Plasticity, exudation and microbiome-association of the root system of Pellitory-of-the-wall plants grown in environments impaired in iron availability.

The investigation of the adaptive strategies of wild plant species to extreme environments is a challenging issue, which favors the identification of new traits for plant resilience. We investigated different traits which characterize the root-soil interaction of Parietaria judaica, a wild plant species commonly known as "Pellitory-of-the-wall". P. judaica adopts the acidification-reduction strategy (Strategy I) for iron (Fe) acquisition from soil, and it can complete its life cycle in highly calcareous environments without any symptoms of chlorosis. In a field-to-lab approach, the microbiome associated with P. judaica roots was analyzed in spontaneous plants harvested from an urban environment consisting in an extremely calcareous habitat. Also, the phenolics and carboxylates content and root plasticity and exudation were analyzed in P. judaica plants grown under three different controlled conditions mimicking the effect of calcareous environments on Fe availability: results show that P. judaica differentially modulates root plasticity under different Fe availability-impaired conditions, and that it induces, to a high extent, the exudation of caffeoylquinic acid derivatives under calcareous conditions, positively impacting Fe solubility.

Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed.

Carbon Fluxes between Primary Metabolism and Phenolic Pathway in Plant Tissues under Stress.

Higher plants synthesize an amazing diversity of phenolic secondary metabolites. Phenolics are defined secondary metabolites or natural products because, originally, they were considered not essential for plant growth and development. Plant phenolics, like other natural compounds, provide the plant with specific adaptations to changing environmental conditions and, therefore, they are essential for plant defense mechanisms. Plant defensive traits are costly for plants due to the energy drain from growth toward defensive metabolite production. Being limited with environmental resources, plants have to decide how allocate these resources to various competing functions. This decision brings about trade-offs, i.e., promoting some functions by neglecting others as an inverse relationship. Many studies have been carried out in order to link an evaluation of plant performance (in terms of growth rate) with levels of defense-related metabolites. Available results suggest that environmental stresses and stress-induced phenolics could be linked by a transduction pathway that involves: (i) the proline redox cycle; (ii) the stimulated oxidative pentose phosphate pathway; and, in turn, (iii) the reduced growth of plant tissues.

Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology.

Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era, originating from charophycean green algae. The successful adaptation to land of these prototypes of amphibious plants - when they emerged from an aquatic environment onto the land - was achieved largely by massive formation of "phenolic UV light screens". In the course of evolution, plants have developed the ability to produce an enormous number of phenolic secondary metabolites, which are not required in the primary processes of growth and development but are of vital importance for their interaction with the environment, for their reproductive strategy and for their defense mechanisms. From a biosynthetic point of view, beside methylation catalyzed by O-methyltransferases, acylation and glycosylation of secondary metabolites, including phenylpropanoids and various derived phenolic compounds, are fundamental chemical modifications. Such modified metabolites have altered polarity, volatility, chemical stability in cells but also in solution, ability for interaction with other compounds (co-pigmentation) and biological activity. The control of the production of plant phenolics involves a matrix of potentially overlapping regulatory signals. These include developmental signals, such as during lignification of new growth or the production of anthocyanins during fruit and flower development, and environmental signals for protection against abiotic and biotic stresses. For some of the key compounds, such as the flavonoids, there is now an excellent understanding of the nature of those signals and how the signal transduction pathway connects through to the activation of the phenolic biosynthetic genes. Within the plant environment, different microorganisms can coexist that can establish various interactions with the host plant and that are often the basis for the synthesis of specific phenolic metabolites in response to these interactions. In the rhizosphere, increasing evidence suggests that root specific chemicals (exudates) might initiate and manipulate biological and physical interactions between roots and soil organisms. These interactions include signal traffic between roots of competing plants, roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil properties to the roots. Plant phenolics can also modulate essential physiological processes such as transcriptional regulation and signal transduction. Some interesting effects of plant phenolics are also the ones associated with the growth hormone auxin. An additional role for flavonoids in functional pollen development has been observed. Finally, anthocyanins represent a class of flavonoids that provide the orange, red and blue/purple colors to many plant tissues. According to the coevolution theory, red is a signal of the status of the tree to insects that migrate to (or move among) the trees in autumn.

Verbascoside, isoverbascoside, and their derivatives recovered from olive mill wastewater as possible food antioxidants.

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, ß-hydroxyverbascoside, ß-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.

Verbascosides from olive mill waste water: assessment of their bioaccessibility and intestinal uptake using an in vitro digestion/Caco-2 model system.

