NMR Metabolite Profiling for the Characterization of Vessalico Garlic Ecotype and Bioactivity against Xanthomonas campestris pv. campestris.

The Italian garlic ecotype "Vessalico" possesses distinct characteristics compared to its French parent cultivars Messidor and Messidrôme, used for sowing, as well as other ecotypes in neighboring regions. However, due to the lack of a standardized seed supply method and cultivation protocol among farmers in the Vessalico area, a need to identify garlic products that align with the Vessalico ecotype arises. In this study, an NMR-based approach followed by multivariate analysis to analyze the chemical composition of Vessalico garlic sourced from 17 different farms, along with its two French parent cultivars, was employed. Self-organizing maps allowed to identify a homogeneous subset of representative samples of the Vessalico ecotype. Through the OPLS-DA model, the most discriminant metabolites based on values of VIP (Variable Influence on Projections) were selected. Among them, S-allylcysteine emerged as a potential marker for distinguishing the Vessalico garlic from the French parent cultivars by NMR screening. Additionally, to promote sustainable agricultural practices, the potential of Vessalico garlic extracts and its main components as agrochemicals against Xanthomonas campestris pv. campestris, responsible for black rot disease, was explored. The crude extract exhibited a MIC of 125 µg/mL, and allicin demonstrated the highest activity among the tested compounds (MIC value of 31.25 µg/mL).

Sustainable Agrochemical Exploitation of Ligurian Aromatic Plant Ecotypes.

The characterization of local improved varieties as well as the reduction of synthetic chemical fertilizers are sustainable approaches in the vision of a new precision Farming. Aim of our study was to improve the geographical characterization of local ecotypes and to identify peculiar features of new crops in terms of bioactive compounds. NMR and LC-MS metabolite profiling approaches followed by multivariate data analysis were applied to characterize local rosemary and garlic ecotypes. With the aim of applying for a protected designation of origin, orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify representative sensory quality indicators for Vessalico garlic and rosemary "Eretto Liguria" local ecotypes, Variable Influence on Projections (VIP) values of OPLS-DA indicated six metabolites as quality indicators for Vessalico garlic and sixteen metabolites as quality indicators for rosemary "Eretto Liguria". Finally, to discover and utilize new ecotypes in a sustainable way, Vessalico garlic extracts antiviral activity, previously evaluated against Tomato brown rugose fruit virus (ToBRFV), a Tobamovirus affecting tomato crops, was extended to Pepino mosaic virus (PepMV) with positive results.

Nonpersistent Nanoarchitectures Enhance Concurrent Chemoradiotherapy in an Immunocompetent Orthotopic Model of HPV+ Head/Neck Carcinoma.

Cisplatin chemoradiotherapy (CRT) is the established standard of care for managing locally advanced human papillomavirus-positive head/neck carcinoma. The typically young patients may suffer serious and long-time side effects caused by the treatment, such as dysphagia, and hearing loss. Thus, ensuring a satisfactory post-treatment quality of life is paramount. One potential replacing approach to the classical CRT involves the combination of standard-dose radiotherapy and radiosensitizers such as noble metal nanoparticles (NPs). However, several concerns about size, shape, and biocompatibility limit the translation of metal nanomaterials to the clinical practice. Here, it is demonstrated that a new model of nonpersistent gold nanoarchitectures containing cisplatin (NAs-Cluster-CisPt) generates, in combination with radiotherapy, a significant in vivo tumor-reducing effect compared to the standard CRT, achieving a complete tumor clearance in 25% of the immunocompetent models that persist for 60 days. These findings, together with the negligible amount of metals recognized in the excretory organs, highlight that the concurrent administration of NAs-Cluster-CisPt and radiotherapy has the potential to overcome some clinical limitations associated to NP-based approaches while enhancing the treatment outcome with respect to standard CRT. Overall, despite further mechanistic investigations being essential, these data support the exploiting of nonpersistent metal-nanomaterial-mediated approaches for oral cancer management.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Targeted metabolite profiling of Salvia rosmarinus Italian local ecotypes and cultivars and inhibitory activity against Pectobacterium carotovorum subsp. carotovorum.

