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Authors would like to thank the financial support of Italian Ministry of University and Research (MIUR) by the project PRIN2015 “Multiple equilibria in natural and Biological fluids: from speciation to selective sequestring” (code 2015MP34H3_004). The authors would like to apologise for any inconvenience caused.

In agreement with all authors of this paper the order of the authors and the contribution “Paola Lunetti[+], Alessandro Romano[+], Chiara Carrisi, Daniela Antonucci, Tiziano Verri, Giuseppe E. De Benedetto, Vincenza Dolce, Francesco P. Fanizzi, Michele Benedetti,* and Loredana Capobianco.* [+] These authors contributed equally to this paper. * Corresponding authors: Michele Benedetti and Loredana Capobianco, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce.” is corrected to read the original order of the first submitted version of the paper: “Chiara Carrisi[+], Alessandro Romano[+], Paola Lunetti, Daniela Antonucci, Tiziano Verri, Giuseppe E. De Benedetto, Vincenza Dolce, Francesco P. Fanizzi, Michele Benedetti,* and Loredana Capobianco.* [+] These authors contributed equally to this paper. * Corresponding authors: Loredana Capobianco and Michele Benedetti, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce.” For completeness the following individual contributions of the authors were added in the Supporting Information: “Author contributions: L.C., M.B., C.C., A.R. designed research; C.C., A.R., P.L. and D.A. performed research; L.C., M.B., C.C., A.R., V.D. and P.L. analysed data; F.P.F., A.R., L.C. and M.B. wrote the paper; L.C., M.B., T.V., G.D.B. and F.P.F. active discussion paper revision.”.

Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants. Graphical abstract

Sooty mold is a plant disease caused by Aleurocanthus spiniferus, an alien insect spread widely in the South of Puglia region (Italy) that causes extensive damage to citrus crops. Sooty mold is a complex disease: the insect sucks the sap of the plant and expels a sugary liquid called honeydew which supports the growth of a black biofilm on the plant. This biofilm is made up of various types of fungi and, while not causing disease, it contributes to plant malaise by inhibiting photosynthesis. In this study, microbiological and chemical methods have been used to characterize two bacterial strains, Bacillus velezensis MT9 and Pseudomonas chlororaphis MT5, which were isolated from soil samples taken from vine-cultivated land and can be used as biocontrol agents (BCA). These bacteria were shown to produce antibacterial and antifungal activities in the form of water-soluble molecules and were effective in vitro in inhibiting the growth of two fungal strains, Penicillium sp. YM1 and YM2, isolated from citrus soot. In addition to water-soluble molecules, P. chlororaphis MT5 was shown to produce antifungal volatile organic compounds (VOCs). The VOCs produced by B. velezensis MT9 and P. chlororaphis MT5 were then identified by gas chromatography – mass spectrometry (GC–MS). Interestingly, some of the identified VOCs have previously been reported to have semiochemical activity in insects and, therefore, can interfere with chemical signaling in plant-insect-microbe interaction. Altogether these in vitro findings and pilot experiments with a limited set of plants indicate that Bacillus velezensis MT9 and Pseudomonas chlororaphis MT5 can effectively combat sooty mold possibly by acting on both the insects and the fungi that inhabit the black biofilm, paving the way for an innovative and sustainable tool to fight this disease.

