A Straightforward Method for the Isolation and Cultivation of Galleria mellonella Hemocytes

Galleria mellonella is an alternative animal model of infection. The use of this species presents a wide range of advantages, as its maintenance and rearing are both easy and inexpensive. Moreover, its use is considered to be more ethically acceptable than other models, it is conveniently sized for manipulation, and its immune system has multiple similarities with mammalian immune systems. Hemocytes are immune cells that help encapsulate and eliminate pathogens and foreign particles. All of these reasons make this insect a promising animal model. However, cultivating G. mellonella hemocytes in vitro is not straightforward and it has many difficult challenges. Here, we present a methodologically optimized protocol to establish and maintain a G. mellonella hemocyte primary culture. These improvements open the door to easily and quickly study the toxicity of nanoparticles and the interactions of particles and materials in an in vitro environment.     

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.     

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact,Efficacy, and Safety

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and ¹H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. ¹H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.     

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.     

Characterisation and stability evaluation of bixin nanocapsules

The aim of this study was to produce bixin nanocapsules by the interfacial deposition of preformed poly-?-caprolactone (PCL). PCL (250 mg), capric/caprylic triglyceride (400 μL), sorbitan monostearate (95 mg) and bixin were dissolved in a mixture of acetone (60 mL) and ethanol (7.5 mL) under stirring (40 °C). This organic solution was added to the aqueous solution (130 mL) containing Tween 80 (195 mg). The size distributions in the formulations with bixin concentration from 11 to 100 μg/mL were evaluated periodically during 3 weeks of storage at ambient temperature. The optimal formulation (bixin concentration of 16.92 ± 0.16 μg/mL) was characterised in terms of particle size distribution, zeta potential, bixin content and encapsulation efficiency, and showed a volume-weighted mean diameter (D_4,3) of 195 ± 27 nm, around 100% of encapsulation efficiency and the nanocapsules were considered physically stable during 119 days of storage at ambient temperature.     

Principal component analysis of proteolytic profiles as markers of authenticity of PDO cheeses

The casein fraction of 13 Portuguese PDO cheeses were analysed using Urea-PAGE and reverse phase-high performance liquid chromatography (RP-HPLC) and then subjected to chemometric evaluation. The chemometric techniques of cluster analysis (CA) and principal component analysis (PCA) were used for the classification studies. Peptide mapping using Urea-PAGE followed by CA revealed two major clusters according to the similarity of the proteolytic profile of the cheeses. PCA results were in accordance with the grouping performed using CA.CA of RP-HPLC results of the matured cheeses revealed the presence of one major cluster comprising samples manufactured with only ovine milk or milk admixtures. When the results of CA technique were compared with the two PCA approaches performed, it was found that the grouping of the samples was similar.Both approaches, revealed the potential of proteolytic profiles (which is an essential aspect of cheese maturation) as markers of authenticity of PDO cheeses in terms of ripening time and milk admixtures not mentioned on the label.     

High resolution melting analysis as a new approach to detect almond DNA encoding for Pru du 5 allergen in foods

Almond is responsible for trigging adverse immune responses in allergic individuals, and since it is present in many processed food, it is considered as a potential hidden allergen. Here we propose a novel, simple and highly specific approach to detect almond in a wide range of processed foods. The method consists of a real-time PCR assay targeting the gene encoding for the Pru du 5 allergen in almond, using the fluorescent EvaGreen® dye combined with high resolution melting (HRM) analysis. The new approach allowed the detection of trace amounts of almond down to the level of 0.005% (w/w) and was successfully applied to processed foods. HRM analysis increased the specificity of the assay and was effective in distinguishing almonds from other plant foods, including the closely related fruits from the Rosaceae family. It was demonstrated for the first time that HRM analysis can provide a powerful tool for the identification of allergens in foods.     

Lysozyme resistance of the ropy strain Pediococcus parvulus IOEB 8801 is correlated with beta-glucan accumulation around the cell

Lactic acid bacteria (LAB) are often exploited to carry out malolactic fermentation in wine. However, a few specific LAB strains and, more precisely, some Pediococcus parvulus strains synthesize a β-glucan, which can be deleterious to wine quality as it confers a ropy texture to the wine that can no longer be commercialized. Although molecular methods exist to detect these unwanted microorganisms, ropy Pediococcus still remain difficult to remove from wine, because of their natural resistance to traditional wine stabilizing treatments. In this work, we show that ropy P. parvulus are resistant to lysozyme. We clearly demonstrate that this resistance may be ascribed to the presence of the β-glucan that forms around the cell a protective barrier against anti-bacteria agents. Moreover, this resistance increases during bacterial growth. We show that using lysozyme with β-glucanase can strongly improve the treatment against ropy strains, in model media as well as red and white wine based media. This work not only brings potential solutions to the wine industry, but also opens interesting perspectives for studying β-glucan producing bacteria which are widespread in the food industry.     

Bioactive compounds from flesh and by-product of fresh-cut watermelon cultivars

BACKGROUND: The fresh‐cut industry produces thousands of tons of waste in non‐edible portions that present an environmental and management problem. These by‐products could be reused, in particular, to obtain bioactive compounds. In this study, five different fresh‐cut watermelon cultivars were assessed for their flesh and by‐product bioactive contents. RESULTS: The amount of by‐product varied between 31.27 and 40.61% of initial fresh weight (f.w.) depending on the cultivar. Watermelon cultivars were poor sources of total antioxidant, and the content was similar between rind and flesh samples (46.96 vs 43.46 mg ascorbic acid equivalent antioxidant capacity kg^?1 f.w.). However, the rind had a moderate total phenolic content higher than that of the flesh (458 vs 389 mg chlorogenic acid equivalent kg^?1 f.w.) and a much higher content of the amino acid citrulline (3.34 vs 2.33 g kg^?1 f.w.), which has potential bioactive properties. CONCLUSION: Watermelon rind offers quantitative interest as a natural source of citrulline, particularly Fashion, a dark‐skinned, seedless cultivar. More research is required on the efficient extraction of citrulline from watermelon rind and its suitability as an additive to drinks, juices or others products to produce new functional food products with valid health claims. Copyright © 2010 Society of Chemical Industry