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In microbial community sequencing, involving bacterial ribosomal 16S rDNA or fungal ITS, the targeted genes are the basis for taxonomical assignment. The traditional bioinformatical procedure has for decades made use of a clustering protocol by which sequences are pooled into packages of shared percent identity, typically at 97%, to yield Operational Technical Units (OTUs). Progress in the data processing methods has however led to the possibility of minimizing technical sequencers errors, which were the main reason for the OTU choice, and to analyze instead the exact Amplicon Sequence Variants (ASV) which is a choice yielding much less agglomerated reads. We have tested the two procedures on the same 16S metabarcoded bacterial amplicons dataset encompassing a series of samples from 17 adjacent habitats, taken across a 700 meter-long transect of different ecological conditions unfolding in a gradient spanning from cropland, through meadows, forest and all successional transitions up to the seashore, within the same coastal area. This design allowed to scan a high biodiversity basin and to measure alpha, beta and gamma diversity of the area, to verify the effect of the bioinformatics on the same data as concerns the values of ten different ecological indexes and other parameters. Two levels of progressive OTUs clustering, (99% and 97%) were compared with the ASV data. The results showed that the OTUs clustering proportionally led to a marked underestimation of the ecological indicators values for species diversity and to a distorted behaviour of the dominance and evenness indexes with respect to the direct use of the ASV data. Multivariate ordination analyses resulted also sensitive in terms of tree topology and coherence. Overall, data support the view that reference-based OTU clustering carries several misleading disadvantageous biases, including the risk of missing novel taxa which are yet unreferenced in databases. Since its alternatives as de novo clustering have on the other hand drawbacks due to heavier computational demand and results comparability, especially for environmental studies which contain several yet uncharacterized species, the direct ASV based analysis, at least for prokaryotes, appears to warrant significand advantages in comparison to OTU clustering at every level of percent identity cutoff., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fasolo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The latest Triassic was characterised by protracted biotic extinctions concluding in the End-Triassic Extinction (~ 200 Ma) and a global carbon cycle perturbation. The onset of declining diversity is closely related to reducing conditions that spread globally from upper Sevatian (uppermost Norian) to across the Norian-Rhaetian boundary, likely triggered by unusually high volcanic activity. We correlate significant organic carbon cycle perturbations to an increase of CO 2 in the ocean-atmosphere system, likely outgassed by the Angayucham igneous province, the onset of which is indicated by the initiation of a rapid decline in 87 Sr/ 86 Sr and 188 Os/ 187 Os seawater values. A possible causal mechanism involves elevated CO 2 levels causing global warming and accelerating chemical weathering, which increased nutrient discharge to the oceans and greatly increased biological productivity. Higher export production and oxidation of organic matter led to a global O 2 decrease in marine water across the Norian/Rhaetian boundary (NRB). Biotic consequences of dysoxia/anoxia include worldwide extinctions in some fossil groups, such as bivalves, ammonoids, conodonts, radiolarians., (© 2024. The Author(s).)

Climate change and anthropogenic disturbances are known to influence soil biodiversity. The objectives of this study were to compare the community composition, species coexistence patterns, and ecological assembly processes of soil microbial communities in a paired setting featuring a natural and an anthropogenic ecosystem facing each other at identical climatic, pedological, and vegetational conditions. A transect gradient from forest to seashore allowed for sampling across different habitats within both sites. The field survey was carried out at two adjacent strips of land within the Po River delta lagoon system (Veneto, Italy) one of which is protected within a natural preserve and the other has been converted for decades into a tourist resort. The anthropogenic pressure interestingly led to an increase in the α-diversity of soil microbes but was accompanied by a reduction in β-diversity. The community assembly mechanisms of microbial communities differentiate in natural and anthropic ecosystems: for bacteria, in natural ecosystems deterministic variables and homogeneous selection play a main role (51.92%), while stochastic dispersal limitation (52.15%) is critical in anthropized ecosystems; for fungi, stochastic dispersal limitation increases from 38.1% to 66.09% passing from natural to anthropized ecosystems. We are on calcareous sandy soils and in more natural ecosystems a variation of topsoil pH favors the deterministic selection of bacterial communities, while a divergence of K availability favors stochastic selection. In more anthropized ecosystems, the deterministic variable selection is influenced by the values of SOC. Microbial networks in the natural system exhibited higher numbers of nodes and network edges, as well as higher averages of path length, weighted degree, clustering coefficient, and density than its equivalent sites in the more anthropically impacted environment. The latter on the other hand presented a stronger modularity. Although the influence of stochastic processes increases in anthropized habitats, niche-based selection also proves to impose constraints on communities. Overall, the functionality of the relationships between groups of microorganisms co-existing in communities appeared more relevant to the concept of functional biodiversity in comparison to the plain number of their different taxa. Fewer but functionally more organized lineages displayed traits underscoring a better use of the resources than higher absolute numbers of taxa when those are not equally interconnected in their habitat exploitation. However, considering that network complexity can have important implications for microbial stability and ecosystem multifunctionality, the extinction of complex ecological interactions in anthropogenic habitats may impair important ecosystem services that soils provide us., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A bacterial metabarcoding approach was used to compare the microbiome composition of caecal and faecal samples from fattening Japanese quails (Coturnix coturnix japonica) fed three different diet regimes. The tested feedstuffs included (1) a commercial diet for fattening quails, (2) a commercial diet containing 12% full-fat silkworm (Bombyx mori) pupae meal, and (3) a commercial diet containing 12% defatted silkworm pupae meal. The aim of the experiment was to verify the relative effect of three variables (diet type, gut tract comparing caecum to rectum, and individual animal) in determining the level of bacterial community dissimilarity to rank the relevance of each of the three factors in affecting and shaping community composition. To infer such ranking, the communities resulting from the high-throughput sequencing from each sample were used to calculate the Bray-Curtis distances in all the pairwise combinations, whereby identical communities would score 0 and totally different ones would yield the maximum distance, equal to 1. The results indicated that the main driver of divergence was the gut tract, as distances between caecal and faecal samples were higher on average, irrespective of diet composition, which scored second in rank, and of whether they had been sampled from the same individual, which was the least effective factor. Simpson's species diversity indexes was not significantly different when comparing tracts or diets, while community evenness was reduced in full-fat silkworm diet-fed animals. The identities of the differentially displayed taxa that were statistically significant as a function of gut tract and diet regimen are discussed in light of their known physiological and functional traits., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bertoldo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The molecular and phenotypic effects of a brown seaweed extract (BSE) were assessed in sugar beet ( Beta vulgaris L.). Transcript levels of BSE-treated and untreated plants were studied by RNA-seq and validated by quantitative real-time PCR analysis (RT-qPCR). Root morphology, sugar yield, and processing quality traits were also analyzed to better elucidate the treatment effects. RNA-seq revealed 1019 differentially expressed genes (DEGs) between the BSE-treated and untreated plants. An adjusted p -value < 0.1 and an absolute value of log2 (fold change) greater than one was used as criteria to select the DEGs. Gene ontology (GO) identified hormone pathways as an enriched biological process. Six DEGs involved in auxin and ABA pathways were validated using RT-qPCR. The phenotypic characterization indicated that BSE treatment led to a significant increase ( p < 0.05) in total root length and the length of fine roots of plants grown under hydroponics conditions. The sugar yield of plants grown under field conditions was higher ( p < 0.05) in the treated field plots compared with the control treatment, without impacting the processing quality. Our study unveiled the relevant effects of BSE application in regulating auxin- and ABA-related gene expression and critical traits related to sugar beet development and yield.

