Artificial ion transporters: synthesis, characterization and ionophoric activity on membrane models

2007/2008Nel corso di alcuni anni il nostro gruppo di ricerca, ispirandosi alla struttura dei composti naturali anfotericina B e squalamina, ha sintetizzato una serie di composti che incorporano gli elementi strutturali fondamentali di questi due ionofori naturali: un’unità idrofobica rigida, una catena idrofilica (modulo di conduzione) e uno o due gruppi terminali polari. L’attività di questi composti nel trasporto di ioni attraverso il doppio strato fosfolipidico è stata investigata utilizzando liposomi come modelli di membrana e metodologie basate sulla spettroscopia NMR e su tecniche spettrofluorimetriche mediante l’utilizzo di probe fluorescenti. Tra i composti sintetizzati, un analogo formato da un ottapeptide di Aib (acido α-amminoisobutirrico) Z-protetto all’ N-terminale (unità idrofobica) e legato tramite il C-terminale ad un gruppo amminico di una diammina derivata dall’esaetilenglicole (modulo di conduzione e gruppo terminale polare), ha mostrato di avere un’attività ionoforica molto elevata nei confronti del trasporto di cationi sodio. La comprensione del motivo di questa alta ed inaspettata attività è stata quindi uno degli scopi principali di questo lavoro di Tesi, in cui sono stati studiati analoghi con catena alchilica a lunghezza variabile al posto del gruppo Z, già precedentemente sintetizzati, per valutare l’effetto idrofobico del sostituente. Dopo aver raccolto una prima serie di dati, per comprendere la rilevanza della presenza dell’anello aromatico sullo ionoforo modello, è stata sintetizzata e studiata una serie di analoghi che portano un diverso gruppo aromatico al posto del gruppo Z. Il secondo e più generale scopo di questo lavoro di Tesi è stato l’inizio di uno studio sull’attività ionoforica di composti strutturalmente diversi al fine di razionalizzare le caratteristiche strutturali necessarie per ottenere composti attivi come trasportatori di ioni. A questo scopo è stata iniziata un’intensa collaborazione con diversi gruppi di ricerca che hanno sintetizzato una serie di possibili ionofori. Il gruppo del Prof. De Riccardis dell’Università di Salerno ha sintetizzato una serie di composti calixarenici sostituiti con catene spermidiniche e una serie di α-ciclopeptoidi formati da un numero variabile di N-benzilossietilglicine. Il gruppo della Prof. Montesarchio dell’Università di Napoli ha preparato degli oligosaccaridi ciclici, legati da legami fosfodiesterei a ponte, chiamati CyPLOS (Cyclic Phosphate-Linked OligoSaccharide macrocycles). Infine il gruppo del Prof. Casnati dell’Università di Parma ha sintetizzato una serie di calixareni sostituiti con un numero variabile di gruppi guanidinio. Tutti questi composti sono stati preparati allo scopo di poter effettuare un ampio studio concernente il rapporto struttura/attività e poter quindi definire i requisiti strutturali minimi per ottenere un composto con attività ionoforica. Accanto ai risultati ottenuti per ogni singola classe di composti, che sono illustrati e discussi in dettaglio nel terzo capitolo della Tesi, la possibilità di studiare un così ampio spettro di composti utilizzando le stesse condizioni sperimentali, ci ha permesso di ottenere delle considerazioni generali sulla natura dell’attività ionoforica ponendo le basi per la progettazione di nuove strutture che possano mostrare maggiore attività e/o selettività. Un altro importante risultato ottenuto durante questo lavoro di Tesi è stato l’acquisizione di esperienza nell’utilizzo di molte tecniche atte ad investigare l’attività ionoforica su liposomi come modelli di membrana. In particolare, è stato approfondita la conoscenza di tecniche comprendenti l’uso di probe fluorescenti che hanno mostrato di essere valide e di facile utilizzo per ottenere dati riguardanti vari aspetti dell’attività ionoforica come ad esempio le sequenze di selettività. Tuttavia, utilizzando esperimenti su liposomi, rimane molto difficile ottenere l’evidenza sperimentale della distinzione tra un meccanismo di trasporto tramite canale e altri tipi di meccanismi (es. carrier). Per questo motivo i dati ottenuti da questo tipo di esperimenti dovrebbero essere integrati da dati ottenuti per via elettrofisiologica e il nostro gruppo di ricerca si sta muovendo proprio in questa direzione.Taking inspiration from the natural ionophores amphotericin B and squalamine, during the past years our group synthesized a series of compounds that incorporate the fundamental structural elements of these natural ion transporters: a rigid hydrophobic unit, a hydrophilic chain (conduction module) and one or two polar head groups. The ionophoric activity of these artificial ionophores was investigated using liposomes as membrane models, and the measurements have been conducted by NMR spectroscopy and spectrofluorimetric techniques involving the use of fluorimetric probes. Among the compounds synthesized, an analogue having an N-terminus Z-protected α-aminoisobutyric acid (Aib) octapeptide (hydrophobic unit) linked, at the C-terminus, to one aminic moiety of a diamine derived by exa(ethylene)glycol (conduction module and polar head), was found to have an impressive activity in Na+ transport. The understanding of the reasons for such high and unexpected activity was therefore one of the main subjects of this Thesis work in which alkyl (instead of the Z-group) analogues of this compound, already prepared in our group, were studied to evaluate the hydrophobic effect of the substituent. After a first collection of data, a new series of analogues carrying a different aromatic group in place of the Z-group were synthesized and studied in order to asses the relevance of the aromatic substitution in the model ionophore. The second and more general subject of this Thesis work was the beginning of an investigation of structurally different compounds aimed to define common structural requirements for ionophoric activity. In this contest we have collaborated with several research groups which have synthesized the putative ionophore. Prof. De Riccardis and co-workers at Salerno University synthesized a series of spermidine calixarenic compounds as well as a series of cyclic α-peptoids formed by a variable number of N-benzyloxyethylglycines. The group of Prof. Montesarchio at Napoli University prepared novel cyclic oligosaccharide analogues, 4,6-linked through phosphodiester bonds, named CyPLOS (Cyclic Phosphate-Linked OligoSaccharide macrocycles). Finally the group of Prof. Casnati at Parma University synthesized a series of guanidinium calixarene conjugates. All these compounds have been prepared in the framework of a long standing collaboration which has as ultimate objective a broad structure/activity investigation aimed to define the structural requirements for ionophoric activity. Beside the results obtained in the study of each single class of compounds which will be illustrated and discussed in detail in the third chapter of this Thesis work, the possibility to investigate the ionophoric properties of such a broad bunch of compounds under the same set of experimental condition allowed us to draw more general considerations and put the basis for the design of new ionophores with enhanced activity and/or selectivity. A further important result of this Thesis work is the acquisition of expertise in several techniques used for the study of the ionophoric activity in liposomes. In particular we have exploited methodologies based on the use of fluorescent probes which have proved to give valid and user friendly entries to important features such as for example selectivity sequences. Ironically, it is the experimental evidence for the existence of ion channels as such (rather than the presence of alternative modes of transport) that remains most difficult to obtain in vesicles. For this reason this type of measurement should be paralleled with electrophysiological data and we are now moving in this direction.198

