature can be applied during ripening. The differentiation of the phenotypic profile reflected the environmental conditions that influenced the isolation source, including those derived by the raw materials. Given the species frequent association with spontaneous fermentations or the ripening microbiota of various products, despite not being intentionally used as starter cultures, the data presented in this study contribute to a deeper comprehension of their role, both advantageous and detrimental, in numerous significant fermented foods.

Metformin potentiates immunosuppressant activity and adipogenic differentiation of human umbilical cord-mesenchymal stem cells.

Metformin, a first-line drug for type-2 diabetes, displays pleiotropic effects on inflammation, aging, and cancer. Obesity triggers a low-grade chronic inflammation leading to insulin resistance, characterized by increased pro-inflammatory cytokines produced by adipocytes and infiltrated immune cells, which contributes to metabolic syndrome. We investigated metformin's differentiation and immunoregulatory properties of human umbilical cord-mesenchymal stem cells (UC-MSC), as cellular basis of its beneficial role in metabolic dysfunctions. Isolation, characterization and multilineage differentiation of UC-MSC were performed using standard protocols and flow-cytometry. Metformin effects on UC-MSC growth was assessed by colony formation and MTT assay, gene and protein expression by qRT-PCR, and western blot analysis. Proliferation of peripheral blood mononuclear cells (PBMCs) co-cultured with metformin-treated UC-MSC-conditioned media was evaluated by dye dilution assay. We show that metformin decreases proliferation and colony formation of UC-MSCs and enhances their adipogenic lineage commitment. Metformin (3 mM) increases PPARγ and downregulates FABP4 mRNA both in basal and in adipogenic culture conditions; however, the modulation of PPARγ expression is unrelated to the antiproliferative effects. Moreover, metformin inhibits UC-MSC inflammatory activity reducing the expression of IL-6, MCP-1, and COX-2. Conditioned media, collected from metformin-treated UC-MSCs, down-regulate CD3+ T lymphocyte growth in stimulated PBMCs and, in particular, reduce the CD8+ T cell population. These results indicate that metformin may favor new adipocyte formation and potentiate immune suppressive properties of UC-MSCs. Thus, adipose tissue regeneration and anti-inflammatory activity may represent possible mechanisms by which metformin exerts its positive effect on lipid metabolism.

Use of two autochthonous bacteriocinogenic strains as starter cultures in the production of salchichónes, a type of Spanish fermented sausages.

In this work, two autochthonous LAB strains (Lactiplantibacillus paraplantarum BPF2 and Pediococcus acidilactici ST6), isolated from spontaneously fermented sausages produced in Spain, were tested to produce Spanish fermented sausages (salchichón) in pilot plants, due to their promising technological and anti-listerial activity. These products were compared with a sample obtained with a commercial starter (RAP) and a spontaneously fermented control sample. Physico-chemical parameters, microbial counts, metagenomic analysis, biogenic amines content and organoleptic profile of the obtained samples were studied to assess the performances of the native starters. In fact, traditional and artisanal products obtained through spontaneous fermentations can represent an important biodiversity reservoir of strains to be exploited as new potential starter cultures, to improve the safety, quality and local differentiation of traditional products. The data underlined that ST6 strain resulted in a final lower percentage if compared with the other LAB used as starter cultures. The use of starters reduced the BA concentration observed in the sausages obtained with spontaneous fermentation and the BPF2 and ST6 strains were able to decrease the level of products rancidity. Moreover, a challenge test against L. monocytogenes were performed. The data confirmed the effectiveness in the inhibition of L. monocytogenes by the two bacteriocinogenic strains tested, with respect to RAP and control samples, highlighting their ability to produce bacteriocins in real food systems. This work demonstrated the promising application in meat industry of these autochthonous strains as starter cultures to improve sensory differentiation and recognizability of typical fermented sausages.

Microbial Spoilage of Traditional Goose Sausages Produced in a Northern Region of Italy.