Material extrusion additive manufacturing enables us to combine more materials in the same nozzle during the deposition process. This technology, called material coextrusion, generates an expanded range of material properties, which can gradually change in the design domain, ensuring blending or higher bonding/interlocking among the different materials. To exploit the opportunities offered by these technologies, it is necessary to know the behavior of the combined materials according to the materials fractions. In this work, two compatible pairs of materials, namely Polylactic Acid (PLA)-Thermoplastic Polyurethane (TPU) and Acrylonitrile Styrene Acrylate (ASA)-TPU, were investigated by changing the material fractions in the coextrusion process. An original model describing the distribution of the materials is proposed. Based on this, the mechanical properties were investigated by analytical and numerical approaches. The analytical model was developed on the simplified assumption that the coextruded materials are a set of rods, whereas the more realistic numerical model is based on homogenization theory, adopting the finite element analysis of a representative volume element. To verify the deposition model, a specific experimental test was developed, and the modeled material deposition was superimposed and qualitatively compared with the actual microscope images regarding the different deposition directions and material fractions. The analytical and numerical models show similar trends, and it can be assumed that the finite element model has a more realistic behavior due to the higher accuracy of the model description. The elastic moduli obtained by the models was verified in experimental tensile tests. The tensile tests show Young's moduli of 3425 MPa for PLA, 1812 MPa for ASA, and 162 MPa for TPU. At the intermediate material fraction, the Young's modulus shows an almost linear trend between PLA and TPU and between ASA and TPU. The ultimate tensile strength values are 63.9 MPa for PLA, 35.7 MPa for ASA, and 63.5 MPa for TPU, whereas at the intermediate material fraction, they assume lower values. In this initial work, the results show a good agreement between models and experiments, providing useful tools for designers and contributing to a new branch in additive manufacturing research.

The fungus Cercospora beticola causes Cercospora Leaf Spot (CLS) of sugar beet (Beta vulgaris L.). Despite the global importance of this disease, durable resistance to CLS has still not been obtained. Therefore, the breeding of tolerant hybrids is a major goal for the sugar beet sector. Although recent studies have suggested that the leaf microbiome composition can offer useful predictors to assist plant breeders, this is an untapped resource in sugar beet breeding efforts. Using Ion GeneStudio S5 technology to sequence amplicons from seven 16S rRNA hypervariable regions, the most recurring endophytes discriminating CLS-symptomatic and symptomless sea beets (Beta vulgaris L.ssp. maritima) were identified. This allowed the design of taxon-specific primer pairs to quantify the abundance of the most representative endophytic species in large naturally occurring populations of sea beet and subsequently in sugar beet breeding genotypes under either CLS symptomless or infection stages using qPCR. Among the screened bacterial genera, Methylobacterium and Mucilaginibacter were found to be significantly (p < 0.05) more abundant in symptomatic sea beets with respect to symptomless. In cultivated sugar beet material under CLS infection, the comparison between resistant and susceptible genotypes confirmed that the susceptible genotypes hosted higher contents of the above-mentioned bacterial genera. These results suggest that the abundance of these species can be correlated with increased sensitivity to CLS disease. This evidence can further prompt novel protocols to assist plant breeding of sugar beet in the pursuit of improved pathogen resistance., (© 2022. The Author(s).)

A meta-analysis was carried out on published literature covering the topic of interactive plant microbiology for botanical species of legumes occurring within the boundary of the Italian island Sardinia, lying between the Tyrrhenian and the western Mediterranean seas. Reports were screened for the description of three types of bacterial occurrences; namely, (a) the nitrogen-fixing symbionts dwelling in root nodules; (b) other bacteria co-hosted in nodules but having the ancillary nature of endophytes; (c) other endophytes isolated from different non-nodular portions of the legume plants. For 105 plant species or subspecies, over a total of 290 valid taxonomical descriptions of bacteria belonging to either one or more of these three categories were found, yielding 85 taxa of symbionts, 142 taxa of endophytes in nodules, and 33 in other plant parts. The most frequent cases were within the Medicago , Trifolium , Lotus , Phaseolus, and Vicia genera, the majority of symbionts belonged to the Rhizobium , Mesorhizobium , Bradyrhizobium, and Sinorhizobium taxa. Both nodular and extra-nodular endophytes were highly represented by Gammaproteobacteria ( Pseudomonas , Enterobacter , Pantoea ) and Firmicutes ( Bacillus , Paenibacillus ), along with a surprisingly high diversity of the Actinobacteria genus Micromonospora . The most plant-promiscuous bacteria were Sinorhizobium meliloti as symbiont and Bacillus megaterium as endophyte. In addition to the microbial analyses we introduce a practical user-friendly software tool for plant taxonomy determination working in a Microsoft Excel spreadsheet that we have purposely elaborated for the classification of legume species of Sardinia. Its principle is based on subtractive keys that progressively filter off the plants that do not comply with the observed features, eventually leaving only the name of the specimen under examination.

