A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

BACKGROUND: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. METHODS: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. RESULTS: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. DISCUSSION: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.

Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen-Ligand Complexes.

BACKGROUND: Protein complexes provide valuable biological information, but can be difficult to handle. Therefore, technical advancements designed to improve their manipulation are always useful. METHODS: We investigated the opportunity to exploit native agarose gels and the contact blot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes. As a simple model of protein-protein interactions, an antigen-ligand complex was used in which both proteins were fused to reporters. RESULTS: At each step, it was possible to visualize both the antigen, fused to a fluorescent protein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers. CONCLUSIONS: The procedure is rapid, inexpensive, and the several detection opportunities allow for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific training.

Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers Is Simplified by Phagemid Design.

Phage display is an effective method to retrieve binders specific for a target epitope from a large clone library. Nevertheless, the panning process allows for the accumulation of some contaminant clones into the selected phage pool and, consequently, each clone requires individual screening to verify its actual specificity. This step is time-consuming, independently on the chosen method, and relies on the availability of reliable reagents. Since phages display a single binder responsible for the antigen recognition but their coat is formed by several repeats of the same proteins, the targeting of coat epitopes is often exploited to amplify the signal. Commercial anti-M13 antibodies are commonly labeled with peroxidase or FITC but customized antibodies might be necessary for specific applications. Here, we report a protocol describing the selection of anti-protoplast Adhirons that relies on the availability of nanobodies fused to a fluorescent protein to use during flow cytometry screening. Specifically, when preparing our Adhiron synthetic library, we designed a new phagemid that allows the expression of the clones fused to three tags. These can interact with a large variety of commercial and home-made reagents, selected according to the needs of the downstream characterization process. In the described case, we combined the ALFA-tagged Adhirons with an anti-ALFAtag nanobody fused with the fluorescent protein mRuby3.

Solid-State Fermentation: Applications and Future Perspectives for Biostimulant and Biopesticides Production.

With the expansion of the green products market and the worldwide policies and strategies directed toward a green revolution and ecological transition, the demand for innovative approaches is always on the rise. Among the sustainable agricultural approaches, microbial-based products are emerging over time as effective and feasible alternatives to agrochemicals. However, the production, formulation, and commercialization of some products can be challenging. Among the main challenges are the industrial production processes that ensure the quality of the product and its cost on the market. In the context of a circular economy, solid-state fermentation (SSF) might represent a smart approach to obtaining valuable products from waste and by-products. SSF enables the growth of various microorganisms on solid surfaces in the absence or near absence of free-flowing water. It is a valuable and practical method and is used in the food, pharmaceutical, energy, and chemical industries. Nevertheless, the application of this technology in the production of formulations useful in agriculture is still limited. This review summarizes the literature dealing with SSF agricultural applications and the future perspective of its use in sustainable agriculture. The survey showed good potential for SSF to produce biostimulants and biopesticides useful in agriculture.

Macroporous Epoxy-Based Monoliths Functionalized with Anti-CD63 Nanobodies for Effective Isolation of Extracellular Vesicles in Urine.

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

Exploring structure, microbiota, and metagenome functions of epigean and hypogean black deposits by microscopic, molecular and bioinformatic approaches.

This study revealed how Bacteria and Archaea communities and their metabolic functions differed between two groups of black deposits identified in gorge and cave environments. Scanning electron microscopy coupled with energy dispersive spectroscopy was used to analyse the presence of microbial biosignatures and the elemental composition of samples. Metabarcoding of the V3-V4 regions of 16S rRNA was used to investigate Bacteria and Archaea communities. Based on 16S rRNA sequencing results, PICRUSt software was used to predict metagenome functions. Micrographs showed that samples presented microbial biosignatures and microanalyses highlighted Mn concretions and layers on Al-Si surfaces. The 16S rRNA metabarcoding alpha-diversity metrics showed similar Simpson's and Shannon indices and different values of the Chao-1 index. The amplicon sequence variants (ASVs) analysis at the different taxonomic levels showed a diverse genera composition. However, the communities of all samples shared the presence of uncultured ASVs belonging to the Gemmatales family (Phylogenesis: Gemmataceae; Planctomycetes; Planctomycetota; Bacteria). The predicted metagenome functions analysis revealed diverse metabolic profiles of the Cave and Gorge groups. Genes coding for essential Mn metabolism were present in all samples. Overall, the findings on structure, microbiota, and predicted metagenome functions showed a similar microbial contribution to epigean and hypogean black deposits Mn metabolism.

