Interpretation of CPTu in “unusual” soils

The paper deals with the interpretation of CPTu in unusual soils, such as shallow clayey layers above the water table and loose, intermediate - permeability soils (loose silt mixtures). The paper shows an approach that could be used for the first type of soil to infer the effective vertical stress from CPTu measurements and in particular from the Ic index. The approach has been checked on a very limited amount of experimental evidence. Moreover, an empirical correction of the Ic index is provided in order to obtain a more realistic soil profiling of loose silt mixtures. The foundation soils of the Serchio River levee system and some dredged sediments, which had been stored in the Port of Livorno, have been considered for the second type of soil

Optical properties of the dibenzothiazolylphenol molecular crystals through ONIOM calculations: the effect of the electrostatic embedding scheme

Periodic density functional theory (DFT) and hybrid ONIOM time-dependent DFT/MM cluster calculations have been carried out to investigate the ground- and excited-state properties of the crystalline structures of the enolic and ketonic tautomeric forms of a propoxy-substituted dibenzothiazolylphenol molecule (OPr), a prototype for systems undergoing the excited-state intramolecular proton transfer process. The crystalline structures of the tautomeric forms are well reproduced and, as expected, at the ground state, the enol polymorph is predicted to be more stable than the keto one. At the excited state, the effect of the environment on time-dependent DFT calculations has been accounted for by including a charge embedding scheme, and the influence of different kinds of point charges (Mulliken, CM5, RESP and QEq) in determining the optical properties of the central molecule has been investigated. The results reveal that, in fair agreement with experimental data, the absorption (emission) energies of the enol (keto) OPr molecule is red-shifted of about 3 (3) nm going from the gas phase to chloroform and blue-shifted of 10 (23) nm going from the gas to the crystal phase when the electronic embedding with Mulliken charges is employed. The electrostatic embedding influences the excited-state properties more severely than the ground-state properties, and apart the QEq charges, all other models provide Stokes shifts in reasonable agreement with experimental data

Safety and efficacy of tranexamic acid for prevention of obstetric haemorrhage. An updated systematic review and meta-analysis

Background. A number of clinical systematic review and meta-analysis have been published on the use of tranexamic in the obstetric setting. The aim of this meta-analysis was to evaluate the safety and effectiveness of tranexamic acid in reducing blood loss when given prior to caesarean delivery. Materials and methods. We searched the Cochrane Wounds Specialized Register, Cochrane Central, MEDLINE (through PUBMED), Embase, and SCOPUS electronic databases. We also searched clinical trials registries for ongoing and unpublished studies, and checked reference lists to identify additional studies. We used no restrictions with respect to language and date of publication. Two review authors independently performed study selection, "Risk of bias" assessment, and data extraction. Initial disagreements were resolved by discussion, or by including a third review author when necessary. Results. We found 18 randomised controlled trials (RCTs) that met our inclusion criteria. Overall, 1,764 women receiving intravenous tranexamic acid for prevention of bleeding following caesarean sections and 1,793 controls receiving placebo were enrolled in the 18 RCTs evaluated. The use of tranexamic acid compared to controls (placebo or no intervention) reduces post-partum haemorrhage >400 mL (risk ratio [RR] 0.40, 95% confidence interval [CI] 0.24-0.65; 5 trials with a total of 786 participants), severe post-partum haemorrhage >1,000 mL (RR 0.32, 95% CI: 0.12-0.84; 5 trials with a total of 1,850 participants), and need for red blood cell transfusion (RR 0.30, 95% CI: 0.18-0.49; 10 trials with a total of 1,873 participants). No particular safety concerns on the use of this antifibrinolytic agent emerged from the analysis of the 18 RCTs included. Discussion. Overall, the results of this meta-analysis support the evidence of a beneficial effect of tranexamic acid in reducing blood loss and need for blood transfusion in pregnant women undergoing caesarean section

Screening and heterologous expression of chitinases from environmental metagenomic libraries.