Background: Non-occupational cases of contact dermatitis to finished wood are occasionally reported from contact with wooden jewellery, musical instruments, knife handles and wooden footwear. Two female patients, 26 and 34 years old, developed acute eczema on the wrists after they worn in the summer a kind of catholic bracelet called “memorabilia” made by single wooden chunks with over-impressed iconic religious images. Objectives: To identify the culprit photoallergen responsible for the reported adverse reactions suspected to be a compound of the wooden bracelets. Methods: Three months after the resolution of the dermatitis, patch test and photo-patch tests were performed with the S.I.D.A.P.A. (Italian Society of Allergic, Occupational and Environmental Dermatology) standard series, and with wood sawdust obtained from both bracelets and tested at 5% in pet. A thin-layer chromatogram (TLC) strip and an extract made from the bracelets were tested in both the patients as patch test and photo-patch test. The extracts of the wooden bracelets were analyzed by gas chromatography – mass spectrometry (GC-MS). Results: Patch tests and photo-patch test with standard series turned out negative. Photo-patch tests with wood sawdust in pet. 5% evidenced a positive reaction (++) in both patients. Two chemicals were detected in the wooden bracelets with GC-MS: dibutylphthalate, commonly used as plasticizer, and 8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-2-one, a coumarin. Patch test with TLC did not elicit positive reactions but the photo-patch test with TLC gave positive reactions in both the patients to the same invisible spot on the strips. Two coumarins (8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-2-one and 8,8-dimethyl-2H,8H-benzo[1,2-b:5,4-b']dipyran-2-one) were detected in the spot with GC-MS. Conclusion: Coumarins are chemicals found in many plants with a known photo-allergic potential. They are probably the main photo-allergens in religious wooden bracelets instead of quinones and psoralens hypothesized in previous reports.

Introduction Xylella fastidiosa (Xf) is a pathogenic bacterium that causes diseases in olive trees. Therefore, analytical methods for both the characterisation of the host/pathogen interaction and infection monitoring are needed. Volatile organic compounds (VOCs) are emitted by plants relate to their physiological state, therefore VOCs monitoring can assist in detecting stress or infection states before visible signs are present. Objective In this work, the headspace-solid phase microextraction-gaschromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the first time to highlight VOCs differences between healthy and Xf-infected olive trees. Methodology VOCs from olive tree twig samples were extracted and analysed by HS-SPME-GC-MS, and hence identified by comparing the experimental linear retention indexes with the reference values and by MS data obtained from NIST library. Data were processed by principal component analysis (PCA) and analysis of variance (ANOVA). Results The HS-SPME step was optimised in terms of adsorbent phase and extraction time. HS-SPME-GC-MS technique was applied to the extraction and analysis of VOCs of healthy and Xf-infected olive trees. More than 100 compounds were identified and the differences between samples were evidenced by the multivariate analysis approach. The results showed the marked presence of methyl esters in Xf-infected samples, suggesting their probable involvement in the mechanism of diffusible signal factor. Conclusion The proposed approach represents an easy and solvent-free method to evaluate the presence of Xf in olive trees, and to evidence volatiles produced by host/pathogen interactions that could be involved in the defensive mechanism of the olive tree and/or in the infective action of Xf.

Concentrated Growth Factors (CGF) represent new autologous (blood-derived biomaterial), attracting growing interest in the field of regenerative medicine. In this study, the chemical, structural, and biological characterization of CGF was carried out. CGF molecular characterization was performed by GC/MS to quantify small metabolites and by ELISA to measure growth factors and matrix metalloproteinases (MMPs) release, structural CGF characterization was carried out by SEM analysis and immunohistochemistry, CGF has been cultured, and its primary cells were isolated for the identification of their surface markers by flow cytometry, Western blot, and real-time PCR, finally, the osteogenic differentiation of CGF primary cells was evaluated through matrix mineralization by alizarin red staining and through mRNA quantification of osteogenic differentiation markers by real-time PCR. We found that CGF has a complex inner structure capable of influencing the release of growth factors, metabolites, and cells. These cells, which could regulate the production and release of the CGF growth factors, show stem features and are able to differentiate into osteoblasts producing a mineralized matrix. These data, taken together, highlight interesting new perspectives for the use of CGF in regenerative medicine.

Over the last few years, different digestion protocols have been proposed to extract microplastics from mussels, an important product from aquaculture and a relevant economic resource, always scrutinized as a potential pollutant concentrator. In this study, a full factorial experimental design technique has been employed to achieve efficiency in removing biological materials while maximizing the recoveries of five common microplastics (polyethylene, polystyrene, polyethylene terephthalate, polypropylene and polyamide). A robust setpoint was calculated, 2.5% potassium hydroxide at 60 °C for 3 h with 5% hydrogen peroxide and 2.7% of methanol, permitting the quantitative digestion of mussel tissues and recovery of microplastics. These experimental conditions were successfully used to digest whole mussels bought from a local market, which possess high levels of microplastic contamination (41 items/g dry weight). The results highlight the importance of optimizing protocols to develop robust, easy to use and cheap quantitative approaches for analysing microplastic accumulation in edible organisms.