The "Infernaccio" Gorges: Microbial Diversity of Black Deposits and Isolation of Manganese-Solubilizing Bacteria.

The present study explored the microbial diversity of black deposits found in the "Infernaccio" gorge. X-ray Powdered Diffraction (XRPD) was used to investigate the crystallinity of the samples and to identify the minerals. Scanning electron microscope and energy-dispersive X-ray spectroscopy (SEM-EDS) were used to detect the bacterial imprints, analyze microbe-mineral interactions, and highlight the chemical element distribution in the black deposits. 16S rRNA gene metabarcoding allowed the study of Archaea and Bacteria communities. Mn-oxide-solubilizing isolates were also obtained and characterized by culturable and molecular approaches. The multidisciplinary approach showed the occurrence of deposits composed of birnessite, diopside, halloysite, and leucite. Numerous bacterial imprints confirmed the role of microorganisms in forming these deposits. The Bacteria and Archaea communities associated with these deposits and runoff waters are dynamic and shaped by seasonal changes. The uncultured and unknown taxa are the most common and abundant. These amplicon sequence variants (ASVs) were mainly assigned to Proteobacteria and Bacteroidetes phyla. Six isolates showed interesting Mn solubilization abilities under microaerophilic conditions. Molecular characterization associated isolates to Brevibacterium, Bacillus, Neobacillus, and Rhodococcus genera. The findings enriched our knowledge of geomicrobiological aspects of one of the Earth's hidden habitats. The study also unveiled the potential of this environment as an isolation source of biotechnologically relevant bacteria.

Fusarium Oxysporum f. sp. Cannabis Isolated from Cannabis Sativa L.: In Vitro and In Planta Biocontrol by a Plant Growth Promoting-Bacteria Consortium.

Industrial hemp (Cannabis sativa L.) is a multipurpose plant used in several fields. Several phytopathogens attack hemp crops. Fusarium oxysporum is a common fungal pathogen that causes wilt disease in nurseries and in field cultivation and causes high losses. In the present study, a pathogenic strain belonging to F. oxysporum f. sp. cannabis was isolated from a plant showing Fusarium wilt. After isolation, identification was conducted based on morphological and molecular characterizations and pathogenicity tests. Selected plant growth-promoting bacteria with interesting biocontrol properties-Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria-were tested against this pathogen. In vitro antagonistic activity was determined by the dual culture method. Effective strains (in vitro inhibition > of 50%) G. diazotrophicus, H. seropedicae and B. ambifaria were combined in a consortium and screened for in planta antagonistic activity in pre-emergence (before germination) and post-emergence (after germination). The consortium counteracted Fusarium infection both in pre-emergence and post-emergence. Our preliminary results show that the selected consortium could be further investigated as an effective biocontrol agent for the management of this pathogen.

Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity.

Biotic stress caused by pathogenic microorganisms leads to damage in crops. Tomato and carrot are among the most important vegetables cultivated worldwide. These plants are attacked by several pathogens, affecting their growth and productivity. Fourteen plant growth-promoting actinomycetes (PGPA) were screened for their in vitro biocontrol activity against Solanum lycopersicum and Daucus carota microbial phytopathogens. Their antifungal activity was evaluated against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and Rhizoctonia solani (RHS). Antibacterial activity was evaluated against Pseudomonas syringae, Pseudomonas corrugata, Pseudomonas syringae pv. actinidiae, and Pectobacterium carotovorum subsp. carotovorum. Strains that showed good in vitro results were further investigated in vitro (cell-free supernatants activity, scanning electron microscope observations of fungal inhibition). The consortium of the most active PGPA was then utilized as biocontrol agents in planta experiments on S. lycopersicum and D. carota. The Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 strains showed the best in vitro biocontrol activities. The diffusible and volatile compounds and cell-free supernatants of these strains showed both antifungal (in vitro inhibition up to 85%, hyphal desegregation and fungicidal properties) and antibacterial activity (in vitro inhibition >25 mm and bactericidal properties). Their consortium was also able to counteract the infection symptoms of microbial phytopathogens during in planta experiments, improving plant status. The results obtained highlight the efficacy of the selected actinomycetes strains as biocontrol agents of S. lycopersicum and D. carota.