Four batches of commercial compost obtained from the organic fraction of municipal solid waste were analyzed from chemical and microbiological standpoints. The working hypothesis was that, being this type of compost derived partly from plant waste, it could contain plant-growth promoting bacterial endophytes, prone to be active again upon its usual delivery as fertilizer. Culturable bacteria were isolated at different temperatures, quantified by colony morphology, identified taxonomically by 16S sequencing and screened for plant-growth promoting phenotypes including auxin and siderophore production, phosphate solubilization and peptide mineralization to ammonia. In parallel, the total community was assessed by culture independent DNA metabarcoding. The capability of plants to select, uptake and internally multiply bacteria from these compost samples was analyzed using grapevine in-vitro rooting cuttings from which acquired bacteria were reisolated, quantified and their identities determined as above. Major differences in compost bacterial composition were observed as function of the season, with the winter sample being rather distinct from the summer ones. Bacillales and Actinomycetales dominated the culturable communities while Alteromonadales, Oceanospirillales and Flavobacteriales prevailed in the total community. In spite of the challenging composting cycle conditions, the plant nature of the main input substrates appeared determinant in guaranteeing that 82% of the culturable bacteria were found endowed with one or more of the plant growth-promoting phenotypes tested. Beside its fertilization role, compost proved to be also a potential inoculant carrier for the in-soil delivery of plant beneficial microorganisms. Furthermore, upon an in vitro passage through grapevine plants under axenic conditions, the subsequently recoverable endophyte community yielded also members of the Rhizobiales order which had not been detectable when culturing directly from compost. This observation further suggests that compost-borne plant-interacting taxa could be also rescued from non-culturable states and/or enriched above detectability levels by a contact with their potential host plants., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in that the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This work aimed to study the effects in tomato ( Solanum lycopersicum L.) of foliar applications of a novel calcium-based biostimulant (SOB01) using an omics approach involving transcriptomics and physiological profiling. A calcium-chloride fertilizer (SOB02) was used as a product reference standard. Plants were grown under well-watered (WW) and water stress (WS) conditions in a growth chamber. We firstly compared the transcriptome profile of treated and untreated tomato plants using the software RStudio. Totally, 968 and 1,657 differentially expressed genes (DEGs) (adj- p -value < 0.1 and |log2(fold change)| ≥ 1) were identified after SOB01 and SOB02 leaf treatments, respectively. Expression patterns of 9 DEGs involved in nutrient metabolism and osmotic stress tolerance were validated by real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Principal component analysis (PCA) on RT-qPCR results highlighted that the gene expression profiles after SOB01 treatment in different water regimes were clustering together, suggesting that the expression pattern of the analyzed genes in well water and water stress plants was similar in the presence of SOB01 treatment. Physiological analyses demonstrated that the biostimulant application increased the photosynthetic rate and the chlorophyll content under water deficiency compared to the standard fertilizer and led to a higher yield in terms of fruit dry matter and a reduction in the number of cracked fruits. In conclusion, transcriptome and physiological profiling provided comprehensive information on the biostimulant effects highlighting that SOB01 applications improved the ability of the tomato plants to mitigate the negative effects of water stress., Competing Interests: FM is employed by Sipcam Italia S.p.A. belonging together with Sofbey SA to the Sipcam Oxon S.p.A. Group (Pero, Italy). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Della Lucia, Baghdadi, Mangione, Borella, Zegada-Lizarazu, Ravi, Deb, Broccanello, Concheri, Monti, Stevanato and Nardi.)

Rhizoctonia solani , causing Rhizoctonia crown and root rot, is a major risk to sugar beet ( Beta vulgaris L.) cultivation. The development of resistant varieties accelerated by marker-assisted selection is a priority of breeding programs. We report the identification of a single-nucleotide polymorphism (SNP) marker linked to Rhizoctonia resistance using restriction site-associated DNA (RAD) sequencing of two geographically discrete sets of plant materials with different degrees of resistance/susceptibility to enable a wider selection of superior genotypes. The variant calling pipeline utilized SAMtools for variant calling and the resulting raw SNPs from RAD sequencing (15,988 and 22,439 SNPs) were able to explain 13.40% and 25.45% of the phenotypic variation in the two sets of material from different sources of origin, respectively. An association analysis was carried out independently on both the datasets and mutually occurring significant SNPs were filtered depending on their contribution to the phenotype using principal component analysis (PCA) biplots. To provide a ready-to-use marker for the breeding community, a systematic molecular validation of significant SNPs distributed across the genome was undertaken to combine high-resolution melting, Sanger sequencing, and rhAmp SNP genotyping. We report that RsBv1 located on Chromosome 6 (9,000,093 bp) is significantly associated with Rhizoctonia resistance ( p < 0.01) and able to explain 10% of the phenotypic disease variance. The related SNP assay is thus ready for marker-assisted selection in sugar beet breeding for Rhizoctonia resistance.

The identification of efficient molecular markers related to low bolting tendency is a priority in sugar beet ( Beta vulgaris L.) breeding. This study aimed to identify SNP markers associated with low bolting tendency by establishing a genome-wide association study. An elaborate 3-year field trial comprising 13 sugar beet lines identified L14 as the one exhibiting the lowest bolting tendency along with an increased survival rate after autumnal sowing. For SNP discovery following phenotyping, contrasting phenotypes of 24 non-bolting and 15 bolting plants of the L14 line were sequenced by restriction site-associated DNA sequencing (RAD-seq). An association model was established with a set of 10,924 RAD-based single nucleotide polymorphism (SNP) markers. The allelic status of the most significantly associated SNPs ranked based on their differential allelic status between contrasting phenotypes ( p < 0.01) was confirmed on three different validation datasets comprising diverse sugar beet lines and varieties adopting a range of SNP detection technologies. This study has led to the identification of SNP&#95;36780842 and SNP&#95;48607347 linked to low bolting tendency and can be used for marker-assisted breeding and selection in sugar beet., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ravi, Campagna, Della Lucia, Broccanello, Bertoldo, Chiodi, Maretto, Moro, Eslami, Srinivasan, Squartini, Concheri and Stevanato.)