The Antarctic silverfish Pleuragramma antarctica is a key species in the Southern Ocean ecosystem, and it is potentially threatened by the climate change affecting Antarctic ecosystems. Assessing the possible exposure to similar or different environmental conditions at early life stages and gathering information about connectivity or segregation between local populations of P. antarctica can be key for planning sound management strategies for this species. By using Laser Ablation Inductively Coupled Plasma Mass Spectrometry, we characterized the otolith chemical composition of 163 adult Antarctic silverfish collected from three areas located thousands of kilometers apart from each other: Cape Hallett, Adelie Land, and Joinville Island. Otoliths were analyzed for chemical composition of both the edge (reflecting the exposure of individuals to environmental conditions at the site where they were sampled) and the core (reflecting exposure to environmental conditions during early life periods after the egg fertilization). We found that the chemical composition along otolith edges was heterogeneous between samples collected at Joinville Island and those collected at the other two sampling areas. In contrast, the chemical composition of otolith cores was homogenous. Our study suggests that adult Antarctic silverfish inhabiting areas very distant from each other have been exposed to similar environmental conditions at early life stages, and could have experienced similar growth rates and physiological processes. This would imply that environmental drivers probably do not play a role in determining potential spatial variability in individual fitness at early life stages, and should not have a major impact on population replenishment.

Coccoid cyanobacteria produce a great variety of secondary metabolites, which may have useful properties, such as antibacterial, antiviral, anticoagulant or anticancer activities. These cyanobacterial metabolites have high ecological significance, and they could be considered responsible for the widespread occurrence of these microorganisms. Considering the great benefit derived from the identification of competent cyanobacteria for the extraction of bioactive compounds, two strains of picocyanobacteria (coccoid cyanobacteria &lt, 3 µm) (Cyanobium sp. ITAC108 and Synechococcus sp. ITAC107) isolated from the Mediterranean sponge Petrosia ficiformis were analyzed. The biological effects of organic and aqueous extracts from these picocyanobacteria toward the nauplii of Artemia&nbsp, salina, sea urchin embryos and human cancer lines (HeLa cells) were evaluated. Methanolic and aqueous extracts from the two strains strongly inhibited larval development, on the contrary, in ethyl acetate and hexane extracts, the percentage of anomalous embryos was low. Moreover, all the extracts of the two strains inhibited HeLa cell proliferation, but methanol extracts exerted the highest activity. Gas chromatography–mass spectrometry analysis evidenced for the first time the presence of β-N-methylamino-l-alanine and microcystin in these picocyanobacteria. The strong cytotoxic activity observed for aqueous and methanolic extracts of these two cyanobacteria laid the foundation for the production of bioactive compounds of pharmacological interest.

Comparison of fine and coarse fractions in terms of sources and dynamics is scarce in southeast Mediterranean countries; differences are relevant because of the importance of natural sources like sea spray and Saharan dust advection, because most of the monitoring networks are limited to PM10. In this work, the main seasonal variabilities of sources and processes involving fine and coarse PM (particulate matter) were studied at the Environmental-Climate Observatory of Lecce (Southern Italy). Simultaneous PM2.5 and PM10 samples were collected between July 2013 and July 2014 and chemically analysed to determine concentrations of several species: OC (organic carbon) and EC (elemental carbon) via thermo-optical analysis, 9 major ions via IC, and 23 metals via ICP-MS. Data was processed through mass closure analysis and Positive Matrix Factorization (PMF) receptor model characterizing seasonal variabilities of nine sources contributions. Organic and inorganic secondary aerosol accounts for 43% of PM2.5 and 12% of PM2.5–10 with small seasonal changes. SIA (secondary inorganic aerosol) seasonal pattern is opposite to that of SOC (secondary organic carbon). SOC is larger during the cold period, sulphate (the major contributor to SIA) is larger during summer. Two forms of nitrate were identified: NaNO3, correlated with chloride depletion and aging of sea-spray, mainly present in PM2.5–10; NH4NO3 more abundant in PM2.5. Biomass burning is a relevant source with larger contribution during autumn and winter because of the influence of domestic heating, however, is not negligible in spring and summer, because of the contributions of fires and agricultural practices. Mass closure analysis and PMF results identify two soil sources: crustal associated to long range transport and carbonates associated to local resuspended dust. Both sources contributes to the coarse fraction and have different dynamics with crustal source contributing mainly in high winds from SE conditions and carbonates during high winds from North direction.