Recently, during the ripening of goose sausage, a defect consisting of ammonia and vinegar smell was noticed. The producer of the craft facility, located in Lombardia, a Northern region of Italy, asked us to identify the cause of that defect. Therefore, this study aimed to identify the potential responsible agents for the spoilage of this lot of goose sausages. Spoilage was first detected by sensory analysis using the "needle probing" technique; however, the spoiled sausages were not marketable due to the high ammonia and vinegar smell. The added starter culture did not limit or inhibit the spoilage microorganisms, which were represented by Levilactobacillus brevis, the predominant species, and by Enterococcus faecalis and E. faecium. These microorganisms grew during ripening and produced a large amount of biogenic amines, which could represent a risk for consumers. Furthermore, Lev. brevis, being a heterofermentative lactic acid bacteria (LAB), also produced ethanol, acetic acid, and a variation in the sausage colour. The production of biogenic amines was confirmed in vitro. Furthermore, as observed in a previous study, the second cause of spoilage can be attributed to moulds which grew during ripening; both the isolated strains, Penicillium nalgiovense, added as a starter culture, and P. lanosocoeruleum, present as an environmental contaminant, grew between the meat and casing, producing a large amount of total volatile nitrogen, responsible for the ammonia smell perceived in the ripening area and in the sausages. This is the first description of Levilactobacillus brevis predominance in spoiled goose sausage.

Development and validation of a GC-MS method for determination of metformin in normal brain and in glioblastoma tissues.

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors.

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

BACKGROUND: Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. METHODS: Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. RESULTS: Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. CONCLUSIONS: We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

Antitumor Potential of Antiepileptic Drugs in Human Glioblastoma: Pharmacological Targets and Clinical Benefits.

Glioblastoma (GBM) is characterized by fast-growing cells, genetic and phenotypic heterogeneity, and radio-chemo-therapy resistance, contributing to its dismal prognosis. Various medical comorbidities are associated with the natural history of GBM. The most disabling and greatly affecting patients' quality of life are neurodegeneration, cognitive impairment, and GBM-related epilepsy (GRE). Hallmarks of GBM include molecular intrinsic mediators and pathways, but emerging evidence supports the key role of non-malignant cells within the tumor microenvironment in GBM aggressive behavior. In this context, hyper-excitability of neurons, mediated by glutamatergic and GABAergic imbalance, contributing to GBM growth strengthens the cancer-nervous system crosstalk. Pathogenic mechanisms, clinical features, and pharmacological management of GRE with antiepileptic drugs (AEDs) and their interactions are poorly explored, yet it is a potentially promising field of research in cancer neuroscience. The present review summarizes emerging cooperative mechanisms in oncogenesis and epileptogenesis, focusing on the neuron-to-glioma interface. The main effects and efficacy of selected AEDs used in the management of GRE are discussed in this paper, as well as their potential beneficial activity as antitumor treatment. Overall, although still many unclear processes overlapping in GBM growth and seizure onset need to be elucidated, this review focuses on the intriguing targeting of GBM-neuron mutual interactions to improve the outcome of the so challenging to treat GBM.

Characterization of Lactic Acid Bacteria Isolated from Spontaneously Fermented Sausages: Bioprotective, Technological and Functional Properties.

Fermentation is one of the most ancient strategies to improve safety and extend shelf-life of the products. Starter cultures are mainly represented by lactic acid bacteria (LAB), which may also be bioprotective agents controlling the fermentation process, the native microbiota and pathogen outgrowth. This work aimed to select new LAB strains from spontaneously fermented sausages produced in different areas of Italy, which can be effective as starter cultures and bioprotective agents in fermented salami. The strains, mainly belonging to the Latilactobacillus sakei species, were characterized for their ability to inhibit major meat pathogens, the presence of antibiotic resistances and amine production. Moreover, technological performances, such as growth and acidification kinetics at increasing NaCl concentrations, were studied. As a result, new autochthonous Lat. sakei strains were obtained, lacking antibiotic resistance, possessing antimicrobial activity against Clostridium sporogenes, Listeria monocytogenes, Salmonella and Escherichia coli and with high growth performance under osmotic pressure. These strains have the potential for future application to improve the safety of fermented meats, even under conditions in which chemical preservatives are reduced or eliminated. Moreover, studies on autochthonous cultures are pivotal for guaranteeing specific characteristics of traditional products that represent an important cultural heritage.

Impact of Lactic Acid Bacteria Fermentation on Phenolic Compounds and Antioxidant Activity of Avocado Leaf Extracts.