Sulfur is an essential plant macronutrient, and its adequate supply allows an efficient root storage and sugar extractability in sugar beets ( Beta &nbsp; vulgaris L.). In this study, we investigated the effect of changes in sulfur availability on the endophytic community structure of sugar beets. Plants were hydroponically grown in a complete nutrient solution (S-supplied), a nutrient solution without MgSO 4 (S-deprived), and a nutrient solution without MgSO 4 for six days and resupplied with 100 μM MgSO 4 for 48 h (S-resupplied). The sulfur status was monitored by inductively coupled plasma ICP-OES, and combustion analysis together with the evaluation of microRNA395 as a biomarker for sulfate status. Metabarcoding of the bacterial 16S rRNA gene was carried out in order to determine leaf endophytic community structure. The Shannon diversity index significantly differed ( p < 0.05) between sulfate-supplied and sulfate-deprived seedlings. Validation by Real-Time PCR showed a significant increase ( p < 0.05) of Burkholderia spp. in sulfate-deprived plants as compared to sulfate-supplied ones. The study sheds new light on the effects of nutrient deficiency on the microbiome of sugar beet plants.

The present study aimed to explore the effects of foliar application of a leonardite-based product on sugar beet ( Beta vulgaris L.) plants grown in the field. The approach concerned the evaluation of the community compositional structure of plant endophytic bacteria through a metabarcoding approach, the expression level of a gene panel related to hormonal metabolism and signaling, and the main sugar beet productivity traits. Results indicated that plants treated with leonardite (dosage of 2,000 ml ha -1 , dilution 1:125, 4 mg C l -1 ) compared with untreated ones had a significant increase ( p < 0.05) in (i) the abundance of Oxalicibacterium spp., recognized to be an endophyte bacterial genus with plant growth-promoting activity; (ii) the expression level of LAX2 gene, coding for auxin transport proteins; and (iii) sugar yield. This study represents a step forward to advance our understanding of the changes induced by leonardite-based biostimulant in sugar beet., Competing Interests: FM was employed by company Sipcam Oxon S.p.A. GCa was employed by company COPROB. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Della Lucia, Bertoldo, Broccanello, Maretto, Ravi, Marinello, Sartori, Marsilio, Baglieri, Romano, Colombo, Magro, Campagna, Concheri, Squartini and Stevanato.)

Objectives: Implantoplasty (IP) is a treatment option for peri-implantitis. Mechanical concerns were raised on fracture resistance of implants subjected to this procedure. This study aimed to compare two methods of IP in terms of implant wear and fracture resistance, and of surface topography., Material and Methods: Eighteen cylindrical screw-shaped dental implants (4 mm diameter, 13 mm length) with an external hexagonal connection were used. IP was performed on the first 6-mm implant surface with a sequence of burs or diamond sonic tips, both followed by an Arkansas finishing. IP duration and implant weight variation were recorded. Micro-computed tomography (micro-CT) was used to evaluate material loss. Implant fracture resistance was assessed by static compression test. Surface topography analysis was performed with a stylus profilometer. Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) was applied for implant surface morphology and elemental characterization., Results: Micro-CT showed less material loss in sonic compared to burs. No statistically significant difference was found between the mean fracture resistance values reached in bur and sonic, both followed by Arkansas, and with respect to control. IP performed with burs led to a smoother surface compared to sonic. Equivalent final surface roughness was found after Arkansas in both IP procedures. SEM-EDS showed a deburring effect associated to sonic and revealed carbon and aluminum peaks attributable to contamination with sonic diamond tips and Arkansas bur, respectively., Conclusions: IP with sonic diamond tips was found to be more conservative in terms of structure loss. This could have a clinical relevance in case of narrow-diameter implants., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Rhizomania is a grave disease affecting sugar beet ( Beta vulgaris L.). It is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), an RNA virus transmitted by the plasmodiophorid vector Polymyxa betae . Genetic resistance to the virus has been accomplished mostly using phenotype-genotype association studies. As yet, the most convenient method to ascertain plant resistance has been the quantification of viral titer in roots through the ELISA test. This method is particularly time-consuming and clashes with the necessities of modern plant breeding. Here, we propose an alternative and successful phenotyping method based on the automatic extraction of the viral RNA from sugar beet roots and its relative and absolute quantification by quantitative real-time PCR (qRT-PCR) and digital PCR (dPCR), respectively. Such a method enables an improved standardization of the study, as well as an accurate quantification of the virus also in those samples presenting low virus titer, with respect to the ELISA test., Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-021-00674-7., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© Indian Virological Society 2021.)

Pangenome analyses reveal major clues on evolutionary instances and critical genome core conservation. The order Rhizobiales encompasses several families with rather disparate ecological attitudes. Among them, Rhizobiaceae, Bradyrhizobiaceae, Phyllobacteriacreae and Xanthobacteriaceae, include members proficient in mutualistic symbioses with plants based on the bacterial conversion of N 2 into ammonia (nitrogen-fixation). The pangenome of 12 nitrogen-fixing plant symbionts of the Rhizobiales was analyzed yielding total 37,364 loci, with a core genome constituting 700 genes. The percentage of core genes averaged 10.2% over single genomes, and between 5% to 7% were found to be plasmid-associated. The comparison between a representative reference genome and the core genome subset, showed the core genome highly enriched in genes for macromolecule metabolism, ribosomal constituents and overall translation machinery, while membrane/periplasm-associated genes, and transport domains resulted under-represented. The analysis of protein functions revealed that between 1.7% and 4.9% of core proteins could putatively have different functions.