Grotta dei Cervi (Apulia, Southern Italy) is the most important European figurative art site for its Neolithic red and brown paintings found in the various galleries of the cave. It was frequented with particular intensity between an advanced phase of the Early Neolithic (first half of the sixth millennium BCE) and continued up to the end of the Bronze Age (last centuries of the second millennium BCE). Among the more than thirty thousand finds, some ceramic shards had black residues on them. These residues were sampled so that molecular and isotopic investigations could be carried out. GC–MS-MS and EA–IRMS after fractionation of the samples allowed us to identify bitumen on some archaeological potsherd remains and to identify its geographic source by comparison with geological samples from Majella and Selenice, two deposits located respectively in Italy and Albania and known to have been exploited since Neolithic times. All the bitumens from Porto Badisco correlate with each other and with the Selenice geological samples. Isotopic fingerprints strongly support the biomolecular evidence and confirm the Albanian origin of the archaeological bitumen. The data presented herein provide the first evidence of export and trade of raw bitumen across the Adriatic Sea from Selenice to Apulia during the Neolithic age thus bringing back to the fifth millennium BCE the transport of bitumen across the Adriatic Sea in the central Mediterranean area.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour. UCP2 is shown in yeast and mammalian cells to transport aspartate out of mitochondria, thus enabling KRAS-mutated pancreatic ductal adenocarcinoma cells to perform glutaminolysis to support cancer growth.

While in recent years the key role of non-coding RNAs (ncRNAs) in the regulation of gene expression has become increasingly evident, their interaction with the global regulatory circuits is still obscure. Here we analyzed the structure and organization of the transcriptome of Streptomyces ambofaciens, the producer of spiramycin. We identified ncRNAs including 45 small-RNAs (sRNAs) and 119 antisense-RNAs (asRNAs I) that appear transcribed from dedicated promoters. Some sRNAs and asRNAs are unprecedented in Streptomyces and were predicted to target mRNAs encoding proteins involved in transcription, translation, ribosomal structure and biogenesis, and regulation of morphological and biochemical differentiation. We then compared ncRNA expression in three strains: (i) the wild-type strain, (ii) an isogenic pirA-defective mutant with central carbon metabolism imbalance, “relaxed” phenotype, and repression of antibiotic production, and (iii) a pirA-derivative strain harboring a “stringent” RNA polymerase that suppresses pirA-associated phenotypes. Data indicated that the expression of most ncRNAs was correlated to the stringent/relaxed phenotype suggesting novel effector mechanisms of the stringent response.

The feasibility of producing durum wheat pasta enriched with a lipophilic phytocomplex, extracted using supercritical carbon dioxide (SC-CO2), from ripe pumpkin, as free oil or as ready-to-mix oil/?-cyclodextrins (?- CDs) powder, was explored. Four types of pasta were prepared: (i) control spaghetti (S-CTRL); (ii) spaghetti supplemented with ?-CDs (S-?-CD); (iii) spaghetti supplemented with pumpkin oil (S-Oil) and (iv) spaghetti supplemented with the pumpkin oil/?-CD powder (S-Oil/?-CD). The chemical, antioxidant, textural and sensory attributes of the different pasta were evaluated and compared. S-Oil and S-Oil/?-CD spaghetti were significantly enriched with phytosterols, squalene, carotenoids, tocochromanols and unsaturated fatty acids. Spaghetti containing ?-CDs were slightly improved in terms of fiber content. Oil chlatration increased the stability of some bioactives during pasta production and ameliorated poor textural and sensory characteristics of the cooked spaghetti compared with S-Oil sample. S-Oil/?-CD spaghetti might be accepted by customers, if the potential health benefits were also explained.

