Correction to: Biodegradation of naphthenic acids: identification of Rhodococcus opacus R7 genes as molecular markers for environmental monitoring and their application in slurry microcosms.

The published online version contains mistake in the author list for the author "M. Cappelletti" was incorrectly presented.

Ingenol mebutate-mediated reduction in p53-positive keratinocytes in skin cancerization field directly correlates with clinical response in patients with multiple actinic keratoses.

BACKGROUND: UV radiation represents the main risk factor for non-melanoma skin cancers. Chronic UV exposure induces 'p53 patches', i.e. clonal outgrowths of keratinocytes with high nuclear expression of mutated p53, which might progress to actinic keratosis (AK) and ultimately squamous cell carcinomas (SCCs). AIMS: Analysis of ingenol mebutate gel (150 and 500 mcg/g) effects in the reduction in 'p53 patches' inside skin cancerization field (CF) in patients with multiple AKs of face/scalp or trunk/extremities, in order to investigate whether the expected reduction in p53+ keratinocytes might have a direct role in the long-term AK reduction in treated areas. RESULTS: We enrolled n = 10 patients, treated with ingenol mebutate and evaluated at 2 and 6 months after treatment. We observed clinical responses in the majority of patients (n = 7), with AK reduction or complete clearance (n = 6 and n = 1, respectively). Notably, two patients did not respond to the treatment, and in one patient, after initial partial response, new lesion was recorded. In untreated skin CF samples (n = 3), we observed numerous p53+ keratinocytes, similar to those observed in invasive SCC samples (53.56 ± 8.79 and 74.34 ± 22.05, respectively; P = 0.2). After treatment, we observed a variable p53+ keratinocyte reduction in CF samples at 2 months (24.67 ± 31.19; P = 0.19). Importantly, the amount of p53+ keratinocytes strongly and directly correlated with AK number (R2  = 0.81). CONCLUSION: Untreated skin CF expresses high level of p53+ keratinocytes as invasive SCC. Ingenol mebutate is able to reduce p53+ keratinocytes with variable efficacy, this reduction degree directly correlating with clinical efficacy.

T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients.

T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39+CD73- nTregs, CD39+CD73+ iTregs, CD39-CD73+ oTregs and CD39-CD73- xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

Cellulose nanocrystals are effective in inhibiting host cell bacterial adhesion.

The use of cellulose nanocrystals (CNCs) as a biomaterial able to inhibit host cell bacterial adhesion is described. Pre-incubation of E. coli ATCC 25922 with a suspension of 0.1% CNCs afforded a significant 2 log reduction of bacterial adhesion to the intestinal cell monolayer HT29. This effect could be attributed to the interaction between the CNCs and the bacterial cell surface as confirmed using fluorescence microscopy experiments.

CD4+FOXP3+ T regulatory cells decrease and CD3+CD8+ T cells recruitment in TILs from melanoma metastases after electrochemotherapy.

Electrochemotherapy (ECT) represents an effective local treatment for skin unresectable melanoma metastases with high overall objective response rate. ECT is based on the combination of anti-neoplastic drugs administration and cancer cells electroporation. Whether ECT can also activate the immune system is a matter of debate, however a significant recruitment of dendritic cells in melanoma treated metastases has been described. Herein we investigated immediate and late effects of ECT treatment on T cell subsets in ECT-treated lesions by fluorescent immunohistochemistry. Biopsies from melanoma patients (n = 10) were taken before ECT (t0), at d1 and d14 from treatment. At t0, CD3+CD4+ T cells were the most represented T cells, well detected in the perilesional dermis, particularly at tumour margin, while CD3+CD8+ T cells were less represented. CD4+FOXP3+ T regulatory (Treg) cells were present in the perilesional dermis and within the lesion. ECT induced a significant decrease of CD4+FOXP3+ Treg cells percentage in the perilesional dermis, observed at d1 and at d14 (p < 0.001). CD3+CD8+ T cells frequency significantly increased at d14 from treatment in the perilesional dermis (p < 0.001). Furthermore calreticulin translocation to the plasma membrane, a hallmark of immunogenic cell death, was observed in metastatic cells after ECT. The data reported here confirm that ECT induces a local response, with a lymphoid infiltrate characterized by CD4+FOXP3+ Treg cells decrease and CD3+CD8+ T cells recruitment in the treated lesions. These results might contribute to design novel combinational therapeutic approaches with ECT and immunotherapy in order to generate a systemic long-lasting anti-melanoma immunity.