Microbial metabarcoding is the standard approach to assess communities' diversity. However reports are often limited to simple OTU abundances for each phylum, giving rather one-dimensional views of microbial assemblages, overlooking other accessible aspects. The first is masked by databases incompleteness; OTU picking involves clustering at 97% (near-species) sequence identity, but different OTUs regularly end up under a same taxon name. When expressing diversity as number of obtained taxonomical names, a large portion of the real diversity lying within the data remains underestimated. Using the 16S sequencing results of an environmental transect across a gradient of 17 coastal habitats we first extracted the number of OTUs hidden under the same name. Further, we observed which was the deepest rank yielded by annotation, revealing for which microbial groups are we missing most knowledge. Data were then used to infer an evolutionary aspect: what is, in each phylum the success of the present time individuals (abundances for each OTU) in relation to their prior evolutionary success in differentiation (number of OTUs). This information reveals whether the past speciation/diversification force is matched by the present competitiveness in reproduction/persistence. The final layer explored is functional diversity, i.e. abundances of groups involved in specific environmental processes., (© FEMS 2020.)

The biomechanics of the abdominal wall depends on muscular activation, tissue mechanical behavior and Intra-Abdominal Pressure (IAP). In this work, a numerical model of a human abdomen is presented, based on abdominal wall geometry from medical images. Specific constitutive formulations describe tissues mechanical behavior. Connective tissues are modelled as hyperelastic fiber-reinforced materials, while muscular tissues are described by means of a three-element Hill's model. The abdominal cavity is represented by a volume region interacting with the abdominal wall. Numerical analyses are developed by applying a muscular contraction, inducing a volume reduction of the abdominal cavity and a simultaneous IAP increase. Numerical results of abdomen displacement at IAP corresponding to an abdominal crunch are compared with experimental results acquired via 3D laser scanning on a healthy subject. Numerical and experimental results are mutually consistent and show that muscular activation induces a raising in the region adjacent to linea alba along the posterior-anterior direction and a lowering along lateral-medial direction of the abdominal wall sides. The numerical model developed in this work allows a coherent representation of the abdominal wall mechanics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Field management practices can alter the physical and chemical properties of the soil, also causing changes to the seed bank. Alterations can also occur to the soil microbial community, which in turn can increase or diminish the process of weed seed decay. In this research, the issue of seed degradation was studied in an undisturbed and a no-till soil, trying not only to uncover where seeds are more degraded, but also to investigate the microbial activities that could be involved in this process. Six different weed species, commonly found in northern Italy, were used: Abutilon theopharsti, Alopecurus myosuroides, Amaranthus retroflexus, Digitaria sanguinalis, Portulaca oleracea and Sorghum halepense . Seed decay was tested in two different sites, a no-till field and the adjacent buffer zone. Soil microbial activity was also measured using the Fertimetro, an approach based on the degradation of cotton and silk threads buried in the soil for one week. Degradation of the buried seeds was higher in the no-till field soil than in the buffer strip for all the studied species as was the microbial cellulolytic activity. Even though the buffer strip soil is an undisturbed habitat and resulted as having higher organic matter, the no-till soil conditions appeared more unfavourable to seed viability. Our findings suggest that no-till management can improve weed seed suppression in the soil. Moreover, cellulolytic microorganisms play an important role in seedbank longevity, so cellulolytic activity surveys could be used as an early monitoring bioindicator for weed seed suppression in soil., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Aims: The purpose of the study was to evaluate the accuracy of a three-dimensional (3D) automated technique (computer-aided design (aCAD)) for the measurement of three canine femoral angles: anatomical lateral distal femoral angle (aLDFA), femoral neck angle (FNA) and femoral torsion angle., Methods: Twenty-eight femurs equally divided intotwo groups (normal and abnormal) were obtained from 14 dogs of different conformations (dolicomorphic and chondrodystrophicCT scans and 3D scanner acquisitions were used to create stereolithographic (STL) files , which were run in a CAD platform. Two blinded observers separately performed the measurements using the STL obtained from CT scans (CT aCAD) and 3D scanner (3D aCAD), which was considered the gold standard method. C orrelation coefficients were used to investigate the strength of the relationship between the two measurements., Results: A ccuracy of the aCAD computation was good, being always above the threshold of R 2 of greater than 80 per cent for all three angles assessed in both groups. a LDFA and FNA were the most accurate angles (accuracy >90 per cent)., Conclusions: The proposed 3D aCAD protocol can be considered a reliable technique to assess femoral angle measurements in canine femur. The developed algorithm automatically calculates the femoral angles in 3D, thus considering the subjective intrinsic femur morphology. The main benefit relies on a fast user-independent computation, which avoids user-related measurement variability. The accuracy of 3D details may be helpful for patellar luxation and femoral bone deformity correction, as well as for the design of patient- specific, custom-made hip prosthesis implants., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Leonardite-based biostimulants are a large class of compounds, including humic acid substances. Foliar application of biostimulants at field level improves plant growth, yield and quality through metabolic changes and stimulation of plant proton pumps. The present study aimed at identifying optimum dosage of BLACKJAK, a humic acid-based substance, which is able to modify genes involved in sugar beet growth. Thirty-three genes belonging to various biochemical pathway categories were tested in leaves of treated sugar beet ( Beta vulgaris L.) samples to assess gene expression profiling in response to BLACKJAK. Seedlings of a diploid and multigerm variety were grown in plastic pots and sprayed with two dilutions of BLACKJAK (dilution 1:500-1.0 mg C L -1 and dilution 1:1000-0.5 mg C L -1 ). Leaf samples were collected after 24, 48, and 72 h treatment with BLACKJAK for each dilution. RNA was extracted and the quantification of gene expression was performed while using an OpenArray platform. Results of analysis of variance demonstrated that, 15 genes out of a total of 33 genes tested with OpenArray qPCR were significantly affected by treatment and exposure time. Analysis for annotation of gene products and pathways revealed that genes belonging to the mitochondrial respiratory pathways, nitrogen and hormone metabolisms, and nutrient uptake were up-regulated in the BLACKJAK treated samples. Among the up-regulated genes, Bv&#95;PHT2;1 and Bv&#95;GLN1 expression exerted a 2-fold change in 1:1000 and 1:500 BLACKJAK concentrations. Overall, the gene expression data in the BLACKJAK treated leaves demonstrated the induction of plant growth-related genes that were contributed almost to amino acid and nitrogen metabolism, plant defense system, and plant growth.