Olive mill wastewater (OMWW) is an agricultural waste material produced in high quantities in the Mediterranean basin. OMWW may be an inexpensive source of health promoting phytochemicals with potential economic value including many low molecular weight compounds such as verbascosides. While promising as antioxidants in vitro, little information is available on the potential absorption of verbascosides by humans. The main objective of the present study was to characterize the verbascoside content and potential for their bioavailability from a partially purified phenolic fraction (IP) of OMWW. The IP was obtained after ultrafiltration step at 5000 Dalton and gel filtration low-pressure chromatography (LH20) of OMWW. RP-HPLC analysis identified several soluble phenolics compounds including verbascoside and isoverbascoside as major components of OMWW fractions. The potential for bioavailability of these polyphenols was estimated by using both in vitro digestion and Caco-2 human intestinal cell models. In vitro digestive recoveries (bioaccessibility) were found to be 35.5%± 0.55% for verbascoside and 9.2% ± 0.94% for isoverbascoside highlighting potential sensitivity of these phenolics to gastric and small intestinal digestive conditions. Accumulation of verbascosides by highly differentiated Caco-2 monolayers was linear between 10 and 100 µM of verbascoside and isoverbascoside from IP extract. Uptake of verbascoside and isoverbascoside was rapid with peak accumulation occurring after 30 min with total accumulation efficiency of 0.1% and 0.2% providing intracellular levels of 130 and 80 pmol/mg cell protein for verbascoside and isoverbascoside, respectively. Combined, these data suggest that verbascosides present in OMWW are bioaccessible and provides a rationale for subsequent in vivo studies on the bioavailability and bioactivity of OMWW components.

Purification, biochemical characterization and cloning of a new cationic peroxidase isoenzyme from artichoke.

A cationic soluble peroxidase isoenzyme (CysPrx) has been purified and characterized from artichoke (Cynara cardunculus subsp. scolymus (L.) Hegi) leaves by combination of aqueous two phase extraction, ion exchange chromatography, and gel filtration. The purification fold was 149 and the activity recovery 5.5%. CysPrx was stable from 5 to 45 °C with a pH optimum around 5.5; the pI was 8.3 and the MW of 37.7 ± 1.5 kDa. MALDI-TOF MS analysis provided partial peptide sequences and resolved CysPrx isoenzyme into two putative isoforms. The presence of these isoforms was confirmed by the isolation of full-length cDNA encoding CysPrx that generate two slightly different sequences coding for two putative CysPrx: CysPrx1 and CysPrx2. The obtained MS peptides showed a 35% coverage with 100% identity with the two CysPrx deduced protein sequences. A molecular modeling analysis was carried out to predict in silico the protein structure and compare it with other plant Prx structures. Considering that CysPrx is quite stable, the study carried out in this paper will offer new insights for the production of the recombinant protein for utilization of CysPrx as an alternative Prx for food technology, biomedical analysis and bioremediation.

Biological activity of high molecular weight phenolics from olive mill wastewater.

Olive oil production generates large amounts of recalcitrant compounds, the olive oil mill wastewater (OMWW), which represent one of the most contaminating effluents among those produced by the agrofood industries. Nowadays, this view has changed to one that recognizes the waste as a low-cost starting material rich in bioactive compounds, particularly biophenols, that can be extracted and applied as natural antioxidants for the food and pharmaceutical industries. The data reported in this paper indicate that the OMWW extracts, besides low molecular weight antioxidant phenolics such as tyrosol and hydroxytyrosol, also contain phenolics with a molecular weight in the range of 600-5000 Da, which exhibit efficient scavenging activities against hydroxyl and peroxyl radicals. This group of phenolics includes, besides verbascoside, isoverbascoside, and an oxidized form of verbascoside, a number of higher molecular weight phenolics arising from oxidative polymerization of hydroxytyrosol and caffeic acid. Overall, these higher molecular weight phenolics prove to be, in some in vitro tests, more efficient scavengers of hydrophilic hydroxyl radicals than hydroxytyrosol, which could be used for industrial applications as natural nontoxic antioxidants.

Sentinel lymph node biopsy in multiple breast cancer using subareolar injection of the tracer.

We performed subdermal injection of (99m)Tc-labelled albumin combined with subareolar (SA) injection of blue dye to axillary lymphatic mapping and sentinel lymph node biopsy (SLNB) in patients with multifocal and multicentric breast cancer to evaluate the feasibility and accuracy of this technique. A retrospective analysis of our experience on 235 SLNB showed that 30(12.7%) had multiple cancer (MC) on final pathologic examination and was considered in relation to the aim of the study. Mean age was 57.19 years (range 24-90). Mean number of SLNs identified was 1.93 (range 1-5). Mean number of axillary LNs examined was 18.10 (range 12-27). Overall successful identification was 100% with a false negative (FN) rate of 6.25%. Overall accuracy of lymphatic mapping and sensitivity was 96.6% and 93.7%, respectively. SLNB using the SA injection technique may be an alternative to complete axillary dissection in patients with multiple breast cancers and a clinically negative axilla.