The growing global consumption of avocados, associated with contents including bioactive compounds with numerous health-promoting properties, is producing a large amount of agro wastes around the world. Different management approaches are available for the recovery of bioactive compounds from wastes as potential ingredients for use in the production of functional foods and nutraceuticals. Lactic acid fermentation can be used to exploit nutritional potential and add value to agro wastes. In this study, fermentations with lactic acid bacteria were carried out in avocado leaves, and the total phenolic content and the antioxidant activity were determined by DPPH and FRAP assays from hydroalcoholic extracts obtained from fermented avocado leaves. Fifteen new phenolic compounds were identified for the first time in avocado leaves by HPLC-ESI-TOF-MS. L. plantarum CECT 748T and P. pentosaceus CECT 4695T showed the highest antioxidant activity. The sum of phenolic compounds was increased by 71, 62, 55 and 21% in fermentations with P. pentosaceus CECT 4695T, L. brevis CECT 5354, P. acidilactici CECT 5765T and L. plantarum CECT 9567, respectively, while it was reduced in the fermentation with L. plantarum 748T by 21% as demonstrated by HPLC-ESI-TOF-MS. Biotransformations induced by bacterial metabolism modified the phenolic compound profile of avocado leaves in a strain-specific-dependent manner. P. pentosaceus CECT 4695T significantly increased kaempferol, P. pentosaceus 4695T, L. brevis 5354 and L. plantarum 9567 increased rutin, and dihydro-p-coumaric acid was increased by the five selected lactic acid bacteria. Total flavonoids were highly increased after fermentations with the five selected lactic acid bacteria but flavonoid glucosides were decreased by L. plantarum 748T, which was related to its higher antioxidant activity. Our results suggest that lactic acid bacteria led the hydrolysis of compounds by enzymatic activity such as glycosidases or decarboxylase and the release of phenolics bound to the plant cell wall, thus improving their bioavailability.

Boron Vehiculating Nanosystems for Neutron Capture Therapy in Cancer Treatment.

Boron neutron capture therapy is a low-invasive cancer therapy based on the neutron fission process that occurs upon thermal neutron irradiation of 10B-containing compounds; this process causes the release of alpha particles that selectively damage cancer cells. Although several clinical studies involving mercaptoundecahydro-closo-dodecaborate and the boronophenylalanine-fructose complex are currently ongoing, the success of this promising anticancer therapy is hampered by the lack of appropriate drug delivery systems to selectively carry therapeutic concentrations of boron atoms to cancer tissues, allowing prolonged boron retention therein and avoiding the damage of healthy tissues. To achieve these goals, numerous research groups have explored the possibility to formulate nanoparticulate systems for boron delivery. In this review. we report the newest developments on boron vehiculating drug delivery systems based on nanoparticles, distinguished on the basis of the type of carrier used, with a specific focus on the formulation aspects.

Safety and technological issues of dry fermented sausages produced without nitrate and nitrite.

The aim of this work was to investigate the possibility to industrially produce fermented sausages without the addition of nitrate and nitrite. Indeed, despite their antimicrobial effect and multiple technological roles, an increasing pressure for their removal has recently raised. To achieve this goal while maintaining an acceptable final product quality, we deeply modified the whole process, that was carried out at 10-15 °C (i.e., temperatures lower than traditional Mediterranean products) and by using bioprotective starter cultures at high concentrations (7 log CFU/g) to lead the fermentation. Different glucose amounts (0.2 or 0.4 % w/w) were also tested to optimize the process. The results showed no significant differences between the control (with nitrate/nitrite) and the sausages without preservatives in terms of aw (value range 0.908-0.914), weight loss (about 38% in all samples), lactic acid bacteria (value range 8.1-8.3 log CFU/g) and coagulase negative cocci (value range 6.8-7.1 log CFU/g). The amount of sugar affected the final characteristics of sausages. Indeed, in the absence of curing salts, lower sugar concentration resulted in better textural features (reduced hardness and gumminess) and lower oxidation (TBARS values 0.80 vs. 1.10 mg MDA/kg of meat product in samples with 0.2% or 0.4% of glucose, respectively). Finally, challenge tests evidenced the inability of selected strains of Listeria innocua, Salmonella enterica sub. enterica and Clostridium botulinum to grow, under the adopted conditions, in fermented sausages. This research highlighted that nitrate/nitrite removal from these meat products requires accurate technological changes to guarantee the final quality.

