Structural characterization and semi-synthetic modification of bacterial glycolipids

The present PhD thesis aims to discover new natural lipid A structures and to obtain semi-synthetic derivatives thereof to be tested as candidates for the pharmacological use as vaccine adjuvants. Recently, many researchers focused on the development of effective innate immunity ligands as vaccine adjuvants. However, most of innate immunity ligands including LPS, one of the bacterial cell surface component that is a main topic in this thesis, involve undesired effects typified by a strong inflammatory induction in addition to useful adjuvant effects. Therefore, the development of novel non-toxic safer LPS is deeply desired. In the first topic of this thesis, we focused on two psychrophilic bacteria to reveal novel non-toxic LPS. As for Colwellia psychrerythraea strain 34H, was accomplished the complete characterization and immunological function analysis of its lipid A having very unique 3-hydroxy unsaturated tetradecenoic acid and acylated phosphoglycerol moiety. As for Psychrobacter arcticus strain 273-4 grown at 4°C, we worked on the lipid A structure investigation and immunological function analysis. In the second topic, a semisynthetic strategy for novel lipid A derivatives from E. coli LPS was reported. The advantages of a fermentative access to complex natural products was combined with the development of site-selective chemical reactions modifying lipid A structures. The selective oxidation method of the primary hydroxyl group at position C-6 of GlcN II enables the introduction of various functional group to lipid A and various lipid A derivatives including clickable lipid As. Semi-synthesized lipid A derivatives showed moderate immunostimulating activity but no-toxicity. In addition, the modification method of the lipid pattern of lipid A was also investigated. These novel strategies will promote structure activity relationship study to reveal non-toxic lipid A. It was also shown by the synthesis of Tn or TF antigen conjugated lipid A derivatives that this strategy is very useful in developing a prospective self-adjuvanting vaccine strategy. Furthermore, this strategy is also useful in the construction of a labeled lipid A that is applicable to elucidation of immune activation mechanism

Characterisation of Woody Necromass in Beech Forests with Different Anthropic Accessibility: The Case of La Rioja (Spain)

In this study, a comprehensive analysis of deadwood was conducted in four macro-areas located in two beech forests of public utility in Enciso (La Rioja, Spain). Dendrometric data, as well as qualitative and quantitative characteristics of deadwood, were collected and analysed with respect to the degree of accessibility to the forest to determine the effect of different levels of forest accessibility on deadwood volume and carbon stocks. All decomposition classes were present except the first, highlighting the development of natural degradation dynamics. Deadwood stored 6.9 t/ha of C in the easy accessibility class, 5.7 t/ha of C in the medium accessibility class and 2.2 t/ha of C in the difficult accessibility class. The average volume of deadwood and carbon stored calculated in this study were higher than the values reported in the Spanish and Italian national forest inventories, including one developed for Riojan beech forests. Deadwood volume was on average 22.5 m3/ha, showing an unequal distribution, with the lowest values found far from the access roads, despite forest accessibility generally being considered a factor that facilitates the human collection of deadwood. The distribution patterns of deadwood in beech forests of La Rioja, apparently counterintuitive, were due to a combination of different factors, including slope, cattle grazing, and weather conditions which might have favoured downward movement of the deadwoodThis research was funded by the Spanish Ministry of Science, Innovation and Universities, AGL2016-76769-C2-2-RS

Structural characterization of core Region in Erwinia amylovora lipopolysaccharide.

Erwinia amylovora (E. amylovora) is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS) core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core), wabH and wabG (outer-LPS core mutants). The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry

Apolipoprotein A-I (ApoA-I) Mimetic Peptide P2a by Restoring CholesterolEsterification Unmasks ApoA-I Anti-Inflammatory Endogenous Activity In Vivo. CO-FIRST AUTHOR

The acute-phase protein haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I) and impairs its action on lecithin-cholesterol acyltransferase, an enzyme that plays a key role in reverse cholesterol transport. We have previously shown that an ApoA-I mimetic peptide, P2a, displaces Hpt from ApoA-I, restoring the enzyme activity in vitro. The aim of this study was to evaluate whether P2a displaces Hpt from ApoA-I in vivo and whether this event leads to anti-inflammatory activity. Mice received subplantar injections of carrageenan. Paw volume was measured before the injection and 2, 4, 6, 24, 48, 72, and 96 h thereafter. At the same time points, concentrations of HDL cholesterol (C) and cholesterol esters (CEs) were measured by high-performance liquid chromatography, and Hpt and ApoA-I plasma levels were evaluated by enzyme-linked immunosorbent assay. Western blotting analysis for nitric-oxide synthase and cyclooxygenase (COX) isoforms was also performed on paw homogenates. CEs significantly decreased in carrageenan-treated mice during edema development and negatively correlated with the Hpt/ApoA-I ratio. P2a administration significantly restored the CE/C ratio. In addition, P2a displayed an anti-inflammatory effect on the late phase of edema with a significant reduction in COX2 expression coupled to an inhibition of prostaglandin E2 synthesis, implying that, in the presence of P2a, CE/C ratio rescue and edema inhibition were strictly related. In conclusion, the P2a effect is due to its binding to Hpt with consequent displacement of ApoA-I that exerts anti-inflammatory activity. Therefore, it is feasible to design drugs that, by enhancing the physiological endogenous protective role of ApoA-I, may be useful in inflammation-based diseases