Introduction: The development of agriculture in terms of sustainability and low environmental impact is, at present, a great challenge, mainly in underdeveloped and marginal geographical areas. The Salvia rosmarinus "Eretto Liguria" ecotype is widespread in Liguria (Northwest Italy), and farmers commonly use it by for cuttings and for marketing. In the present study, this ecotype was characterized in comparison with other cultivars from the same geographical region and Campania (Southern Italy), with a view to application and registration processes for the designation of protected geographical indications. Moreover, the possibility of using the resulting biomass after removing cuttings or fronds as a source of extracts and pure compounds to be used as phytosanitary products in organic farming was evaluated. Specifically, the potential of rosemary extracts and pure compounds to prevent soft rot damage was then tested. Methods: A targeted NMR metabolomic approach was employed, followed by multivariate analysis, to characterize the rosemary accessions. Bacterial soft rot assay and disk diffusion test were carried out to evaluate the activity of extracts and isolated compounds against Pectobacterium carotovorum subsp. carotovorum. Enzymatic assay was performed to measure the in vitro inhibition of the pectinase activity produced by the selected pathogen. Molecular docking simulations were used to explore the possible interaction of the selected compounds with the pectinase enzymes. Results and Discussion: The targeted metabolomic analysis highlighted those different geographical locations can influence the composition and abundance of bioactive metabolites in rosemary extracts. At the same time, genetic factors are important when a single geographical area is considered. Self-organizing maps (SOMs) showed that the accessions of "Eretto Liguria" appeared well characterized when compared to the others and had a good content in specialized metabolites, particularly carnosic acid. Soft rotting Enterobacteriaceae belonging to the Pectobacterium genus represent a serious problem in potato culture. Even though rosemary methanolic extracts showed a low antibacterial activity against a strain of Pectobacterium carotovorum subsp. carotovorum in the disk diffusion test, they showed ability in reducing the soft rot damage induced by the bacterium on potato tissue. 7-O-methylrosmanol, carnosol and isorosmanol appeared to be the most active components. In silico studies indicated that these abietane diterpenoids may interact with P. carotovorum subsp. carotovorum pectate lyase 1 and endo-polygalacturonase, thus highlighting these rosemary components as starting points for the development of agents able to prevent soft rot progression.

Development and validation of a GC-MS method for determination of metformin in normal brain and in glioblastoma tissues.

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors.

Phytochemicals and Cancer Treatment: Cell-Derived and Biomimetic Vesicles as Promising Carriers.

The majority of anticancer agents currently used derive from natural sources: plants, frequently the ones employed in traditional medicines, are an abundant source of mono- and diterpenes, polyphenols, and alkaloids that exert antitumor activity through diverse mechanisms. Unfortunately, many of these molecules are affected by poor pharmacokinetics and limited specificity, shortcomings that may be overcome by incorporating them into nanovehicles. Cell-derived nanovesicles have recently risen to prominence, due to their biocompatibility, low immunogenicity and, above all, targeting properties. However, due to difficult scalability, the industrial production of biologically-derived vesicles and consequent application in clinics is difficult. As an efficient alternative, bioinspired vesicles deriving from the hybridization of cell-derived and artificial membranes have been conceived, revealing high flexibility and appropriate drug delivery ability. In this review, the most recent advances in the application of these vesicles to the targeted delivery of anticancer actives obtained from plants are presented, with specific focus on vehicle manufacture and characterization, and effectiveness evaluation performed through in vitro and in vivo assays. The emerging overall outlook appears promising in terms of efficient drug loading and selective targeting of tumor cells, suggesting further engrossing developments in the future.

Towards a better understanding of thermally treated polycarbophil matrix tablets for controlled release.

Polycarbophil (POL), a polyacrylic acid cross-linked with divinyl glycol, is widely used in semisolid and solid dosage forms. When undergoing a thermal treatment in the range 120-160 °C, POL shows interesting morphological modifications, related to changes in physical properties, such as swelling of the powder granules, or hardening and matrix formation if included in the composition of a tablet. Thermal analysis conducted on POL highlighted a thermal event (Z) that can be correlated both to the shrinking of the powder granules and to the matrix formation in compacted POL powder. Modulated differential scanning calorimetry (MDSC) allowed to distinguish, inside event Z, an irreversible process overlapping with a reversible glass transition, attributable to the volatilization of residual solvents identified, through a complex TGA-FTIR-GC-MS interface, as acetate esters used for the polymer production as very fine powder. A specific interaction between acetates and POL, capable of stabilizing the polymer chains in a given conformation, was highlighted. The molecular rearrangement of the POL chains, following the volatilization of the solvent-stabilizers, is therefore ascribable to a loss of energetic stability of this material, which justifies the shrinking phenomena in the granules of the powder and the matrix formation when POL is compacted.