Allium cepa L. Inoculation with a Consortium of Plant Growth-Promoting Bacteria: Effects on Plants, Soil, and the Autochthonous Microbial Community.

The present work was aimed at investigating the effects of a four bacterial strain consortium-Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, and Burkholderia ambifaria-on Allium cepa L. and on soil health. The bacterial consortium was inoculated on seeds of two different onion varieties; inoculated and Control seeds (treated with autoclaved inoculum) were sown in an open-field and followed until harvest. Plant growth development parameters, as well as soil physico-chemical and molecular profiles (DNA extraction and 16S community sequencing on the Mi-Seq Illumina platform), were investigated. The results showed a positive influence of bacterial application on plant growth, with increased plant height (+18%), total chlorophylls (+42%), crop yields (+13%), and bulb dry matter (+3%) with respect to the Control. The differences between Control and treatments were also underlined in the bulb extracts in terms of total phenolic contents (+25%) and antioxidant activities (+20%). Soil fertility and microbial community structure and diversity were also positively affected by the bacterial inoculum. At harvest, the soil with the presence of the bacterial consortium showed an increase in total organic carbon, organic matter, and available phosphorus, as well as higher concentrations of nutrients than the Control. The ecological indexes calculated from the molecular profiles showed that community diversity was positively affected by the bacterial treatment. The present work showed the effective use of plant growth-promoting bacteria as a valid fertilization strategy to improve yield in productive landscapes whilst safeguarding soil biodiversity.

In vitro and in planta antagonistic effects of plant growth-promoting rhizobacteria consortium against soilborne plant pathogens of Solanum tuberosum and Solanum lycopersicum.

Potatoes (Solanum tuberosum L.) and tomatoes (Solanum lycopersicum L.), among the main crops belonging to the Solanaceae family, are attacked by several pathogens. Among them Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani are very common and cause significant losses. Four plant growth-promoting rhizobacteria, Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria were tested against these pathogens. In vitro antagonistic activities of single strains were assessed through dual culture plates. Strains showing antagonistic activity (G. diazotrophicus, H. seropedicae and B. ambifaria) were combined and, after an in vitro confirmation, the consortium was applied on S. lycopersicum and S. tuberosum in a greenhouse pot experiment. The bioprotection was assessed in pre-emergence (infection before germination) and post-emergence (infection after germination). The consortium was able to successfully counteract the infection of both F. oxysporum and R. solani, allowing a regular development of plants. The biocontrol of the fungal pathogens was highlighted both in pre-emergence and post-emergence conditions. This selected consortium could be a valid alternative to agrochemicals and could be exploited as biocontrol agent to counteract losses due to these pathogenic fungi.

Molecular Characterization and Antimicrobial Susceptibility of C. jejuni Isolates from Italian Wild Bird Populations.