Microencapsulation of new probiotic formulations for gastrointestinal delivery: in vitro study to assess viability and biological properties.

The paper describes the preparation of new probiotic formulations based on chitosan-coated alginate microcapsules containing three different probiotic strains, Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070, and Bifidobacterium animalis subsp. lactis PBS075 taken individually and as a mixture of them. The effects of microencapsulation on the viability of the strains in conditions simulating the gastrointestinal tract and under industrial processes conditions were studied. In addition, an evaluation of their probiotic properties was also investigated by in vitro tests on the human intestinal cell line HT-29 to explore the effect of microencapsulation on health beneficial effect of the considered strains. Non-encapsulated cells were completely destroyed when exposed to simulated gastric juice and other stress conditions, while encapsulated cells exhibited a significantly higher resistance to artificial intestinal juice and heat and osmotic treatment. Moreover, in this study, the effect of the various microencapsulated probiotic strain formulations was compared with analogous formulations also containing the ß-glucan Pleuran. The microencapsulation effectively protected the selected bacteria, as single strain and as a mixture of the three strains in both the formulations with and without Pleuran, from simulating gastrointestinal tract and industrial process conditions in delivering the viable cells without any significant adverse effect on their functionalities. The comparative study of the immunomodulatory properties of each single strain and the mixture of the three strains revealed a synergistic effect of the probiotic mixture, but no appreciable difference between the two kinds of formulations could be detected, as the effect of Pleuran is covered by the higher potential of the probiotic strains.

Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect.

Probiotic ingestion is recommended as a preventive approach to maintain the balance of the intestinal microbiota and to enhance the human well-being. During the whole life of each individual, the gut microbiota composition could be altered by lifestyle, diet, antibiotic therapies and other stress conditions, which may lead to acute and chronic disorders. Hence, probiotics can be administered for the prevention or treatment of some disorders, including lactose malabsorption, acute diarrhoea, irritable bowel syndrome, necrotizing enterocolitis and mild forms of inflammatory bowel disease. The probiotic-mediated effect is an important issue that needs to be addressed in relation to strain-specific probiotic properties. In this work, the probiotic properties of new Lactobacillus and Bifidobacterium strains were screened, and their effects in vitro were evaluated. They were screened for probiotic properties by determining their tolerance to low pH and to bile salts, antibiotic sensitivity, antimicrobial activity and vitamin B8, B9 and B12 production, and by considering their ability to increase the antioxidant potential and to modulate the inflammatory status of systemic-miming cell lines in vitro. Three out of the examined strains presenting the most performant probiotic properties, as Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070 and Bifidobacterium animalis subsp. lactis PBSO75, were evaluated for their effects also on human intestinal HT-29 cell line. The obtained results support the possibility to move to another level of study, that is, the oral administration of these probiotical strains to patients with acute and chronic gut disorders, by in vivo experiments.

Genome Sequence of Rhodococcus opacus Strain R7, a Biodegrader of Mono- and Polycyclic Aromatic Hydrocarbons.

Rhodococcus opacus strain R7 (CIP107348) degrades several mono- and polycyclic aromatic hydrocarbons. Here, we present the high-quality draft genome sequence of strain R7, consisting of 10,118,052 bp, with a G+C content of 67.0%, 9,602 protein-coding genes, and 62 RNAs genes.

Genome Sequence of Rhodococcus sp. Strain BCP1, a Biodegrader of Alkanes and Chlorinated Compounds.

Rhodococcus sp. strain BCP1 cometabolizes chlorinated compounds and mineralizes a broad range of alkanes, as it is highly tolerant to them. The high-quality draft genome sequence of Rhodococcus sp. strain BCP1, consisting of 6,231,823 bp, with a G+C content of 70.4%, 5,902 protein-coding genes, and 58 RNA genes, is presented here.