Background and Objective: The biomechanical analysis of the abdominal wall should take into account muscle activation and related phenomena, such as intra-abdominal pressure variation and abdomen surface deformation. The geometry of abdominal surface and its deformation during contraction have not been extensively characterized, while represent a key issue to be investigated., Methods: In this work, the antero-lateral abdominal wall surface of ten healthy volunteers in supine position is acquired via laser scanning in relaxed conditions and during abdominal muscles contraction, repeating each acquisition six times. The average relaxed and contracted abdominal surfaces are compared for each subject and displacements measured., Results: Muscular activation induces raising in the region adjacent to linea alba along the posterior-anterior direction and a simultaneous lowering along lateral-medial direction of the abdominal wall sides. Displacements reach a maximum value of 12.5 mm for the involved subjects. The coefficient of variation associated to the abdomen surface measurements in the same configuration (relaxed or contracted) is below 0.75%. Non-parametric Mann-Whitney U test highlights that the differences between relaxed and contracted abdominal wall surfaces are significant (p < 0.01)., Conclusions: Laser scanning is an accurate and reliable method to evaluate surface changes on the abdominal wall during muscular contraction. The results of this experimental activity can be useful to validate numerical models aimed at describing abdominal wall biomechanics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Humic substances extracted from leonardite are widely considered to be bioactive compounds, influencing the whole-plant physiology and the crop yield. The aim of this work was to evaluate the effect of a new formulate based on leonardite in the early stage of growth of sugar beet ( Beta vulgaris L.). A commercial preparation of leonardite (BLACKJAK) was characterized by ionomic analysis, solid-state 13 C MAS NMR spectroscopy. Seedlings of sugar beet were grown in Hoagland's solution under controlled conditions. After five days of growth, an aliquot of the concentrated BLACKJAK was added to the solution to obtain a final dilution of 1:1000 (0.5 mg C L -1 ). The sugar beet response in the early stage of growth was determined by evaluating root morphological traits as well as the changes in the expression of 53 genes related to key morphophysiological processes. Root morphological traits, such as total root length, fine root length (average diameter < 0.5 mm), and number of root tips, were significantly ( p < 0.001) increased in plants treated with BLACKJAK, compared to the untreated plants at all sampling times. At the molecular level, BLACKJAK treatment upregulated many of the evaluated genes. Moreover, both Real Time PCR and digital PCR showed that genes involved in hormonal response, such as PIN, ARF3, LOGL 10, GID1, and BRI1, were significantly ( p < 0.05) upregulated by treatment with BLACKJAK. Our study provides essential information to understand the effect of a leonardite-based formulate on plant growth hormone metabolism, although the molecular and physiological basis for these complicated regulatory mechanisms deserve further investigations., Competing Interests: The authors declare no conflict of interest.

The worldwide growing consumption of proteins to feed humans and animals has drawn a considerable amount of attention to insect rearing. Insects reared on organic wastes and used as feed for monogastric animals can reduce the environmental impact and increase the sustainability of meat/fish production. In this study, we designed an environmentally closed loop for food supply in which fruit and vegetable waste from markets became rearing substrate for Hermetia illucens (BSF- black soldier fly). A vegetable and fruit-based substrate was compared to a standard diet for Diptera in terms of larval growth, waste reduction index, and overall substrate degradation. Morphological analysis of insect organs was carried out to obtain indications about insect health. Processing steps such as drying and oil extraction from BSF were investigated. Nutritional and microbiological analyses confirmed the good quality of insects and meal. The meal was then used to produce fish feed and its suitability to this purpose was assessed using trout. Earthworms were grown on leftovers of BSF rearing in comparison to a standard substrate. Chemical analyses of vermicompost were performed. The present research demonstrates that insects can be used to reduce organic waste, increasing at the same time the sustainability of aquaculture and creating interesting by-products through the linked bio-system establishment., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

This study investigated a novel bioreactor configuration coupled with a side-stream dynamic membrane (DM) for Anammox enrichment as an alternative for conventional membrane. Bioreactor was fed with synthetic feed and seeded with a mix of anaerobic and aerobic sludge. In situ mechanical cleaning was employed for DM cleaning. DM development and performance was analysed over two polyamide-nylon meshes (200 and 52 µm). Solid-liquid separation of 52 µm mesh outperformed 200 µm with an average effluent turbidity of 2.4 ± 0.1 NTU. The system was operated at a maximum nitrogen loading rate of 696 mg-N L -1  d -1 and achieved a maximum nitrogen removal rate of 611.6 mg-N L -1  d -1 . At steady state, the average ammonium, nitrite and total nitrogen removal efficiencies were 87 ± 0.6%, 98.5 ± 0.15% and 87.5 ± 0.56% respectively. Digital realtime PCRSequence analysis showed that Planctomycetales belonging to ascertained Anammox-specific genera progressively increased their presence in the reactor consistently with its nitrogen removal performance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Advances in additive manufacturing technologies facilitate the fabrication of cellular materials that have tailored functional characteristics. The application of solid freeform fabrication techniques is especially exploited in designing scaffolds for tissue engineering. In this review, firstly, a classification of cellular materials from a geometric point of view is proposed; then, the main approaches on geometric modeling of cellular materials are discussed. Finally, an investigation on porous scaffolds fabricated by additive manufacturing technologies is pointed out. Perspectives in geometric modeling of scaffolds for tissue engineering are also proposed.