Poultry is considered a major reservoir of human campylobacteriosis. It also been reported that not only poultry, but also wild birds, are capable of carrying C. jejuni, thus demonstrating to be a risk of spreading the bacteria in the environment. To gain insight into the population structure and investigate the antimicrobial resistance genotypes and phenotypes, we analyzed a collection of 135 C. jejuni from 15 species of wild birds in Italy. MLST revealed the presence of 41 sequence types (STs) and 13 clonal complexes (CCs). ST-179 complex and the generalist ST-45 complex were the most prevalent. Core genome MLST revealed that C. jejuni from ST-45 complex clustered according to the bird species, unlike the ST-179 complex which featured 3 different species in the same cluster. Overall we found a moderate prevalence of resistance to tetracycline (12.5%), ciprofloxacin and nalidixic acid (10%). The novel ST isolated from one pigeon showed resistance to all the antibiotics tested. The ST-179 complex (33.3%) was identified with significantly higher nalidixic acid resistance relative to other tested STs. Nine AMR genes (tet(O), cmeA, cmeB, cmeC, cmeR, aad, blaOXA-61, blaOXA-184 and erm(B)) and 23S rRNA and gyrA-associated point mutations were also described, indicating a concordance level between genotypic and phenotypic resistance of 23.3%, 23.4% and of 37.5% for streptomycin, tetracycline and quinolones/fluoroquinolones, respectively. We recommend that particular attention should be given to wild birds as key sentinel animals for the ecosystem contamination surveillance.

Dynamical model for sympatric speciation in an ecological niche.

The speciation phenomenon is the process used by the evolution to allow populations to become distinct species. The speciation is the primary cause of the complexity of the ecological network. Sympatric speciation concerns the rise of a new species from a surviving ancestral species while both continue to inhabit the same ecological niche or geographical region. In sympatric speciation, reproductive isolation evolves within a population in an ecological niche without the aid of geographic barriers. Different models have been proposed for alternative modes of sympatric speciation. The most popular was first put forward by John Maynard Smith in 1966 who suggested that in a given population homozygous individuals may, under particular environmental conditions, have a greater fitness than those with alleles heterozygous for a certain trait, eventually leading to speciation in the population. In this framework we assume an effective description of the speciation process based on a dynamical model for the populations in an ecological system. Our basic assumption is the existence of an ancestral population in an ecological niche that can express two phenotypes. In presence of certain environmental conditions one of the phenotypes has the propensity to separate from the original population in the reproduction process. Then new individuals may give rise to a new species in the ecosystem realizing a sympatric speciation. Due to the finite resources in the niche the populations are continuously competing each other's, and their numerousness fluctuates according to the changes of the environmental conditions. The effect of natural selection is introduced in the model by stochastic perturbations, that decrease the reproduction rate of the populations in the niche. We show some the dynamical properties of the system and we prove the existence of a threshold values in the environmental stress in order to observe the speciation process. We also discuss some biological implications of the model and the validation problem using empirical data.

Microbiological and biochemical aspects of inland Pecorino Abruzzese cheese.

Little is known on physicochemical and biochemical characteristics of "Pecorino" Abruzzese cheese in L'Aquila province, an artisanal cheese produced from ewe raw full-cream milk. Three batches of inland "Pecorino" Abruzzese cheese were examined for microbiological, compositional, biochemical and sensory characteristics at the aim of isolating and storing in a bacterial collection, indigenous strain to preserve the microbial biodiversity present in this cheese, to a possible definition of a PDO. Cheese samples from three dairies, at different stages of production were collected and 148 colonies were characterized. Physicochemical assays, species-specific PCR and 16S rRNA gene sequencing revealed that the majority of the lactic acid bacteria (LAB) isolates were Enterococcus faecium and En. faecalis. They were highly prevalent, accounting for 48% of the isolates. The lactic microflora consisted of lactobacilli and lactococci from the species Lactobacillus plantarum (12.2%), Lactobacillus brevis (10.1%), Lactococcus lactis subsp. cremoris (11.5%), respectively. Urea-PAGE electrophoresis showed extensive degradation of αS1-casein (CN) and moderate hydrolysis of ß-CN. Formation of γ-CNs from ß-CN were highlighted. RP-HPLC profiles of the ethanol-soluble and ethanol-insoluble fractions of the pH 4.6-soluble nitrogen showed only minor differences between the three farms: lower proteolysis in the soluble fraction than the insoluble. Leucine, glutamic acid, lysine, valine were the free amino acids present at the highest levels in all the cheeses. Flavour and texture profile were characterized through a sensory analysis.

A study of chlorinated solvent contamination of the aquifers of an industrial area in central Italy: a possibility of bioremediation.