17ß-estradiol protects human skin fibroblasts and keratinocytes against oxidative damage.

BACKGROUND: Reactive oxygen species (ROS) cause severe damage to extracellular matrix and to molecular structure of DNA, proteins and lipids. Accumulation of these molecular changes apparently constitutes the basis of cell ageing. 17b-estradiol (E2) has a key role in skin ageing homeostasis as evidenced by the accelerated decline in skin appearance seen in the perimenopausal years. Oestrogens improve many aspects of the skin such as skin thickness, vascularization, collagen content and quality. Despite these clinical evidences, the effects of oestrogens on skin at the cellular level need further clarification. MATERIALS AND METHODS: HaCaT and human fibroblasts were cultured under various conditions with E2 and H2 O2 ; then were subjected to immunofluorescence and western blot analysis. Lipoperoxidation was investigated using BODIPY. RESULTS: In human fibroblasts oxidative stress decreases procollagen-I synthesis, while E2 significantly increases it. Fibroblasts and HaCaT cells viability in the presence of E2 demonstrates a notably increased resistance to H2 O2 effects. Furthermore E2 is able to counteract H2 O2 -mediated lipoperoxidation and DNA oxidative damage in skin cells. DISCUSSION: In this study we highlight that the menopause-associated oestrogens decline is involved in reduced collagen production and that E2 could counteract the detrimental effects of oxidative stress on the dermal compartment during skin aging. Furthermore, our data show that physiological concentrations of oestrogens are able to interfere with ROS-mediated cell viability reduction and to protect human skin cells against oxidative damage to cellular membranes and nucleic acids structure. CONCLUSION: Our experimental data show that the presence of 17ß-estradiol may protect skin cells against oxidative damage and that the dramatic lowering of oestrogen levels during menopause, could render skin more susceptible to oxidative damage.

Development of microbial engineered whole-cell systems for environmental benzene determination.

This paper reports the development of two recombinant bacterial systems that can be used to monitor environmental benzene contamination based on Escherichia coli, which carry genes coding for benzene dioxygenase and benzene dihydrodiol dehydrogenase from Pseudomonas putida MST. E. coli strains express these two enzymes under the control of the Ptac promoter or without any induction. These activities can be detected electrochemically or colorimetrically and used to monitor benzene pollution in environmental air samples collected from an oil refinery assessing benzene by different laboratory experimental procedures. The procedures involving whole-cell bioassays determine the concentration of benzene through benzene dioxygenase activity, which allows for direct correlation of oxygen consumption, and through the benzene dihydrodiol dehydrogenase that causes catechol accumulation and restores NADH necessary for the activity of the first enzyme. Oxygen consumption and catechol production deriving from both enzymatic activities are related to benzene concentration and their measurements determined the sensitivity of the system. The results indicated that the sensitivity was enough to detect the benzene vapor at a lower concentration level of 0.01 mM in about 30 min. The possibility for on-line monitoring of benzene concentration by our new recombinant cells results from the fact that no particular treatment of environmental samples is required. This is a major advantage over other biosensors or assays. Moreover, the development of microbial cells that did not require any addition or effectors for the transcription of the specific enzymes, allowed these systems to be more versatile in automated environmental benzene monitoring.

Selection of surfactants for enhancing diesel hydrocarbons-contaminated media bioremediation.

The use of surfactants represents a valuable method to enhance the access of the microorganisms to low-soluble and recalcitrant compounds in bioremediation techniques. The choice of surfactants is the first step of feasibility studies for this application. So far, no defined procedures are present in literature to select the most suitable surfactant for the treatment of a specific contaminated site. Furthermore, the characterisation of physico-chemical parameters is important to understand the reason of successes and failures. In this paper a step procedure to select and characterise a commercial surfactant to be used in bioremediation enhancement of hydrocarbon-contaminated media was developed. Among the commercial surfactants, the procedure was applied to alkyl polyethoxylates (Brij family) and sorbitan derivates (Tween family). The selection resulted in the application of Brij 56 and Tween 80 as biodegradation-enhancer in different lab scale systems for remediation of diesel contamination. In liquid systems, Tween 80 greatly increased biodegradation of highly branched and high-molecular weight hydrocarbons, while Brij 56 enhanced degradation of highly branched hydrocarbons. Based on these results, the potential applications and the limitations of these surfactants at full scale level were estimated.