Taxonomical Identification and Safety Characterization of Lactobacillaceae from Mediterranean Natural Fermented Sausages.

Fermented meat products represent an important industrial sector in Europe, particularly in the Mediterranean Countries (MC), where the presence of numerous local productions, still obtained through spontaneous fermentation, is recognized as a formidable treasure chest of unexplored microbial biodiversity. Lactobacillaceae naturally occurring in fifteen spontaneously fermented sausages from MC (Italy, Spain, Croatia, and Slovenia) were isolated and taxonomically characterized using molecular techniques. Additionally, a safety assessment for the presence of antibiotic resistances and biogenic amine (BA) production was performed to determine their suitability as autochthonous starter cultures. Molecular typing, performed using REP-PCR, discriminated 151 strains belonging to Latilactobacillus sakei (59.6%), Latilactobacillus curvatus (26.5%) and Companilactobacillus alimentarius (13.9%). The minimum inhibitory concentrations (MICs) of eight different antibiotics revealed a high resistance to streptomycin (27%), tetracycline (16%), followed by gentamycin (14%) and kanamycin (13%). Interestingly, the results showed a geographical distribution of resistant biotypes. tetM/tetS or ermB genes were identified in only six strains. The amino-biogenic potential of the strains was assessed, confirming the absence of this trait among L. sakei, while a high number of producer strains was found among L. curvatus. On the 151 analyzed strains, 45 demonstrated safety traits for their future use as starter food cultures. These results open the way to further studies on the technological properties of these promising autochthonous strains, strongly linked to the Mediterranean environment.

Effects of Rubus fruticosus and Juniperus oxycedrus derivatives on culturability and viability of Listeria monocytogenes.

The consumers' demand for safe foods without chemical additives increased the research for green solutions, based on natural antimicrobials. Plants can be an important source of bioactive compounds able to prevent the development of foodborne pathogens and spoilage microflora. This paper aimed to characterize phenolic extracts (PEs) and essential oils (EOs) obtained from Mediterranean Rubus fruticosus leaves and Juniperus oxycedrus needles and to evaluate their antimicrobial effects against Listeria monocytogenes Scott A. The growth dynamics with sub-lethal concentrations of plant derivatives were modeled and flow cytometry was used to better evidence the effect on cell viability and culturability. The results showed that these plant derivatives affected the growth of L. monocytogenes, increasing lag phase (about 40 h in the presence of PEs vs. 8 h in the control) and decreasing the final cell load of at least 1 log cycle with respect to the control. R. fruticosus EO was the most effective, determining an initial decrease of cell counts of about 6 log cycles, followed by a restart of growth after 10 h, with rate similar to the control (0.08 with R. fruticosus EO vs. 0.09 ((log CFU/ml)/h in the control) but significantly lower final cell load (7.33 vs. 8.92 log CFU/ml). According to flow cytometry, only R. fruticosus EO induced a relevant increase of dead cells, while the other plant derivatives determined different extent of sub-lethal cell injury. The discrepancy observed in some cases between viability and culturability could indicate the presence of cells not able to grow in culture media, whose fate needs to be further investigated to assess their potential recovery, thus bringing to an overestimation of the antimicrobial effect of these substances. This research contributed to increase the knowledge of these underused raw materials such as blackberry leaves and juniper needles that can be exploited in food and other industries.

Insights into the Metabolomic Diversity of Latilactobacillus sakei.