Perchloroethene, trichloroethene, and other chlorinated solvents are widespread groundwater pollutants. They form dense non-aqueous phase liquids that sink through permeable groundwater aquifers until non-permeable zone is reached. In Italy, there are many situations of serious contamination of groundwater that might compromise their use in industry, agriculture, private, as the critical case of a Central Italy valley located in the province of Teramo ("Val Vibrata"), characterized by a significant chlorinated solvents contamination. Data from the various monitoring campaigns that have taken place over time were collected, and new samplings were carried out, resulting in a complete database. The data matrix was processed with a multivariate statistic analysis (in particular principal component analysis, PCA) and was then imported into geographic information system (GIS), to obtain a model of the contamination. A microcosm anaerobic study was utilized to assess the potential for in situ natural or enhanced bioremediation. Most of the microcosms were positive for dechlorination, particularly those inoculated with a mineral medium. This indicate the presence of an active native dechlorinating population in the subsurface, probably inhibited by co-contaminants in the groundwater, or more likely by the absence or lack of nutritional factors. Among the tested electron donors (i.e., yeast extract, lactate, and butyrate) lactate and butyrate enhanced dechlorination of chlorinated compounds. PCA and GIS studies allowed delimiting the contamination; the microcosm study helped to identify the conditions to promote the bioremediation of the area.

In vitro and in vivo inoculation of four endophytic bacteria on Lycopersicon esculentum.

Four bacteria selected on the basis of their capability of fixing atmospheric nitrogen, stimulating plant-growth, and protecting the host plant from pathogens - Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Burkholderia ambifaria - were inoculated on tomato seeds either singularly, in couple and in a four bacteria mixer. Aim of this research was to evaluate: (1) effect of single and mixed cultures on the inoculated plant - plant growth, dry weight, root length and surface, number of leaves, among others; (2) colonization and interactions of the bacteria inside the host plant; (3) localization inside the host of single bacterial strains marked with the gusA reporter gene. The results obtained indicate that all selected microbial strains have colonized Lycopersicon esculentum but in a different way, depending on the single species. A. brasilense, G. diazotrophicus inoculated in vitro singularly and together were the best plant colonizers. In vivo essays, instead, B. ambifaria and the four-bacteria mixer gave the best results. It was possible to localize both A. brasilense and H. seropedicae inside the plant by the gusA reporter gene. The bacterial strains occur along the root axis from the apical zone until to the basal stem, on the shoot from the base up to the leaves. The four bacteria actively colonize tomato seeds and establish an endophytic community inside the plant. This review gives new information about colonization processes, in particular how bacteria interact with plants and whether they are likely to establish themselves in the plant environment after field application as biofertilizers or biocontrol agents.

Calcium carbonate mineralization: involvement of extracellular polymeric materials isolated from calcifying bacteria.

This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

[Role of surgery in patients with metastases from melanoma. A case report]. / Ruolo della chirurgia in pazienti con metastasi da melanoma. Un caso clinico.

INTRODUCTION: The prognosis of patients with melanoma varies according to the staging of disease at the moment of diagnosis. Melanoma can metastasize to every organ or tissue, but the most common site involved is locoregional. In selected patients surgery plays a central role with the possibility of changing the prognosis at distance. CASE REPORT: A 65-year-old man with a diagnosis of neoplasm of the left thyroid lobe and metastatic melanoma with unknown primitive localization. Since 2003 the patient has undergone many operations to remove metastatic melanoma. Currently he is in good conditions and performs neoadjuvant palliative treatments. DISCUSSION: The indication for surgery in cases of patients with distant disease (IV stage) or metastases in transit (IIIc stage) is linked to the possibility of surgical removal of individual lesions. In particular are candidates for surgery patients who have a visceral localization less than or equal to 2 sites, a number less than or equal to 8 metastases, in good health and having a melanoma-specific survival estimated more than 3 months; surgical approach requires an accurate and early identification by imaging study. The case report shows that in selected cases, some patients may benefit from aggressive surgery, especially in terms of survival at distance.

Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides.

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25-40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.