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis

Bombesin peptide antagonist for target-selective delivery of liposomal doxorubicin on cancer cells

Purpose: This study addresses novel peptide modified liposomal doxorubicin to specifically target tissues overexpressing bombesin (BN) receptors. Methods: DOTA-(AEEA)2-peptides containing the [7–14]bombesin and the new BN-AA1 sequence have been synthesized to compare their binding properties and in serum stabilities. The amphiphilic peptide derivative (MonYBN- AA1) containing BN-AA1, a hydrophobic moiety, polyethylenglycole (PEG), and diethylenetriaminepentaacetate (DTPA), has been synthesized. Liposomes have been obtained by mixing of MonY-BN-AA1 with 1,2-distearoylsn-glycero-3-phosphocholine (DSPC). Results: Both 111In labeled peptide derivatives present nanomolar Kd to PC-3 cells. 177Lu labeled peptide DOTA- (AEEA)2-BN-AA1 is very stable (half-life 414.1 h), while DOTA-(AEEA)2-BN, shows a half-life of 15.5 h. In vivo studies on the therapeutic efficacy of DSPC/MonY-BN-AA1/Dox in comparison to DSPC/MonY-BN/Dox, were performed in PC-3 xenograft bearing mice. Both formulations showed similar tumor growth inhibition (TGI) compared to control animals treated with non-targeted DSPC/Dox liposomes or saline solution. For DSPC/MonY-BN-AA1/Dox the maximum effect was observed 19 days after treatment. Conclusions: DSPC/MonY-BN-AA1/Dox nanovectors confirm the ability to selectively target and provide therapeutic efficacy in mice. The lack of receptor activation and possible acute biological side effects provided by using the AA1 antagonist bombesin sequence should provide safe working conditions for further development of this class of drug delivery vehicles

Shifts in wood anatomical traits after a major hurricane

•1. Trait variation across individuals and species influences the resistance and resilience of ecosystems to disturbance, and the ability of individuals to capitalize on postdisturbance conditions. In trees, the anatomical structure of xylem directly affects plant function and, consequently, it is a valuable lens through which to understand resistance and resilience to disturbance. •2. To determine how hurricanes affect wood anatomy of tropical trees, we characterized a set of anatomical traits in wood produced before and after a major hurricane for 65 individuals of 10 Puerto Rican tree species. We quantified variation at different scales (among and within species, and within individuals) and determined trait shifts between the pre- and posthurricane periods. We also assessed correlations between traits and growth rates. •3. While the majority of anatomical trait variation occurred among species, we also observed substantial variation within species and individuals. Within individuals, we found significant shifts for some traits that generally reflected increased hydraulic conductivity in the posthurricane period. We found weak evidence for an association between individual xylem anatomical traits and diameter growth rates. •4. Ultimately, within-individual variation of xylem anatomical traits observed in our study could be related to posthurricane recovery and overall growth (e.g. canopy filling). Other factors, however, likely decouple a relationship between xylem anatomy and diameter growth. While adjustments of wood anatomy may enable individual trees to capitalize on favourable postdisturbance conditions, these may also influence their future responses or vulnerability to subsequent disturbances

