Elisabetta Carata

Clonothrix fusca Roze 1896, a Filamentous, Sheathed, Methanotrophic γ-Proteobacterium

ABSTRACT Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing γ-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohns Crenothrix and Rozes Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing γ-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic γ-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase α subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase α subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

BackgroundThere is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.ResultsSpontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.ConclusionRifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

Nanomaterials and autophagy: new insights in cancer treatment.

Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies.

In Vitro Analysis of the Anti-Inflammatory Effect of Inhomogeneous Static Magnetic Field-Exposure on Human Macrophages and Lymphocytes

The effect of inhomogeneous static magnetic field (SMF)-exposure on the production of different cytokines from human peripheral blood mononuclear cells (PMBC), i.e., lymphocytes and macrophages, was tested in vitro. Some cultures were activated with lipopolysaccharide (LPS) at time point −3 h and were either left alone (positive control) or exposed to SMF continuously from 0 until 6, 18, or 24 h. The secretion of interleukin IL-6, IL-8, tumor necrosis factor TNF-α, and IL-10 was tested by ELISA. SMF-exposure caused visible morphological changes on macrophages as well as on lymphocytes, and also seemed to be toxic to lymphocytes ([36.58; 41.52]%, 0.308≤p≤0.444), but not to macrophages (<1.43%, p≥0.987). Analysis of concentrations showed a significantly reduced production of pro-inflammatory cytokines IL-6, IL-8, and TNF-α from macrophages compared to negative control ([56.78; 87.52]%, p = 0.031) and IL-6 compared to positive control ([45.15; 56.03]%, p = 0.035). The production of anti-inflammatory cytokine IL-10 from macrophages and from lymphocytes was enhanced compared to negative control, significantly from lymphocytes ([−183.62; −28.75]%, p = 0.042). The secretion of IL-6 from lymphocytes was significantly decreased compared to positive control ([−115.15; −26.84]%, p = 0.039). This massive in vitro evidence supports the hypotheses that SMF-exposure (i) is harmful to lymphocytes in itself, (ii) suppresses the release of pro-inflammatory cytokines IL-6, IL-8, and TNF-α, and (iii) assists the production of anti-inflammatory cytokine IL-10; thus providing a background mechanism of the earlier in vivo demonstrated anti-inflammatory effects of SMF-exposure.

Sphingomonas cynarae sp. nov., a proteobacterium that produces an unusual type of sphingan.

Strain SPC-1(T) was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered to be the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-stain-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide, formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30 °C in the presence of 0-0.5 % NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1(T) clustered together with species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, 2-hydroxymyristic acid (C(14 : 0) 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomic position. 16S rRNA gene sequence analysis showed that SPC-1(T) was most closely related to Sphingomonas hankookensis ODN7(T), Sphingomonas insulae DS-28(T) and Sphingomonas panni C52(T) (98.19, 97.91 and 97.11 % sequence similarities, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed; the type strain is SPC-1(T) ( = JCM 17498(T) = ITEM 13494(T)). A component analysis of the exopolysaccharide suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells

This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×103 or 2×104 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag+ release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×104 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag+ release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation.

Silver and carbon nanoparticles toxicity in sea urchin Paracentrotus lividus embryos

Abstract Environment hazards and risks of engineered NanoParticles (NPs) have been debated in recent years. In this paper, the effects of silver (Ag) and carbon (C) NPs were explored in sea urchin Paracentrotus lividus (P. lividus) development. Fertilization and development of P. lividus up to the pluteus stage were assayed in the presence of increasing amounts of NPs. The embryotoxicity test performed on sea urchin P. lividus, from fertilization until the larva stage, revealed that both AgNPs and CNPs were embryotoxic since they caused embryo malformations and alteration of the normal progression through the development stages. Embryonic development was slowed down by AgNPs and sped up by CNPs. Interestingly, AgNPs-induced malformations led embryos to die in a concentration-dependent manner; while embryos bearing CNPs-induced malformations survived for a longer time.

Potential effects of RFID systems on biotechnology insulin preparation: A study using HPLC and NMR spectroscopy

The item-level traceability is a very important requirement for many practical application scenarios, where it needs to guarantee perfect transparency for products flow along the whole supply chain. Among these, the pharmaceutical distribution is a very interesting scenario, characterized by many challenges, where, the Radio Frequency Identification (RFID) technology will play a very important role. Unfortunately, there are still some technical barriers that are retarding the deployment of these innovative technologies in large-scale. For the pharmaceutical supply chain, there have been concerns raised regarding the potential effects on the quality of drugs due to electromagnetic fields exposure. This work aimed to evaluate potential effects of tracing RFID systems on the molecular structure of biological drugs. In particular, some samples of a commercial human insulin preparation have been exposed for different periods to electromagnetic fields generated by RFID devices. In order to evaluate possible alterations on the molecular structure, the following diagnostic techniques were used: High Pressure Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). HPLC analysis demonstrated that there is are no differences between the RFID exposed samples and the control. On the contrary, a first and partial NMR analysis detected some changes on the insulin molecule spectra after one hour of exposition to the electromagnetic field. Unfortunately, this approach did not allow us to verify possible damages on the protein because of presence of expicients and low drug concentration. Further investigations, e.g. in vitro functional analysis, are required.