Slurry phase bioremediation of PAHs in industrial landfill samples at laboratory scale.

The effect of Tween 80 and selected bacteria additions on the bioremediation of PAH contaminated landfill soil (70.38mgkg(-1)) was evaluated in a slurry phase bioreactor. A phenanthrene-degrading consortium was selected by enrichment cultures and used as autochthonous inoculum. The Tween 80 addition increased the aqueous concentration of both high and low molecular weight PAHs. In the experiment with Tween 80 and inoculum addition, added microorganisms improved (>90%) the biodegradation of two- and three-ring PAHs as well as of the four-ring PAHs pyrene and fluoranthene. Biodegradation of the higher molecular weight PAHs was about 30% in experiments with Tween 80 addition, with and without inoculum addition.

Dioxygenation of naphthalene by Pseudomonas fluorescens N3 dioxygenase: optimization of the process parameters.

The bioconversion of naphthalene to the 1,2-dihydro-1,2-dihydroxy derivative was performed in good yield using an Escherichia coli recombinant strain carrying Pseudomonas fluorescens N3 dioxygenase. However, the efficiency of such transformation is affected by many process parameters, and their optimization is essential to the scaling up of the process. The following process parameters were considered for optimization: cell concentration together with the corresponding glucose concentration (DCW/L); pH of medium; temperature; stirring speed; air flow; substrate concentration; Fe(2+) concentration; microelements concentration; reaction volume. We used a two-step multivariate experimental design to select important variables and assign them optimal values. The most significant parameters were selected by adopting a Plackett-Burman design, and were then correlated, using a full factorial design, with the experimental results. The experimental results illustrate that the optimized process of recombinant whole cell biotransformation in two-liquid phase systems enhances the naphthalene dihydrodiol yield threefold. This biotransformation opens the way to future experiments involving different substrates.

Naphthalene biodegradation kinetics in an aerobic slurry-phase bioreactor.

The research was focused on the slurry-phase biodegradation of naphthalene in soil. Among ex situ techniques, the slurry phase offers the advantage of increased availability of contaminants to bacteria. From naphthalene contaminated soil, a Pseudomonas putida M8 strain capable to degrade naphthalene was selected. Experiments were performed in a stirred and oxygenated reactor. In this study, the influence of air flow rate and agitation rate on volatilisation and biodegradation of naphthalene was investigated. The hydrocarbon disappearance, the carbon dioxide production, and the ratio of total heterotrophic and naphthalene-degrading bacteria was monitored. The results obtained confirm that the selected bioremediation technology is successful in the treatment of contaminated soils.

Bioremediation of diethylhexyl phthalate contaminated soil: a feasibility study in slurry- and solid-phase reactors.

The aim of the research was to verify the possibility of applying bioremediation as a treatment strategy on a poly(vinyl chloride) (PVC) manufacturing site in the north of Italy contaminated by diethylhexyl phthalate (DEHP) at a concentration of 5.51 mg/g of dry soil. Biodegradation kinetic experiments with DEHP contaminated soil samples were performed in both slurry- and solid-phase systems. The slurry-phase results showed that the cultural conditions, such as N and P concentrations and the addition of a selected DEHP degrading strain, increased the natural DEHP degradation rate. On the basis of these data, experiments to simulate bioventing on contaminated soil columns were performed. The DEHP concentration reached 0.63 mg/g of dry soil in 76 days (89% of degradation). A kinetic equation was developed to fit the experimental data and to predict the concentration of contaminant after treatment. The data obtained are encouraging for a future in situ application of the bioventing technology.

Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil.