Bioaccumulation of polycyclic aromatic hydrocarbons and survival of earthworms (Eisenia andrei) exposed to biochar amended soils

Biochar has a charcoal polycyclic aromatic structure which allows its long half-life in soil, making it an ideal tool for C sequestration and for adsorption of organic pollutants, but at the same time raises concerns about possible adverse impacts on soil biota. Two biochars were tested under laboratory-controlled conditions on Eisenia andrei earthworms: a biochar produced at low temperature from wine tree cuttings (WTB) and a commercial low tar hardwood lump charcoal (HLB). The avoidance test (48-h exposure) showed that earthworms avoid biochar-treated soil with rates higher than 16 t ha 121 for HLB and 64 t ha 121 for WTB. After 42 days, toxic effects on earthworms were observed even at application rates (100 t ha 121) that are generally considered beneficial for most crops. The concentration of HLB and WTB required to kill half of earthworms\u2019 population (LC50; 95 % confidence limits) in the synthetic OECD soil was 338 and 580 t ha 121, respectively. Accumulation of polycyclic aromatic hydrocarbons (PAH) in earthworms exposed to the two biochar types at 100 t ha 121 was tested in two soils of different texture. In biochar-treated soils, the average earthworm survival rates were about 64 % in the sandy and 78 % clay-loam soils. PAH accumulation was larger in the sandy soil and largest in soils amended with HLB. PAH with less than four rings were preferentially scavenged from the soil by biochars, and this behaviour may mask that of the more dangerous components (i.e. four to five rings), which are preferentially accumulated. Earthworms can accumulate PAH as a consequence of exposure to biochar-treated soils and transfer them along the food chain. Soil type and biochar quality are both relevant in determining PAH transfer

assessment and comparison of multi annual size profiles of particulate matter monitored at an urban industrial site by an optical particle counter by a chemometric approach