My PhD thesis has been developed in the frame of the EU-funded Metaexplore project. This project, conceived in collaboration with eighteen research groups all over Europe, concerns the development of new metagenomic techniques to detect enzymes involved in the biodegradation of recalcitrant natural molecules such as chitin and lignin. The final goal is the heterologous expression and characterization of the newly discovered chitinases and ligninases to exploit them in industrial bioprocesses. My thesis deals with chitinases that include a group of enzymes acting in a synergic and consecutive way, responsible for the complete hydrolysis of chitin into the constitutive monomers of N-acetylglucosamine. An alternative pathway involves chitin deacetylases for the deacetylation of chitin into chitosan, which is then further hydrolyzed by chitosanases in glucosamine units. Chitin derivatives have a multiplicity of industrial applications, which make chitinases attractive at industrial level. University of Insubria participates at the Metaexplore project in different work-packages. In the first part, my work is devoted to the validation of the functional screening methods to detect chitin degrading activities from metagenomic libraries. The following task regards the preliminary analysis of the most promising clones and the heterologous expression of the genes of interest in conventional and alternative hosts. At the moment, protein expression is the major bottleneck for the biotechnological success of the metagenomic approach to discover new enzymes. The optimization of the heterologous expression of metagenome-sourced genes will allow a complete biochemical characterization of the new enzymes and their production at industrial level. The following chapters report Material and Methods, Results and Discussion related to the experimental work performed for the Metaexplore project, during the three years of my PhD course. The first chapter, Screening for chitinase activities in actinomycetes, consist in an exploration of the University of Insubria’s actinomycete culture collection for chitin degrading activities. This culture collection comprises microorganisms belonging to Streptomyces genus, but also to other genera of uncommon actinomycetes named “rare”, since difficult to be isolated, cultivated, and manipulated. All the assayed microorganisms are degrading chitin when it is used as a sole carbon and nitrogen source in plate assay, but only some of them seem to possess chitosan and chitin deacetylase activities. Induction and repression of the chitinolytic system has been investigated in representative strains. Enzymatic assays to detect the extracellular and intracellular levels of chitin depolymerase, chitobiosidase, N-acetylglucosaminidase activities have been developed, validated and transferred to the Metaexplore partners to be applied at the screening of the metagenomic libraries. Interestingly, the rare actinomycete Nonomuraea sp. ATCC 39727, previously never explored for its chitin degrading activity, results positive in the chitin deacetylase plate assay and produces chitosan. To my knowledge, this is the first report on chitin deacetylase enzyme discovered from actinomycetes. The second paper, Bacterial chitinase with biocontrol capacity from suppressive soil revealed by functional metagenomics, presents the research work performed in collaboration with the School of Life Sciences of Södertörn University (Huddinge, Sweden), on the heterologous expression of the first chitinase isolated from a metagenomic library within the Metaexplore consortium. The metagenomic library originates from a soil considered suppressive for clubroot disease of cabbage. Genetic screening using degenerate primers for the conserved central catalytic domain of family 18 chitinases has selected a putative chitobiosidase gene, called chi18H8. The gene is cloned in BL21(DE3) E. coli cells, in transcriptional fusion with the gluthathione-S-transferase tag. In this host, most of the protein accumulates in the insoluble fraction as inclusion bodies, so expression optimization in E. coli soluble fractions is required for protein purification by affinity chromatography. On the pure enzyme, recovered at very low concentration, preliminary analysis confirms its chitinolytic nature and its antifungal properties against common plant phytopathogens. In the third chapter, entitled Expression of a metagenome-sourced chitinase in Streptomyces lividans, this actinomycete is presented as alternative candidate to E. coli for the heterologous expression of the chi18H8 chitinase. Streptomyces spp. are typical degraders of biomasses in soil habitats. They possess a developed secretory pathway for the extracellular release of their hydrolytic enzymes. As described in the first paper on screening, they usually produce different chitinases, whose activity can be detected also when cultivated in rich media not containing chitin as carbon source. To use S. lividans as a host for heterologous chitinase expression, the chitinolytic system is metabolically repressed by adding glucose to the culture media, and the chi18H8 gene is cloned in a multicopy plasmid under the control of a heterologus constitutive promoter which is not repressed by the presence of glucose. The transformation of S. lividans TK 24 occurs by E. coli - S. lividans integeneric conjugation after a DNA demethylation step. Enzymatic activity assay and zymogram analysis confirm that the recombinant protein is secreted in the extracellular broth with an estimated volumetric productivity of 66 U/L which represents a good starting point for the further attempts of protein production and purification of Chi18H8 in S. lividans host

Screening and heterologous expression of chitinases from environmental metagenomic libraries.