Rhodococcus opacus R7 was isolated from a soil contaminated with polycyclic aromatic hydrocarbons for its ability to grow on naphthalene. The strain was also able to degrade o-xylene, the isomer of xylenes most recalcitrant to microbial degradation. The catabolic pathways for naphthalene and o-xylene were investigated by identification of metabolites in R. opacus R7 cultures performed with the two hydrocarbons and by evaluation of some enzymes involved in the metabolism of these compounds. 1,2-Dihydro-1,2-dihydroxynaphthalene, salicylic and gentisic acids were identified as metabolites in cultures exposed to naphthalene. This suggests that the degradation occurs through the dioxygenation of the aromatic ring with the formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehydrogenated to the corresponding 1,2-dihydroxy derivative which is further oxidized to salicylic acid, a key intermediate of naphthalene metabolism; this compound is converted to gentisic acid cleaved by a gentisate 1,2-dioxygenase. From R. opacus R7 cultures supplied with o-xylene, 2,3-dimethylphenol and 3,4-dimethylcatechol were observed. The pathway of o-xylene involves the monooxygenation of the benzene nucleus leading to dimethylphenol which is further metabolised to 3,4-dimethylcatechol, followed by a meta cleavage reaction, catalyzed by the catechol 2,3-dioxygenase. R. opacus R7 is the first strain thus far described both in Gram-negative and Gram-positive bacteria which has the ability to degrade both a polycyclic aromatic hydrocarbon such as naphthalene and a monocyclic aromatic hydrocarbon such as o-xylene.

Efficient polycyclic aromatic hydrocarbons dihydroxylation in direct micellar systems.

Optimization of whole-cell bioconversion of the polycyclic aromatic hydrocarbons (PAHs) anthracene, phenanthrene, and naphthalene to the enantiomerically pure corresponding cis-dihydroxydihydro derivatives by the Escherichia coli JM109 (pPS1778) recombinant strain, carrying the naphthalene dioxygenase and corresponding regulatory genes cloned from Pseudomonas fluorescens N3, in micellar systems, is presented. We show that direct microemulsion systems, where a nonionic surfactant such as 1.5% (v/v) Triton X-100 plus 0.6% to 1.0% (v/v) selected oils are able to solubilize the PAHs tested at relatively high concentrations (initial concentrations in the reaction medium > or =10 mM for naphthalene and phenanthrene and > or =2 mM for anthracene), and allow for more efficient substrate bioconversion. These media, while not affecting bacteria viability and performance, provide increased efficiency and final product yields (100% for naphthalene, >30% for anthracene, >60% for phenanthrene). The phase behavior of the direct microemulsion systems for the different substrates and oils utilized was monitored as a function of their volume fraction by light scattering experiments, and related to the bioconversion results. For anthracene and phenanthrene, the dihydroxylated products have an inhibitory effect on the conversion reactions, thus hindering complete turnover of the substrates. We ascertain that such inhibition is reversible because removal of the products formed allowed the process to start over at rates comparable to initial rates. To allow for complete conversion of the PAHs tested a stepwise or continuous separation of the product formed from the micellar reaction environment is being developed.

Direct micellar systems as a tool to improve the efficiency of aromatic substrate conversion for fine chemicals production.

Whole-cell bioconversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Escherichia coli JM109(pPS1778) recombinant strain, carrying naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3, in direct micellar systems is optimized as an example of fine chemicals bioproduction from scarcely water-soluble substrates. The oxygen insertion into the aromatic substrate, which stops at the enantiomerically pure cis dihydroxylated product, is performed in direct microemulsion systems, where a non-ionic surfactant stabilizes naphthalene containing oil droplets in an aqueous medium. These media provide an increased substrate solubility so that a homogeneous reaction can be carried out, while not affecting bacteria viability and performances. The influence of the chemical nature of the oil is investigated. The phase behavior of the direct microemulsion system was monitored for three different oils as a function their volume fraction and characterized through light scattering. The addition of isopropyl palmitate, oleic acid, or glyceryl trioleate, 0.6-1.2% v/v to the micellar systems, led to an increase of the substrate concentration in the solution and particularly its bioavailability, allowing faster catalytic conversions. All these systems resulted in being suitable for catalytic conversions of aromatic compounds. Although the nature of the oil does have a deep effect on the phase behavior of the micellar systems, in the present investigation no differences in the yields and in the rates of product formation of the enzymatic system were observed on changing the oil, thus showing that in this case the substrate concentration or bioavailability is not the rate-limiting step.