Two Novel PET Radiopharmaceuticals for Endothelial Vascular Cell Adhesion Molecule-1 (VCAM-1) Targeting.

Atherosclerosis is a chronic progressive disease involving inflammatory events, such as the overexpression of adhesion molecules including the endothelial Vascular Cell Adhesion Molecule-1 (VCAM-1). VCAM-1 is rapidly overexpressed in the first stages of atherosclerosis, thus representing a promising target for early atheroma detection. Two novel Positron Emission Tomography (PET) radiopharmaceuticals (MacroP and NAMP), based on the VCAM-1-binding peptide having sequence VHPKQHRGGSKGC, were synthesized and characterized. MacroP is derived from the direct conjugation of a DOTA derivative with the peptide, while NAMP is a biotin derivative conceived to be employed in a three-step pretargeting system, involving the use of a double-chelating derivative of DOTA. The identity of the newly synthesized radiopharmaceuticals was confirmed by mass spectrometry and, after radiolabeling with 68Ga, both showed high radiochemical purity; in vitro tests on human umbilical vein endothelial cells evidenced their VCAM-1 binding ability, with higher radioactive uptake in the case of NAMP. Moreover, NAMP might also be employed in a theranostic approach in association with functionalized biotinylated nanoparticles.

Trace Elements in Edible Flowers from Italy: Further Insights into Health Benefits and Risks to Consumers.

The use of edible flowers in cooking dates back to ancient times, but recently it is gaining success among the consumers, increasingly attentive to healthy and sustainable foods of high quality, without neglecting taste, flavour, and visual appeal. The present study aims to deepen the knowledge regarding the mineral composition of edible flowers, an aspect not widely investigated in scientific literature. The concentrations of Cd, Co, Cu, Fe, Mn, Ni, Pb, Sr, V, and Zn have been determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) in flowers belonging to a wide variety of species. The study highlights that some floral species are characterized by significantly higher concentrations of certain trace elements, e.g., the flowers of Acmella oleracea for Mn, those of basil (Ocimum basilicum) and of pumpkins (Cucurbita moschata and C. pepo) for Cu and Sr, and those of orange daylily (Hemerocallis fulva) for Ni. Potentially toxic elements are present at low concentrations, often below the limit of the detection for Cd, Co, Ni, V. In all samples, Cd and Pb are well below the maximum permitted levels in foodstuffs. It can be concluded that the edible flowers analyzed can be considered a good source of essential elements and do not present risks for the consumer health as for the mineral composition.

Author Correction: A new microdispersed albumin derivative potentially useful for radio-guided surgery of occult breast cancer lesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mineral composition of organic and conventional white wines from Italy.

Despite of the increased interest of consumers for organic agro-food products and of the growing demand for organic wines, scientific literature reports a limited number of studies aimed to evaluate the chemical composition of organic wine with respect to conventional wine in terms of major and trace metals. In the present study the concentrations of 19 elements (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Sr, V, and Zn) were determined in samples of white wines from Italy, conventionally and organically produced. No significant difference in the mineral composition was found between the two groups, except for Ni, which showed a higher concentration in organic wines. By comparing our data with data from literature it can be pointed out that there is no agreement among the results presented in the different studies referring to comparisons between organically and conventionally produced wines, concluding that the mineral composition of wines depends on factors different from organic/conventional production method.

A new microdispersed albumin derivative potentially useful for radio-guided surgery of occult breast cancer lesions.