In this study, a system based on omics profiling was set-up for sugar beet ( Beta vulgaris L. subsp. vulgaris ) evaluation after changes in sulfate availability. Seedlings were grown on sulfate-deprived Hoagland solution. Six days after germination, 100 μM MgSO 4 was added to the solution. Root samples were collected 36 h after treatments. WinRHIZO root-scanning approach was used for the automated image analysis of plant root morphology. Inductively Coupled Plasma Spectrometry (ICP-OES) and quadrupole-time-of-flight mass spectrometry (Q-TOF) were used for ionomic and metabolic analysis, respectively. Nanofluidic real-time PCR (OpenArray system) was used for molecular profiling. OpenArray chips were designed with TaqMan probes for 53 sugar beet genes putatively involved in sulfate nutrition. At morphological level treated seedlings showed significantly higher values ( P < 0.01) than untreated plants for root traits related to soil exploration and nutrient uptake, such as total root length, fine roots length and root tips number. ICP-OES, Q-TOF and transcriptomic data revealed changes due to sulfate availability in sugar beet samples. Two key results are highlighted in sulfate-supplied roots and leaves. Firstly, high expression levels of auxin efflux carrier component 1 (PIN) and 5-phosphoribosyl-anthranilate, precursor of tryptophan and auxin synthesis, were observed in roots. Secondly, high levels of 2-Cys peroxiredoxin BAS1, chloroplastic, thioredoxin reductase (NADPH) and cysteine synthase, chloroplastic/chromoplastic, O- acetylserine sulfhydrylase, involved in protection against oxidative stress and cysteine synthase activity, respectively, were observed in leaves. Based on our findings, the combination of evaluated omics approaches could become a key system for the evaluation of the nutritional status of sugar beet under different nutrient availability conditions.

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet ( Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets ( Beta vulgaris L. subsp. maritima ) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8&#95;184910&#95;pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet., Competing Interests: The authors declare no conflict of interest.

The unique environment of a 4m-thick, free-floating peat island within the Posta Fibreno lake (Central Italy) was analyzed using DNA-based techniques to assess bacterial and fungal community members identity and abundance. Two depths were sampled at 41 and 279 cm from the surface, the former corresponding to an emerged portion of Sphagnum residues accumulated less than 30 yrs ago, and the latter mainly consisting of silty peat belonging to the deeply submerged part of the island, dating back to 1520-1660 AD. The corresponding communities were very diverse, each of them dominated by a different member of the Delta-proteobacteria class for prokaryotes. Among Eukaryotes, Ascomycota prevailed in the shallow layer while Basidiomycota were abundant in the deep sample. The identity of taxa partitioning between acidic surface layer and neutral core is very reminiscent of the differences reported between bogs and fens respectively, supporting the view of Posta Fibreno as a relic transitional floating mire. Moreover, some microbial taxa show an unusual concurrent species convergence between this sub-Mediterranean site and far Nordic or circumpolar environments. This study represents the first report describing the biotic assemblages of such a peculiar environment, and provides some insights into the possible mechanisms of its evolution.

Objective: To define and validate a method for the measurement of 3-dimensional (3D) morphometric parameters in polygonal mesh models of canine femora., Study Design: Ex vivo/computerized model., Sample Population: Sixteen femora from 8 medium to large-breed canine cadavers (mean body weight 28.3 kg, mean age 5.3 years)., Methods: Femora were measured with a 3D scanner, obtaining 3D meshes. A computer-aided design-based (CAD) software tool was purposely developed, which allowed automatic calculation of morphometric parameters on a mesh model. Anatomic and mechanical lateral proximal femoral angles (aLPFA and mLPFA), anatomic and mechanical lateral distal femoral angles (aLDFA and mLDFA), femoral neck angle (FNA), femoral torsion angle (FTA), and femoral varus angle (FVA) were measured in 3D space. Angles were also measured onto projected planes and radiographic images., Results: Mean (SD) femoral angles (degrees) measured in 3D space were: aLPFA 115.2 (3.9), mLPFA 105.5 (4.2), aLDFA 88.6 (4.5), mLDFA 93.4 (3.9), FNA 129.6 (4.3), FTA 45 (4.5), and FVA -1.4 (4.5). Onto projection planes, aLPFA was 103.7 (5.9), mLPFA 98.4 (5.3), aLDFA 88.3 (5.5), mLDFA 93.6 (4.2), FNA 132.1 (3.5), FTA 19.1 (5.7), and FVA -1.7 (5.5). With radiographic imaging, aLPFA was 109.6 (5.9), mLPFA 105.3 (5.2), aLDFA 92.6 (3.8), mLDFA 96.9 (2.9), FNA 120.2 (8.0), FTA 30.2 (5.7), and FVA 2.6 (3.8)., Conclusion: The proposed method gives reliable and consistent information about 3D bone conformation. Results are obtained automatically and depend only on femur morphology, avoiding any operator-related bias. Angles in 3D space are different from those measured with standard radiographic methods, mainly due to the different definition of femoral axes., (© Copyright 2016 by The American College of Veterinary Surgeons.)

The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies.

Tree colonization and feeding activity of the invasive wood-borer Asian longhorn beetle (Anoplophora glabripennis), an Asian pest introduced into North America and Europe, was studied in a newly invaded area in Italy. The hypothesis being tested was that the reproductive success of the insect depend on habitat type and tree suitability. Adult beetles were caged on branches of host and nonhost species, in both urban and forest habitats. Two months later, number and size of feeding patches on plant tissues, eggs laid, and surviving larvae were assessed. Bark concentration of C and N was also measured from the same trees. Results indicated that the mean area of plant tissues consumed by adult feeding was significantly larger on trees growing in forest than in urban habitat, although within the same habitat there were no differences between susceptible and nonsusceptible trees. ALB tree colonization, in terms of number of eggs laid and young larvae survival, was not affected by habitat while it was higher on susceptible trees. Although trees growing in forests had a lower nitrogen concentration, they allowed colonization rates similar to those of trees growing in the urban habitat. Hence, the amount of carbon and nitrogen did not fully explain tree suitability or habitat selection. We suggest compensatory feeding as a potential mechanism that might explain this peculiar situation, as supported by a more intensive feeding activity recorded on trees in the forest. Suitability of different trees may be due to other factors, such as secondary chemical compounds., (© 2014 Institute of Zoology, Chinese Academy of Sciences.)

