Sandra Citterio

Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining.

ABSTRACT The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria

BACKGROUND Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. METHODS We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. RESULTS AND CONCLUSIONS With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Heavy metal tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L.

Experiments in semi-natural conditions were undertaken to assess hemp metal tolerance and its ability to accumulate cadmium, nickel and chromium. Cannabis sativa was grown in two soils, S1 and S2, containing 27, 74, 126 and 82, 115, 139 μg g−1 of Cd, Ni and Cr, respectively. After two months from germination and at ripeness, no significant alteration in plant growth or morphology was detected. On the contrary, a high hemp reactivity to heavy metal stress with an increase in phytochelatin and DNA content was observed during development, suggesting the Cannabis sativa ability to avoid cell damage by activating different molecular mechanisms. Metals were preferentially accumulated in the roots and only partially translocated to the above-ground tissues. The mean shoot Cd content was 14 and 66 μg g−1 for S1 and S2 soil, respectively. Although not negligible concentrations they were about 100 times lower than those calculated for the hyperaccumulator Thlaspi caerulescens. Similarly Ni uptake was limited if compared with that of the Ni-hyperaccumulator Alyssum murale. Chromium uptake was negligible. As expected on the base of the metal concentration detected in ripe plants, no statistically significant variation in soil metal content was detected after one crop of hemp. Nevertheless, a consistent amount (g) of Cd and Ni is expected to be extracted by 1 ha biomass of hemp (about 10 t) per year and along the time a slow restoration of deeper soil portions can be obtained by its wide root system (at least 0,5 m deep). In addition, the possibilities of growing hemp easily in different climates and using its biomass in non-food industries can make heavy metal contaminated soils productive. This means economical advantage along with a better quality of soil.

Ragweed pollen collected along high‐traffic roads shows a higher allergenicity than pollen sampled in vegetated areas

Pollutants may affect pollen allergenicity and thus the prevalence of allergies. Although a few studies are available in literature, the connection between pollution and the allergenic potential of pollen has yet to be clearly defined. The objective of this study was to evaluate the effect of traffic‐related pollution on the allergenicity of ragweed (Ambrosia artemisiifolia L.) pollen through a field‐based experiment.

Localization of stilbene synthase in Vitis vinifera L. during berry development

Summary.The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.

A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ∼20 N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10 N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.

Genetic relationships in Opuntia Mill. genus (Cactaceae) detected by molecular marker

The Opuntia genus includes over 181 species comprising, on the basis of morphological traits, a total of 29 series [The Cactaceae (1919)]. Starting from this classification, several authors have investigated the Opuntia genus taxonomy but the large morphological variation within different species, suggests that phenotypical characteristics will not serve to produce a stable classification. In this work chloroplastic simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP) were used to evaluate the usefulness of molecular markers in Opuntia species characterization and to study the relationships among different species. Results show that the combination of cpSSR and AFLP markers provide a quantitative estimation of genetic relationships among several Opuntia species. Both molecular analyses reveal a genetic similarity among species of series 20 and 21 [The Cactaceae (1919)] as suggested also by morphological traits. Particular attention was focused on the genetic relationship between Opuntia ficus-indica and Opuntia megacantha: individuals from different populations of the two species were analyzed with both molecular markers. A common genetic constitution of O. ficus-indica and O. megacantha was detected. On the basis of molecular data, morphological traits and biogeographical distribution, we suggest that O. ficus-indica should be considered as a domesticated form of O. megacantha. Our results suggest the importance of a revision of Opuntia genus classification using several tools: molecular, morphological and biogeographical analysis.

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida.

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the proteins subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.

The rotation of white lupin (Lupinus albus L.) with metal-accumulating plant crops: A strategy to increase the benefits of soil phytoremediation

Most of the plants employed to remove metals from contaminated soils are annuals and have a seed-to-seed life cycle of a few months, usually over spring and summer. Consequently, for most of the year, fields are not actively cleaned but are completely bare and subject to erosion by water and wind. The objective of this study was to evaluate the benefits of using Lupinus albus as a winter crop in a rotation sequence with a summer crop ideally selected for phytoextraction, such as industrial hemp. Lupin plants were grown in two alkaline soil plots (heavy metal-contaminated and uncontaminated) of approximately 400 m(2) each after the cultivation and harvest of industrial hemp. A smaller-scale parallel pot experiment was also performed to better understand the lupin behavior in increasing concentrations of Cd, Cu, Ni and Zn. White lupin grew well in alkaline conditions, covering the soil during the winter season. In few months plants were approximately 40-50 cm high in both control and contaminated plots. In fields where the bioavailable fraction of metals was low (less than 12%), plants showed a high tolerance to these contaminants. However, their growth was affected in some pot treatments in which the concentrations of assimilable Cu, Zn and Ni were higher, ranging from approximately 40-70% of the total concentrations. The lupins ability to absorb heavy metals and translocate them to shoots was negligible with respect to the magnitude of contamination, suggesting that this plant is not suitable for extending the period of phytoextraction. However, it is entirely exploitable as green manure, avoiding the application of chemical amendments during phytoremediation. In addition, in polluted fields, white lupin cultivation increased the soil concentration of live bacteria and the bioavailable percentage of metals. On average live bacteria counts per gram of soil were 65×10(6)±18×10(6) and 99×10(6)±22*10(6) before and after cultivation, respectively. The percentages of bioavailable Cu, Pb, Ni, Zn and Cr, which were 5.7±0.7, 5.3±1.7, 1.2±0.1, 12±1.5 and 0.1±0.02%, respectively, before lupin growth, increased to 9.6±1.6, 7±2, 2±0.3, 14±1.5 and 0.1±0.02% after lupin harvest. On the whole, our results indicate that the winter cultivation of white lupin in sequence with a metal-accumulator summer crop can improve the recovery of soil quality during the phytoextraction period. It improves the safety of the area, limiting additional ecological and human health problems, and enhances soil health by avoiding the use of chemical amendments and by increasing the levels of viable microorganisms.

Concomitant sensitization to ragweed and mugwort pollen: who is who in clinical allergy?

BACKGROUND In many areas of Europe, double sensitization to ragweed and mugwort is common, and because of the overlapping flowering periods of the 2 plants, it is not possible to diagnose the primary sensitizing allergen source and hence to determine the proper immunotherapy. OBJECTIVES To elucidate whether double-sensitized patients are cosensitized or cross-sensitized and, in the latter case, to define the primary sensitizer. METHODS Serum samples from 34 patients with late summer respiratory allergy underwent skin prick testing with whole ragweed, and mugwort extracts were analyzed for their reactivity to recombinant Art v 1 and Amb a 1 by ImmunoCAP and then to Amb a 1, Art v 6, and Art v 1 isoforms by a proteomic approach. In double reactors, the primary sensitizing sources were detected by inhibition experiments. RESULTS Serum samples from patients monosensitized to ragweed contained IgE to epitopes specific of all Amb a 1 isoforms. In contrast, serum samples from double reactors found to be primarily sensitized to mugwort reacted to Art v 1 and Art v 6 and cross-reacted to a few Amb a 1 isoforms. Finally, serum samples from double reactors found to be primarily sensitized to ragweed contained IgE reacting to all Amb a 1 isoforms, part of which cross-reacted to Art v 6. We did not find cosensitized patients. CONCLUSION This study found that Art v 6 plays an important role in mugwort allergy and that the cross-reactivity between Art v 6 and Amb a 1 is frequent, bidirectional, and clinically relevant in the area of Milan.