This paper describes a new nuclear imaging agent, 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid of human albumin (HAC), potentially suitable for application in the Radio-guided Occult Lesion Localization (ROLL) of non-palpable mammalian cancerous lesions, as a tool to overtake the short radio-signal half-life of the technetium-99m based radiopharmaceutical currently used. This conjugate is a microsized powder aggregate, water-insoluble between pH 3 and 8.5, obtained by conjugating the protein with the macrocyclic chelating agent DOTA through a one-pot reaction in aqueous medium. The product has been fully characterized and is stable to the thermal conditions adopted for labeling; after radiolabeling with longer half-life radionuclides such as 177Lu or 111In, it has shown radiochemical purity (RCP) >90% and resulted stable when stored in saline or plasma for 6 days at 37 °C. A µPET/CT study, performed in vivo on adult female rats, showed that the radioactivity of HAC labeled with 64Cu remained located in the mammary glands for at least 40 h, without diffusion or drainage in healthy tissues or in the lymphatic circulation. This new imaging agent might make the ROLL procedure more accessible, safe and flexible, promoting a significant time and cost reduction of this intervention. Moreover, HAC might also be used in other radio-guided surgical procedures in oncology.

Trace elements in Plantago lanceolata L., a plant used for herbal and food preparations: new data and literature review.

Plantago lanceolata L. is a common grassland and roadside plant, widely used in many countries in food and herbal preparations. In this study, samples of this wild plant were collected from rural, suburban/urban, and industrial environments; the concentrations of As, Cd, Co, Cu, Fe, Mn, Ni, P, Pb, V, and Zn were measured in the edible parts of the plant (leaves), in the roots, and in the soils in order to calculate the bioaccumulation and translocation factors. The data obtained were compared with literature data available. Except for samples collected near mines and smelting plants, where Cd, Pb, and Zn concentrations were up to 15 times higher, in all other cases, no differences were observed with respect to samples from rural areas, except for Pb concentration, which was 3 times higher in urban areas. In the samples collected in our study area, the metal content does not pose particular health risks; however, even within a quite restricted region like the investigated area, high metal concentrations, possibly due to the presence of particular substrates, were observed in some samples collected from areas considered "clean" and suitable for wild food plant gathering.

Development of an Injectable Slow-Release Metformin Formulation and Evaluation of Its Potential Antitumor Effects.

Metformin is an antidiabetic drug which possesses antiproliferative activity in cancer cells when administered at high doses, due to its unfavorable pharmacokinetics. The aim of this work was to develop a pharmacological tool for the release of metformin in proximity of the tumor, allowing high local concentrations, and to demonstrate the in vivo antitumor efficacy after a prolonged metformin exposition. A 1.2% w/w metformin thermoresponsive parenteral formulation based on poloxamers P407 and P124, injectable at room temperature and undergoing a sol-gel transition at body temperature, has been developed and optimized for rheological, thermal and release control properties; the formulation is easily scalable, and proved to be stable during a 1-month storage at 5 °C. Using NOD/SCID mice pseudo-orthotopically grafted with MDA-MB-231/luc+ human breast cancer cells, we report that multiple administrations of 100 mg of the optimized metformin formulation close to the tumor site cause tissue accumulation of the drug at levels significantly higher than those observed in plasma, and enough to exert antiproliferative and pro-apoptotic activities. Our results demonstrate that this formulation is endowed with good stability, tolerability, thermal and rheological properties, representing a novel tool to be pursued in further investigations for adjuvant cancer treatment.

Chemical and magnetic analyses on tree bark as an effective tool for biomonitoring: A case study in Lisbon (Portugal).

Tree bark has proven to be a reliable tool for biomonitoring deposition of metals from the atmosphere. The aim of the present study was to test if bark magnetic properties can be used as a proxy of the overall metal loads of a tree bark, meaning that this approach can be used to discriminate different effects of pollution on different types of urban site. In this study, the concentrations of As, Cd, Co, Cu, Fe, Mn, Ni, P, Pb, V and Zn were measured by ICP-OES in bark samples of Jacaranda mimosifolia, collected along roads and in urban green spaces in the city of Lisbon (Portugal). Magnetic analyses were also performed on the same bark samples, measuring Isothermal Remanent Magnetization (IRM), Saturation Isothermal Remanent Magnetization (SIRM) and Magnetic Susceptibility (χ). The results confirmed that magnetic analyses can be used as a proxy of the overall load of trace elements in tree bark, and could be used to distinguish different types of urban sites regarding atmospheric pollution. Together with trace element analyses, magnetic analyses could thus be used as a tool to provide high-resolution data on urban air quality and to follow up the success of mitigation actions aiming at decreasing the pollutant load in urban environments.