STRUCTURAL PATTERNS, GROWTH PROCESSES, CARBON STOCKS IN AN ITALIAN NETWORK OF OLD-GROWTH BEECH FORESTS

In the framework of the Project of National Interest (PRIN) “Climate change and forests - Dendroecological and ecophysiological responses, productivity and carbon balance on the Italian network of old-growth beech forests”, 9 old beech (Fagus sylvatica L.) forests were sampled in the eastern Alps and in the central Apennines to assess: i) the degree of ‘old-growthness’ on a structural and dendroecological basis; ii) the carbon (C) stocks in the different ecosystem compartments; iii) some ecophysiological traits using stable isotopes. Live and dead tree structure, soil features and C stocks were examined in some among the oldest and less disturbed beech forests in Italy. Furthermore, leaves, litter and wood cores were sampled for concurrent dendroecological, nutrient and stable isotope analysis, to study age structure, disturbance history and medium- and long-term response to climate and ecophysiological traits. In all sampled stands, values of basal area, volume and large trees density reached or exceeded control values reported for European and North American old-growth forests, while total amount of deadwood was generally low, except in two sites. Diameter distribution showed a remarkable differentiation from bimodal to ‘rotated-sigmoid’ curve. In accordance with structural analyses, disturbance chronologies revealed the importance of frequent low-to-moderate events in generating a fine-scale structure, confirming previous results on beech forests. Different degrees of ‘old-growthness’ were identified for Alpine and Apennine beech stands through the ‘structural-based approach’, which proved to be a valid tool for old-growth forests detection. Preliminary results on carbon stocks on a subset of sites indicate that these old-growth forests are relevant carbon reservoirs, with 192-268 MgC ha-1 of total biomass (67-73% aboveground; 27-33% belowground, 4 stands) and 7-21 MgC ha-1 of deadwood. In these stands, also forest floor (excluding deadwood) and soils are stores of relevant amount of carbon (5-9 MgC ha-1 litter layer, 4 stands; 168-420 MgC ha-1 mineral soil, 3 stands). Finally, carbon isotope discrimination (Δ) analysis, a proxy of wateruse efficiency, was applied on tree-ring cores from a subset of sites. The perspectives of this technique for a retrospective ecophysiological interpretation of climate-change impact on old-growth forests were described. Preliminary results obtained within this PRIN project indicate that the reported ecological indicators can be used to produce a description of forest structures and processes driving stand dynamics (‘structural-based approach’). Furthermore, the use of multiple sampling and research techniques and the integration of research groups with complementary expertise can foster deeper understanding of the ecology and dynamics of old-growth forests.L'articolo è disponibile sul sito dell'editore http://www.aisf.it

Reply to Elmendorf and Ettinger: Photoperiod playsa dominantand irreplaceable role in triggering secondary growth resumption

In their Letter, Elmendorf and Ettinger (1) question the dominant role of photoperiod in driving secondary growth resumption (hereafter referred to as xylem formation onset) of the Northern Hemisphere conifers, recently reported by Huang et al. (2). Their opinions are grounded on the following three aspects, including 1) the seasonality of the photoperiod, 2) the dependence of the predictor variables (e.g., photoperiod, forcing, and chilling) on the response variable (the date of onset of xylem formation, day of the year [DOY]), and 3) the limited value of the obtained models for interannual forecasting. We think they bring up an interesting issue that deserves further discussion and clarification. Photoperiod is acknowledged to regulate spring bud swelling while wood formation starts (3, 4). Although photoperiod seasonality occurs at each site, its influence is marginal in our study given that the analysis involved comparisons among sites across the Northern Hemisphere. Our conclusion that photoperiod plays a dominant role was built upon the combination of several coherent pieces of evidence, rather than “the crux of Huang et al….” as they pointed out. First, we clearly stated that model 2, which modeled DOY as a function of the mean annual temperature of the site (MAT), forcing, chilling, and soil moisture, was considered the best model in terms of parsimony according to minimum Akaike information criterion and Bayesian information criterion, rather than R2 as referred to in their Letter. Second, photoperiod interacted with MAT and can explain 61.7% of the variance of MAT alone (2). Therefore, we concluded that secondary growth resumption was driven primarily by MAT and photoperiod or by their interaction, which is challenging to be disentangled without experimental data, we agree. In terms of biological functioning, they play an ..

Tree-ring isotopes suggest atmospheric drying limits temperature–growth responses of treeline bristlecone pine

Altitudinally separated bristlecone pine populations in the White Mountains (California, USA) exhibit differential climate-growth responses as temperature and tree-water relations change with altitude. These populations provide a natural experiment to explore the ecophysiological adaptations of this unique tree species to the twentieth century climate variability. We developed absolutely dated annual ring-width chronologies, and cellulose stable carbon and oxygen isotope chronologies from bristlecone pine growing at the treeline (~3500 m) and ~200 m below for the period AD 1710-2010. These chronologies were interpreted in terms of ecophysiological adaptations to climate variability with a dual-isotope model and a leaf gas exchange model. Ring widths show positive tree growth anomalies at treeline and consistent slower growth below treeline in relation to the twentieth century warming and associated atmospheric drying until the 1980s. Growth rates of both populations declined during and after the 1980s when growing-season temperature and atmospheric vapour pressure deficit continued to increase. Our model-based interpretations of the cellulose stable isotopes indicate that positive treeline growth anomalies prior to the 1980s were related to increased stomatal conductance and leaf-level transpiration and photosynthesis. Reduced growth since the 1980s occurred with a shift to more conservative leaf gas exchange in both the treeline and below-treeline populations, whereas leaf-level photosynthesis continued to increase in response to rising atmospheric CO2 concentrations. Our results suggest that warming-induced atmospheric drying confounds positive growth responses of apparent temperature-limited bristlecone pine populations at treeline. In addition, the observed ecophysiological responses of attitudinally separated bristlecone pine populations illustrate the sensitivity of conifers to climate change