A new Italian earthworm morphologically close to the similarly large and anecic Eophila tellinii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750 km2 from East to West on the Asiago Plateau and Vittorio Veneto Hills, from North to South on mounts Belluno Prealps (Praderadego and Cesen), Asiago, Grappa and onto the Montello foothills. This range abuts that of Eophila tellinii in northern Friuli Venezia Giulia region. Known localities of both E. tellinii and E. crodabepis sp. nov. are mapped. mtDNA barcoding definitively separates the new western species from classical Eophila tellinii (Rosa, 1888).

Background: Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches., Results: Abundant microbial cell masses were observed on the external appendages. Cansiliella's midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals' digestive traits., Conclusions: Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.

The fermented manure derivative known as Preparation 500 is traditionally used as a field spray in biodynamic agriculture for maintaining and increasing soil fertility. This work aimed at characterizing the product from a microbiological standpoint and at assaying its bioactive properties. The approach involved molecular taxonomical characterization of the culturable microbial community; ARISA fingerprints of the total bacteria and fungal communities; chemical elemental macronutrient analysis via a combustion analyzer; activity assays for six key enzymes; bioassays for bacterial quorum sensing and chitolipooligosaccharide production; and plant hormonelike activity. The material was found to harbor a bacterial community of 2.38 × 10(8) CFU/g dw dominated by Grampositives with minor instances of Actinobacteria and Gammaproteobacteria. ARISA showed a coherence of bacterial assemblages in different preparation lots of the same year in spite of geographic origin. Enzymatic activities showed elevated values of beta-glucosidase, alkaline phosphatase, chitinase, and esterase. The preparation had no quorum sensing-detectable signal, and no rhizobial nod gene-inducing properties, but displayed a strong auxin-like effect on plants. Enzymatic analyses indicated a bioactive potential in the fertility and nutrient cycling contexts. The IAA activity and microbial degradation products qualify for a possible activity as soil biostimulants. Quantitative details and possible modes of action are discussed.

The present paper describes the design features, potential indications and a clinical application of a newly designed device for jaw motion rehabilitation, the Spring-Bite. This device is characterized by a first class lever mechanism, which allows performing passive jaw motion rehabilitation at constant load without an active participation by the patient. Spring-Bite was developed for the management of temporomandibular joint (TMJ) hypomobility and its application may be much useful in the post-operatory phases of TMJ or orthognathic surgeries as well as in patients with reduced muscular force.

This paper describes the accumulation pattern of 42 mineral elements in Vitis vinifera L. berries during development and ripening and their distribution in berry skin, seeds, and flesh around harvest time. Grape berries were sampled in two different vineyards with alkaline soil and analyzed using a ICP-MS. Although elemental amounts were significantly different in the grapes from the two vineyards, the accumulation pattern and percentage distribution in different parts of the berries were generally quite similar. Ba, Eu, Sr, Ca, Mg, Mn, and Zn accumulate prior to veraison. Al, Ce, Dy, Er, Ga, Gd, Ho, La, Nd, Pr, Sm, Sn, Zr, Th, Tm, U, Y, and Yb accumulate mainly prior to veraison but also during ripening. Ag, As, B, Cd, Cs, Cu, Fe, Ge, Hg, K, Li, Na, P, Rb, Sb, Se, and Tl accumulate progressively during growth and ripening. With regard to distribution, Ba, Ca, Eu, Fe, Mn, P, Sr, and Zn accumulate mainly in the seeds, Al, B, Ga, Sn, and the rare earths analyzed, except for Eu, accumulate mainly in the skin, and Ag, As, Cd, Cs, Cu, Ge, Hg, K, Li, Mg, Na, Rb, Sb, Se, Th, Tl, U, and Zr accumulate mainly in the flesh. A joint representation of the accumulation and distribution patterns for the elements in the berry is also given.

Background: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial., Methodology/principal Findings: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA)., Conclusions: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

Background: Current methodologies in the prevision of post-surgical features of the face in orthognathic surgery are mainly 2-D. An improvement is certainly given by the introduction of CT, but its acceptance is controversial due to its high biological cost. As an alternative, in this study an effective procedure for the construction of a 3-D textured digital model of the face and dental arches of patients with dentofacial malformations using a 3-D laser scanner at no biological cost is presented., Methods: A 3-D Laser scanner Konica-Minolta VIVID 910 is used to obtain multiple scans from different perspectives of the face of patients with dentofacial malocclusions requiring orthognathic surgery. These multiple views are then recombined, integrating also the maxillary and mandibular arch plaster casts, to obtain the 3-D textured model of the face and occlusion with minimal error., Results: A viable methodology was identified for the face and occlusal modeling of orthognathic patients and validated in a test case, confirming its effectiveness: the 3-D model created accurately describes the actual features of the patient's face; the proposed methodology can be easily applied in the clinical routine to accurately record the steps of the surgical treatment and to perform accurate anthropometric analyses of the facial morphology, and thus constitute the necessary database for the development of previsional tools in orthognathic surgery., Conclusions: The proposed method is effective in recording all the morphological facial features of patients with dentofacial malformations, to develop a facial modification database and tools for virtual surgery.

This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ss-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement ("self-impact"), and how much the two rhizobia would additively contribute to nodulation.

To gain further insight into the macromolecular behaviour of humic substances we treated a humic material with simple organic compounds and followed the change in molecular size distribution. Monocarboxylic, dicarboxylic, and tricarboxylic acids shifted the humic absorbance from high to low molecular sizes in size exclusion chromatograms. Mineral acids, phenol, alcohols, were not able to produce the same shift and gave total absorbance at the column void volume as in the case of humic substances alone. Our results are evidence of the micellar behaviour of humic substances in solution and of the importance of hydrophobic bondings in holding humic molecules together. The organic acids enter in the interior of the humic micelle and alter its stereochemical hydrophobic arrangement. In alkaline conditions, the developed negative charges disrupt the apparent high molecular size configuration and disperse the humic material into small-size micelles. This macromolecular property, that we report for the first time, may be of great importance in understanding the biological activity and the overall environmental behaviour of humic substances.