Is there a risk of trace element contamination in herbal preparations? A test study on the lichen Cetraria islandica.

Lichens are a source of unique secondary metabolites, which have been proved to have many biological properties with possible pharmaceutical roles, including e.g. antioxidant, antimicrobial, antifungal or anti-inflammatory activities and to be worth of consideration for potential human use. However, lichens lack cuticolar tissues and are exposed to several atmospheric contaminants, including trace elements. This work aims at exploring the potential toxicity of herbal preparations derived by the lichen Cetraria islandica due to trace element contamination, testing whether different concentrations may be observed, depending on the origin of the raw material. Fourteen samples of C. islandica, as cut dried thalli occurring on the European market, have been bought from different providers. For each sample, the concentrations of As, Cd, Co, Cu, Fe, Mn, Ni, P, Pb, V and Zn were measured on comminuted herbal substance and on the corresponding decoction, using atomic emission spectrometry (ICP-OES). The elemental concentrations in decoctions were significantly lower than those measured in raw materials, dropping down to levels of negligible health concern. Differences observed in raw materials were not observed anymore in the corresponding decoctions. Also, the elemental transfer rate from dried lichen to decoction was extremely element-specific, ranging from 2% for Cu to 95% for Zn.

The bark of the branches of holm oak (Quercus ilex L.) for a retrospective study of trace elements in the atmosphere.

Tree bark has proved to be a useful bioindicator for trace elements in the atmosphere, however it reflects an exposure occurring during an unidentified period of time, so it provides spatial information about the distribution of contaminants in a certain area, but it cannot be used to detect temporal changes or trends, which is an important achievement in environmental studies. In order to obtain information about a known period of time, the bark collected from the annual segments of tree branches can be used, allowing analyses going back 10-15 years with annual resolution. In the present study, the concentrations of As, Cd, Co, Cu, Fe, Mn, Ni, Pb, V and Zn were measured by atomic emission spectrometry in a series of samples covering the period from 2001 to 2013 in an urban environment. Downward time trends were significant for Cd, Pb and Zn. The only trace element showing an upward time trend was V. The concentrations of the remaining six trace elements were constant over time, showing that their presence in bark is not simply proportional to the duration of exposure. This approach, which is simple, reliable and widely applicable at a low cost, allows the "a posteriori" reconstruction of atmospheric trace element deposition when or where no monitoring programme is in progress and no other natural archives are available.

Branch bark of holm oak (Quercus ilex L.) for reconstructing the temporal variations of atmospheric deposition of hexavalent chromium.

The bark from the annual segments of the branches of holm oak (Quercus ilex L.) is exposed to trace element deposition for a known period of time and thus it is a possible candidate as a bioindicator for reconstructing historical changes in pollution. A series of samples were analysed for Cr(VI) concentration by electrothermal atomic absorption spectrometry (ET-AAS) after selective extraction in a sodium carbonate solution. In this way the atmospheric deposition of Cr(VI) was reconstructed from 2001 to 2010 in an area where an industrial plant produced Cr(VI) compounds until 2003. The present study shows the potential of this type of sample as a natural archive for persistent pollutants, useful for monitoring changes that occur before a monitoring programme is established, with the advantage of being easy to collect almost everywhere.

Human contribution to trace elements in urban areas as measured in holm oak (Quercus ilex L.) bark.

The effect of human activities on the presence of trace elements in the atmosphere was evaluated by analyzing samples of holm oak bark, collected in Italy in a large city, in a small town, and in a reference area, scarcely inhabited. In all cases, point sources of pollution were excluded (e.g., industries and incinerators). The concentrations of As, Cd, Co, Cu, Fe, Mn, Ni, Pb, V, and Zn were measured using inductively coupled plasma optical emission spectrometry (ICP-OES). The element concentrations in the small town are not different from the reference area, except for Pb and Cu, while the samples collected in the large city show higher concentrations of Co, Cu, Fe, Ni, Pb, V, and Zn with respect to the rural area. In particular, the Pb levels in the large city are approximately 16 times higher than in the reference site, and five times higher than in the small town. Most element concentrations are correlated in the large city, while in the reference site, only a few significant correlations between elements were found. Even in the absence of specific sources of pollution, populations living in big cities are exposed to higher concentrations of trace elements than those living in rural environments or in small urban centers.