The ancient Latin city of Gabii is situated 18 km (11.2 miles) to the east of Rome (Central Italy) along the modern Via Prenestina. Gabii was a renowned city in Roman times, particularly during the Republican period and there are various influences in the site that can be identified in Roman culture itself. Gabii is also one of the most significant and important archaeological sites in the territory of the Municipality of Rome and due to its characteristics, it represents today an extraordinary research context. From the excavations carried out in the past it is possible to see how, under the soil, the main structures and buildings of the ancient city are still largely preserved. Among the various testimonies of the past, the tombs, and the micro and macro remains that these contain, represent an opportunity to investigate such practices in the context of Early Iron Age and Orientalizing Latium. In particular, the finds from the Area D baby burials of Gabii enriched the existing dataset so far significantly, allowing us to explore funerary ritual behavior in a more systematic way. This work reports the results of the detailed examination of four tombs (Tombs 30, 50, 51 and 52) of archaeological site. The field strategy for the excavation of the tombs was geared from the start towards both the systematic retrieval of archaeobotanical and zooarchaeological remains and the sampling for organic residue analysis. Aiming for total recovery, the sediments from the tomb fills were sifted in their entirety as their stratigraphic excavation progressed, and samples were taken for flotation. This careful screening allowed for the detection of concentrations of organic material that represent plant and/or animal depositions. The excavation and removal of the grave goods was carried out following strict protocols for residue sampling, minimizing the risk of organic contamination. Samples were analysed by High Temperature Gas Chromatography/Mass Spectrometry (HTGC/MS) and Gas chromatography/Combustion/Isotope ratio mass spectrometry (GC-C-IRMS). For each burial, a subset of vessels including both closed and open shapes was selected, such as cups, open bowl without foot, amphoretta, amphora with dots, Kantharos, plate on a foot, olla, and olpe in bucchero. The results demonstrate the still largely unexploited potential of this sort of integrated studies, encouraging us to expand the application of chemical methods to contexts from other well–controlled excavations.

Lecce stone belongs to the group of Miocene limestone and is distributed in many areas of the Salento peninsula. The great part of the historical buildings, both religious and civil, in this area have been constructed with this porous and soft material. In the past, various methods and recipes have been used to protect the surfaces and ensure their longevity. However, these ancient "recipes" are unidentified, because these techniques were known only by the artisans who used them [2]. For this purpose, selected religious and civil buildings in the territory of the province of Lecce, made of Lecce stone, not yet restored and possibly treated with some of these unknown recipes because of their good conservation state, were studied and the presence of surface treatments was investigated. Samples were analysed by Py/GC-MS with and without thermally assisted hydrolysis and methylation using tetramethylammonium hydroxide (TMAH). The results of the analyses showed that these buildings have been protected with different natural products. Moreover, for the first time, chemical biomarkers demonstrating the use of prickly pear leaves (Opuntia ficus indica [3]) as protective surface finishes have been identified.

Here we report the simple and rapid synthesis of three-dimension Pt flower-like nanostructures (PtNFs) on a polypyrrole nanowires (PPyNWs) matrix. Both PtNFs and PPyNWs are prepared by an electrochemical approach without using any seed, template or surfactant. The morphology and chemical composition of the resulting PtNF/PPyNWs hybrids are characterized by scanning electron microscopy and by X-ray photoelectron spectroscopy, respectively. Taking methanol oxidation as a model catalysis reaction, the electrocatalytic performance of the as-prepared PtNF/PPyNWs system has been evaluated by cyclic voltammetry and chronoamperometry, evidencing that these 3D materials exhibit excellent electrocatalytic activity and high level of poisoning tolerance to the carbonaceous oxidative intermediates. Such electrocatalytic performances can be ascribed to the combined effect of the flower-like structure promoting the exposure of more sites and the polymer nanowires matrix endorsing high dispersion of PtNF on a high electrochemically active surface area, besides the removal of sub-products from electrocatalytic sites.