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Seasonal variations of PAHs content and distribution patterns in a mixed land use area: A case study in NE Italy with the transplanted lichen Pseudevernia furfuracea

The seasonal differences of the polycyclic aromatic hydrocarbons (PAHs) content in lichen transplants were investigated in an area of ca. 40 square kilometers in NE Italy characterized by different land use. Two sets of samples of the epiphytic lichen Pseudevernia furfuracea were collected in a pristine area of the Carnic Alps and transplanted to 40 exposure sites for a period of two months, respectively in late winter and in summer. Results revealed a pronounced difference between the two seasons in terms of PAH content and distribution patterns. After the summer exposure the PAH concentrations in the transplants were more than one order of magnitude lower than after the winter exposure (ranging from 48.22 to 272.73 ng g 121 dw and from 289.73 to 1575.85 ng g 121 dw in the summer and winter samples respectively). Also the main emission sources changed, mostly due to the drastic reduction in the emissions by wood burning for domestic heating and to the different meteorological conditions. In summer PAHs degradation was enhanced by intense UV radiation, high temperatures, and presence of ozone. The implications of these findings for the biomonitoring of PAHs pollution are addressed

SARS-CoV-2 RNA Recovery from Air Sampled on Quartz Fiber Filters: A Matter of Sample Preservation?

The airborne route of transmission of SARS-CoV-2 was confirmed by the World Health Organization in April 2021. There is an urge to establish standardized protocols for assessing the concentration of SARS-CoV-2 RNA in air samples to support risk assessment, especially in indoor environments. Debates on the airborne transmission route of SARS-CoV-2 have been complicated because, among the studies testing the presence of the virus in the air, the percentage of positive samples has often been very low. In the present study, we report preliminary results on a study for the evaluation of parameters that can influence SARS-CoV-2 RNA recovery from quartz fiber filters spotted either by standard single-stranded SARS-CoV-2 RNA or by inactivated SARS-CoV-2 virions. The analytes were spiked on filters and underwent an active or passive sampling; then, they were preserved at −80 °C for different numbers of days (0 to 54) before extraction and analysis. We found a mean recovery of 2.43%, except for the sample not preserved (0 days) that showed a recovery of 13.51%. We found a relationship between the number of days and the recovery percentage. The results presented show a possible issue that relates to the quartz matrix and SARS-CoV-2 RNA recovery. The results are in accordance with the already published studies that described similar methods for SARS-CoV-2 RNA field sampling and that reported non-detectable concentrations of RNA. These outcomes could be false negatives due to sample preservation conditions. Thus, until further investigation, we suggest, as possible alternatives, to keep the filters: (i) in a sealed container for preservation at 4 °C; and (ii) in a viral transport medium for preservation at a temperature below 0 °C.This research was funded by University of Trieste Atheneum Fund for scientific research (2021) and IRCCS Burlo Garofolo (RC47/20)

SARS-CoV-2 RNA Recovery from Air Sampled on Quartz Fiber Filters: A Matter of Sample Preservation?