Contrast-enhanced breast MRI in patients with suspicious microcalcifications on mammography: results of a multicenter trial.

OBJECTIVE: The objective of our study was to test dynamic MRI in evaluating mammographically detected suspicious microcalcifications. MATERIALS AND METHODS: One hundred twelve patients with mammographically detected microcalcifications with BI-RADS category 5 (n = 78) or 4 (n = 34) lesions were studied at 17 centers a using 3D gradient-echo dynamic coronal technique (< or = 3 mm thickness) and 0.1 mmol/kg of gadoteridol. A pathologic sample was obtained in all cases. Agreement between the major diameter measured on mammography, MRI, or both and the major diameter measured at pathologic examination was calculated in 62 cases. RESULTS: Of the 112 lesions, pathologic examination revealed 37 benign lesions, 33 ductal carcinoma in situ (DCIS), and 42 invasive carcinomas. The specificity of MRI for benign lesions was 68%. Considering the subgroups of calcifications alone and calcifications associated with masses, the specificity values became 79% and 33%, respectively. The sensitivity of MRI for DCIS was 79%. Analysis of the two subgroups showed sensitivity values of 68% for calcifications alone and of 1% for calcifications associated with masses. The sensitivity for invasive carcinomas was 93%. Analysis of the two subgroups showed sensitivity values to be 92% for calcifications alone and 94% for calcifications associated with masses. Considering the overall results, the sensitivity of MRI was 87%; specificity, 68%; positive predictive value, 84%; negative predictive value, 71%; and accuracy, 80%. Considering the subgroups of calcifications alone and calcifications associated with masses, the sensitivity values became 80% and 97%; the positive predictive values, 86% and 82%; the negative predictive values, 71% and 75% (95% confidence interval [CI], 0.19-0.99); and the accuracy values, 80% and 82% (95% CI, 0.66-0.92), respectively. An odds ratio (OR) of 13.54 (95% CI, 5.20-35.28) showed a raised risk of malignant breast tumor in subjects with positive MR examination of mammographically detected suspicious clusters of microcalcifications. The statistical analysis on each subgroup showed an OR of 15.07 (95% CI, 4.73-48.08) for calcifications alone and an OR of 14.00 (95% CI, 1.23-158.84) for calcifications associated with masses. Any significant improvement in the predictive ability of dynamic MRI depending on the extent of calcifications on mammography was not proved. Considering the 62 cases of proved malignancy with measured maximal diameter at pathologic examination, both mammography and MR examination seem to overestimate tumor extent. CONCLUSION: The not-perfect sensitivity of MRI (87%), when applying our interpretation criteria and imaging sequences, is a crucial point that prevents us from clinical use of MRI in the diagnosis of mammographically detected microcalcifications.

Sensitivity of MRI versus mammography for detecting foci of multifocal, multicentric breast cancer in Fatty and dense breasts using the whole-breast pathologic examination as a gold standard.

OBJECTIVE: Our aim was to compare the effectiveness of mammography and MRI in the detection of multifocal, multicentric breast cancer. SUBJECTS AND METHODS: Ninety patients with planned mastectomies (nine bilateral) underwent mammography and dynamic gadolinium-enhanced MRI. Off-site reviewers aware of the entry criterion (planned mastectomy) evaluated both examinations for the presence of malignant foci, recording the density pattern on mammography. The gold standard was pathologic examination of the whole excised breast (slice thickness, 5 mm). RESULTS: Of 99 breasts, pathologic findings revealed 52 unifocal, 29 multifocal, and 18 multicentric cancers for a total of 188 malignant foci (158 invasive and 30 in situ). Overall sensitivity was 66% (124/188) for mammography and 81% (152/188) for MRI (p < 0.001); 72% (113/158) and 89% (140/158) for invasive foci (p < 0.001); and 37% (11/30) and 40% (12/30) for in situ foci (p > 0.05, not significant), respectively. Mammography and MRI missed 64 and 36 malignant foci, respectively, with median diameters of 8 and 5 mm (p = 0.033) and an invasive-noninvasive ratio of 2.4:1 (45:19) and 1.0:1 (18:18) (p = 0.043), respectively. The overall positive predictive value (PPV) was 76% (124/164) for mammography and 68% (152/222) for MRI (not significant). In breasts with an almost entirely fatty pattern, sensitivity was 75% for mammography and 80% for MRI (not significant), and the PPV was 73% and 65% (not significant), respectively. In breasts with fibroglandular or dense pattern, the sensitivity was 60% and 81% (p < 0.001), and the PPV was 78% and 71% (not significant), respectively. CONCLUSION: MRI was more sensitive than mammography for the detection of multiple malignant foci in fibroglandular or dense breasts. Mammography missed larger and more invasive cancer foci than MRI. A relatively low PPV was a problem for both techniques.