My PhD thesis has been developed in the frame of the EU-funded Metaexplore project. This project, conceived in collaboration with eighteen research groups all over Europe, concerns the development of new metagenomic techniques to detect enzymes involved in the biodegradation of recalcitrant natural molecules such as chitin and lignin. The final goal is the heterologous expression and characterization of the newly discovered chitinases and ligninases to exploit them in industrial bioprocesses. My thesis deals with chitinases that include a group of enzymes acting in a synergic and consecutive way, responsible for the complete hydrolysis of chitin into the constitutive monomers of N-acetylglucosamine. An alternative pathway involves chitin deacetylases for the deacetylation of chitin into chitosan, which is then further hydrolyzed by chitosanases in glucosamine units. Chitin derivatives have a multiplicity of industrial applications, which make chitinases attractive at industrial level. University of Insubria participates at the Metaexplore project in different work-packages. In the first part, my work is devoted to the validation of the functional screening methods to detect chitin degrading activities from metagenomic libraries. The following task regards the preliminary analysis of the most promising clones and the heterologous expression of the genes of interest in conventional and alternative hosts. At the moment, protein expression is the major bottleneck for the biotechnological success of the metagenomic approach to discover new enzymes. The optimization of the heterologous expression of metagenome-sourced genes will allow a complete biochemical characterization of the new enzymes and their production at industrial level. The following chapters report Material and Methods, Results and Discussion related to the experimental work performed for the Metaexplore project, during the three years of my PhD course. The first chapter, Screening for chitinase activities in actinomycetes, consist in an exploration of the University of Insubria’s actinomycete culture collection for chitin degrading activities. This culture collection comprises microorganisms belonging to Streptomyces genus, but also to other genera of uncommon actinomycetes named “rare”, since difficult to be isolated, cultivated, and manipulated. All the assayed microorganisms are degrading chitin when it is used as a sole carbon and nitrogen source in plate assay, but only some of them seem to possess chitosan and chitin deacetylase activities. Induction and repression of the chitinolytic system has been investigated in representative strains. Enzymatic assays to detect the extracellular and intracellular levels of chitin depolymerase, chitobiosidase, N-acetylglucosaminidase activities have been developed, validated and transferred to the Metaexplore partners to be applied at the screening of the metagenomic libraries. Interestingly, the rare actinomycete Nonomuraea sp. ATCC 39727, previously never explored for its chitin degrading activity, results positive in the chitin deacetylase plate assay and produces chitosan. To my knowledge, this is the first report on chitin deacetylase enzyme discovered from actinomycetes. The second paper, Bacterial chitinase with biocontrol capacity from suppressive soil revealed by functional metagenomics, presents the research work performed in collaboration with the School of Life Sciences of Södertörn University (Huddinge, Sweden), on the heterologous expression of the first chitinase isolated from a metagenomic library within the Metaexplore consortium. The metagenomic library originates from a soil considered suppressive for clubroot disease of cabbage. Genetic screening using degenerate primers for the conserved central catalytic domain of family 18 chitinases has selected a putative chitobiosidase gene, called chi18H8. The gene is cloned in BL21(DE3) E. coli cells, in transcriptional fusion with the gluthathione-S-transferase tag. In this host, most of the protein accumulates in the insoluble fraction as inclusion bodies, so expression optimization in E. coli soluble fractions is required for protein purification by affinity chromatography. On the pure enzyme, recovered at very low concentration, preliminary analysis confirms its chitinolytic nature and its antifungal properties against common plant phytopathogens. In the third chapter, entitled Expression of a metagenome-sourced chitinase in Streptomyces lividans, this actinomycete is presented as alternative candidate to E. coli for the heterologous expression of the chi18H8 chitinase. Streptomyces spp. are typical degraders of biomasses in soil habitats. They possess a developed secretory pathway for the extracellular release of their hydrolytic enzymes. As described in the first paper on screening, they usually produce different chitinases, whose activity can be detected also when cultivated in rich media not containing chitin as carbon source. To use S. lividans as a host for heterologous chitinase expression, the chitinolytic system is metabolically repressed by adding glucose to the culture media, and the chi18H8 gene is cloned in a multicopy plasmid under the control of a heterologus constitutive promoter which is not repressed by the presence of glucose. The transformation of S. lividans TK 24 occurs by E. coli - S. lividans integeneric conjugation after a DNA demethylation step. Enzymatic activity assay and zymogram analysis confirm that the recombinant protein is secreted in the extracellular broth with an estimated volumetric productivity of 66 U/L which represents a good starting point for the further attempts of protein production and purification of Chi18H8 in S. lividans host

A novel Markov model for the computation of the Continuity Risk in maritime applications

In this paper we focused on the analysis of the continuity risk for a maritime user and on the derivation of the receiver implementation scheme that fulfills IMO [2] requirements. We started our analysis by considering the model derived in [6], which is accurate for aviation applications, while it is not guaranteed to work in other environments. To take into account the time evolution of the continuity risk, we propose in this paper to introduce Markov models. The derived models can be used to compute the continuity risk when a receiver not implementing exclusion is used, as well as when a receiver implements both snapshot and sequential exclusion. The derived conclusion is that without exclusion, it is not possible to achieve the required performance. It is shown that, considering only satellite faults, over the 3 hours of operation, in principle a snapshot exclusion mechanism is sufficient. When implementing sequential exclusion, at the cost of an increased complexity, the continuity risk is furthermore reduced. We also showed that, under some temporal limitations and with some assumptions on the exclusion mechanism, the results provided by the Markov models and by the model derived in [6] coincide. This final consideration shows that the proposed approach can be seen as a natural extension of the state of the art model from the avionic environment to the maritime environment