The airborne route of transmission of SARS-CoV-2 was confirmed by the World Health Organization in April 2021. There is an urge to establish standardized protocols for assessing the concentration of SARS-CoV-2 RNA in air samples to support risk assessment, especially in indoor environments. Debates on the airborne transmission route of SARS-CoV-2 have been complicated because, among the studies testing the presence of the virus in the air, the percentage of positive samples has often been very low. In the present study, we report preliminary results on a study for the evaluation of parameters that can influence SARS-CoV-2 RNA recovery from quartz fiber filters spotted either by standard single-stranded SARS-CoV-2 RNA or by inactivated SARS-CoV-2 virions. The analytes were spiked on filters and underwent an active or passive sampling; then, they were preserved at −80 °C for different numbers of days (0 to 54) before extraction and analysis. We found a mean recovery of 2.43%, except for the sample not preserved (0 days) that showed a recovery of 13.51%. We found a relationship between the number of days and the recovery percentage. The results presented show a possible issue that relates to the quartz matrix and SARS-CoV-2 RNA recovery. The results are in accordance with the already published studies that described similar methods for SARS-CoV-2 RNA field sampling and that reported non-detectable concentrations of RNA. These outcomes could be false negatives due to sample preservation conditions. Thus, until further investigation, we suggest, as possible alternatives, to keep the filters: (i) in a sealed container for preservation at 4 °C; and (ii) in a viral transport medium for preservation at a temperature below 0 °C

Sustainable domestic burning of residual biomasses from the Friuli Venezia Giulia region

The implementation of short supply chain of available biomass from forest and agriculture residues locally available aims to gain environmental benefits deriving from reduced transportation, avoided abiotic depletion of resources and reduced net emissions of green-house gases. The main objective of the present study was to analyze possible environmental impacts arising from domestic burning of residual biomasses, in terms air pollution. Three different types of woody residual biomasses (Robinia pseudoacacia, Picea abies and Vitis vinifera) were collected within the Friuli Venezia Giulia region in the form of wood chips, converted into pellets and used in controlled combustion tests in a domestic stove. The efficiency of the combustion process and the amount of selected emitted pollutants (carbon monoxide, organic gaseous carbon, nitrogen oxides, benzene, polycyclic aromatic hydrocarbons and total suspended particulate matter) were evaluated. Moreover, the same analysis and combustion tests were carried out on three commercially available pellets (Abies alba, Fagus sylvatica and softwood mix) to achieve a better interpretation and contextualization of the results obtained. Picea abies developed significantly high concentrations of organic gaseous carbon, benzene and polycyclic aromatic hydrocarbons and rather high carbon monoxide emissions. Vitis vinifera was characterized by the highest total suspended particulate matter and carbon monoxide emissions. Robinia pseudoacacia was a very promising fuel showing both combustion and emission properties similar to the commercial pellets. Thus, with the outcomes of our work we highlighted that possible environmental drawbacks due to air pollution arising from residual biomass burning should be evaluated. Moreover, the scientific community of life cycle assessment scientists may benefit from having updated experimental measurements of air pollutants emitted by a domestic stove loaded with different locally available residual biomasses

Advances in TD/GC-MS untargeted compound analysis on particulate matter beyond PAHs routine monitoring

The monitoring of some micro-pollutants is routinary because of their potential toxic activity and of normative regulation, but focusing only on some targeted compounds can divert analysts and researchers from identifying pollution macro-events associated to untargeted compounds. Routine analysis of micro-pollutants, as PAHs, in fast scan GC-MS produces Single Ion Monitoring (SIM) chromatograms used for quantitative determination of target compounds but also a Total Ion Current (TIC) signal which can potentially contain interesting unrevealed information. This is specially true when thermal desorption from solid samples as air particulate matter filters is used as pretreatment method; TD/GC-MS transfers analytes to the separation column without discarding some/part of the compounds. The aim of this study is to report the setup of an experimental and data analysis procedure adequate for disclosing interesting data from raw GCMS data collections acquired during routine monitoring. In this communication we present a 3 month daily PAHs routine monitoring near an incinerator in a Friuli Venezia Giulia chair production district in which we collected 120 samples of PM10 by quartz filter sampling accordingly to EN12341. A series of raw data (retention time, peak area, peak height, peak width, MW, CAS number) were acquired directly from the data analysis software (meanwhile quantifying PAHs) using the \u201cautointegration\u201d and \u201cMS library search\u201d functions. These data were handled within R free statistical computing environment (http://cran.rproject. org), using a home-made script to filter and intercomparing chromatograms by peak properties. In this way we were able, starting from over 5000 peaks to discriminate, beyond PAHs, 21 relevant peaks present in a great number of samples. Using MS library search reports we were able to identify three molecules with high correspondence to the MS database, which can be related to activities in the sampling site: a plasticizer, a phtalate and an erbicide. These compounds were quantified using corresponding commercial standards