Latilactobacillus sakei (L. sakei), widely used as a starter culture in fermented sausages, is a species adapted to meat environments. Its ability to survive for a long time in such products is due to the exploitation of different metabolic pathways to gain energy (hexose and pentose sugar fermentation, amino acids catabolism, etc.). Since L. sakei demonstrates high phenotypic and metabolic strain biodiversity, in this work, a metabolomic approach was used to compare five strains of different origins. They were cultivated in a defined medium with glucose or ribose at two concentrations, and analyzed through nuclear magnetic resonance (1H-NMR) spectroscopy to monitor amino acid consumptions and accumulation of organic acids and aroma compounds. The results showed that all the strains were able to use arginine, especially when cultivated with ribose, while serine was consumed mainly in the presence of glucose. Aroma compounds (i.e., diacetyl and acetoin) were mainly accumulated in samples with ribose. These aspects are relevant for starter cultures selection, to confer specific features to fermented sausages, and to optimize the fermentations. Moreover, the use of 1H-NMR allowed the fast identification of different classes of compounds (without derivatization or extraction procedures), providing a powerful tool to increase the knowledge of the metabolic diversity of L. sakei.

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives.

BACKGROUND: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. METHODS: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). RESULTS: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. CONCLUSIONS: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

Survival, growth, and biogenic amine production of Enterococcus faecium FC12 in response to extracts and essential oils of Rubus fruticosus and Juniperus oxycedrus.

Introduction: Enterococci are lactic acid bacteria (LAB) usually found as food contaminants in fermented products such as cheeses and fermented sausages. Due to their antibiotic resistance, the presence of virulence factors, and the ability to produce biogenic amines (BAs), the determination of these bacteria is crucial to assure food quality and safety. BAs production and consequent accumulation in foods can cause toxicological effects on human health. Plant phenolic compounds are promising alternatives to chemical preservatives and reflect consumers' demand for "green" solutions. In this study, the antimicrobial effect of blackberry (Rubus fruticosus) leaves and prickly juniper (Juniperus oxycedrus) needles, both as phenolic extracts (PE) and essential oils (EO), were evaluated against Enterococcus faecium FC12, a known tyramine-producing strain. Methods: The growth kinetics in the presence of sub-lethal concentrations of such plant derivatives were modeled (Gompertz equation) and BA production was monitored over time by HPLC. Moreover, flow cytometry (FCM) was used to study the effects of EOs and PEs on cell viability. Results: The EOs showed a higher antimicrobial effect (especially R. fruticosus added at 0.75 mg/ml), determining an initial decrease of culturable cells followed by a recovery, even if with lower growth rates and final cell loads. Different rates of BA formation were observed, with tyramine concentrations ranging from 120 to 160 mg/l after 96 h of incubation, and 2-phenylethylamine was produced in lower amounts, usually after reaching the peak of tyramine. FCM confirmed the higher efficacy of R. fruticosus EO that induced cell membrane injury in 93% of the total population. However, complete recovery occurred in the following incubation, demonstrating transient damage. Discussion: Although further research is required to better investigate this recovery and to assess the suitability of this approach in a real food system, the present study showed the potential antimicrobial activity of plant derivatives, especially R. fruticosus EO, against the tyramine-producing E. faecium FC12.

Mediterranean Spontaneously Fermented Sausages: Spotlight on Microbiological and Quality Features to Exploit Their Bacterial Biodiversity.

The wide array of spontaneously fermented sausages of the Mediterranean area can represent a reservoir of microbial biodiversity and can be an important source of new technological and functional strains able to preserve product properties, counteracting the impoverishment of their organoleptic typical features due to the introduction of commercial starter cultures. We analysed 15 artisanal salamis from Italy, Spain, Croatia and Slovenia to evaluate the microbiota composition, through culture-dependent and culture-independent techniques (i.e., metagenomic analysis), chemical-physical features, biogenic amines and aroma profile. The final pH varied according to origin and procedures (e.g., higher pH in Italian samples due to long ripening and mold growth). Lactic acid bacteria (LAB) and coagulase-negative cocci (CNC) were the dominant population, with highest LAB counts in Croatian and Italian samples. Metagenomic analysis showed high variability in qualitative and quantitative microbial composition: among LAB, Latilactobacillus sakei was the dominant species, but Companilactobacillus spp. was present in high amounts (45-55% of the total ASVs) in some Spanish sausages. Among staphylococci, S. epidermidis, S. equorum, S. saprophyticus, S. succinus and S. xylosus were detected. As far as biogenic amines, tyramine was always present, while histamine was found only in two Spanish samples. These results can valorize the bacterial genetic heritage present in Mediterranean products, to find new candidates of autochthonous starter cultures or bioprotective agents.