Nicola Landi

Seven-Tesla micro-MRI in early detection of acute arterial ischaemia: evolution of findings in an in vivo rat model

Purpose. Acute mesenteric ischaemia (AMI) is a lifethreatening vascular emergency with a high mortality rate. Early diagnosis is the key to reducing its mortality rate and improving the quality of life. Although computed tomography (CT) is still the gold standard for acute intestinal disorders, over the last few years, magnetic resonance imaging (MRI) has become a useful alternative tool. An animal model of AMI was developed in order to study the effectiveness of MRI in early detection of this condition and to observe lesion evolution. Methods. Thirty Sprague Dawley rats were randomly divided into two groups (n=15): in the fi rst group, after laparotomy, the animals underwent ligation of the superior mesenteric artery (SMA), followed by macroscopic monitoring and histological evaluation; in the second, ischaemia was induced by squeezing a loop around the SMA 3 days before evaluation with 7-T micro-MRI. Results. Macroscopically, a refl ex spastic ileus followed by refl ex hypotonic ileus and colour changes in some of the loops were detected. MRI evidenced luminal dilatation with air‐fl uid levels, free intraperitoneal fl uid and bowelwall oedema. Histological analysis confi rmed ischaemia and earlier damage involving the central portion of thePurposAcute mesenteric ischaemia (AMI) is a life-threatening vascular emergency with a high mortality rate. Early diagnosis is the key to reducing its mortality rate and improving the quality of life. Although computed tomography (CT) is still the gold standard for acute intestinal disorders, over the last few years, magnetic resonance imaging (MRI) has become a useful alternative tool. An animal model of AMI was developed in order to study the effectiveness of MRI in early detection of this condition and to observe lesion evolution.MethodsThirty Sprague Dawley rats were randomly divided into two groups (n=15): in the first group, after laparotomy, the animals underwent ligation of the superior mesenteric artery (SMA), followed by macroscopic monitoring and histological evaluation; in the second, ischaemia was induced by squeezing a loop around the SMA 3 days before evaluation with 7-T micro-MRI.ResultsMacroscopically, a refl ex spastic ileus followed by refl ex hypotonic ileus and colour changes in some of the loops were detected. MRI evidenced luminal dilatation with air-fluid levels, free intraperitoneal fluid and bowelwall oedema. Histological analysis confirmed ischaemia and earlier damage involving the central portion of the ileum.ConclusionsThis model shows the correct sequence of events during arterial AMI and demonstrates that MRI can be recommended for early diagnosis of these lesions.RiassuntoObiettivoL’ischemia mesenterica acuta (IMA) rappresenta un’emergenza vascolare ad elevata mortalità. La diagnosi precoce è imprescindibile per migliorare sopravvivenza e qualità di vita dei pazienti. Sebbene attualmente la tomografia computerizzata (TC) rimane la metodica di scelta nelle patologie intestinali acute, negli ultimi anni la risonanza magnetica (RM) si è proposta come valida alternativa. Per valutare l’efficacia della RM nella diagnosi precoce di IMA ed investigare l’evoluzione delle lesioni è stato utilizzato un modello animale di ischemia mesenterica.Materiali e metodiTrenta ratti Sprague-Dawley sono stati divisi casualmente in 2 gruppi (n=15): nel primo, dopo legatura dell’arteria mesenterica superiore (AMS) in laparotomia, le lesioni sono state monitorate macroscopicamente e istopatologicamente; nel secondo, grazie ad un cappio posizionato all’emergenza dell’AMS tre giorni prima, è stata indotta l’ischemia e valutata con micro-RM a 7 T.RisultatiMacroscopicamente si è osservato un ileo rifl esso spastico, transitato poi in ipotono, ed il viraggio cromatico di alcune anse. La RM ha documentato la dilatazione luminale con livelli idro-aerei, liquido libero intraperitoneale ed edema parietale. L’analisi istopatologica ha confermato l’ischemia con danno più precoce a carico dell’ileo centrale.ConclusioniIl modello documenta la corretta sequenza degli eventi dell’ischemia mesenterica arteriosa acuta (IMAA) e dimostra che la RM può essere proposta per la sua diagnosi precoce.

7T μMRI of mesenteric venous ischemia in a rat model: Timing of the appearance of findings

OBJECTIVES The aim of this study is to analyze the chronological development of macroscopic, microscopic and magnetic resonance imaging (MRI) findings in a rat model of Superior Mesenteric Venous (SMV) ligation, and to evaluate the role of MRI in the diagnosis of mesenteric venous thrombosis. METHODS Thirty adult Sprague-Dawley rats were used and divided in two different groups that underwent a different surgical model and a different monitoring of ischemic damage. Group I underwent macroscopical and histological observation; Group II underwent 7T μMRI evaluation and histological analysis. RESULTS The first alterations occurred 30 min after SMV ligation and progressively worsened until the eighth hour. The morphological and MRI findings showed the same course. CONCLUSIONS This study provides a systematic evaluation of early anatomopathological and MRI findings following the SMV ligation. MRI allows to identify the early pathological findings of venous mesenteric ischemia and allows to correlate those to the histopathological features. Our data suggest a relevant role of MRI in the diagnostic management of mesenteric venous thrombosis, allowing to non-invasively identify and characterize the histopathologic findings. So, thanks to these skills, its future application in early diagnosis of human mesenteric venous ischemia is supposable.

A new active antimicrobial peptide from PD-L4, a type 1 ribosome inactivating protein of Phytolacca dioica L.: A new function of RIPs for plant defence?

We investigated the antimicrobial activity of PD‐L4, a type 1 RIP fromPhytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440–65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full‐length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha‐helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440–65 is the first antimicrobial peptide identified in a type 1 RIP.

Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from Phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.

Immobilised peroxidases from Asparagus acutifolius L. seeds for olive mill waste water treatment

This work compares the main enzymatic parameters of a cationic peroxidase (AaP-1-4), purified and characterized from Asparagus acutifolius L. seeds (Mol. Biotechnol., 2014, 56, 738–746) and its immobilised form (Eup-AaP-1-4), on Eupergit® CM with Horseradish peroxidase. The optimum in the pH-activity profile was pH 4.0 and pH 3.0 for AaP-1-4 and Eup-AaP-1-4, respectively. Ca2+ cation enhanced both enzymatic activities, however, when submitted to a temperature stress (120 min at 50 °C) Eup-AaP-1-4 lost only 20% activity while AaP-1-4 70%. Furthermore, AaP-1-4 was proved to be able to remove (poly)phenols in olive mill waste water (OMW), with hydrogen peroxide electron donor. The Eup-AaP-1-4 kinetic proprieties were investigated and the operational stability evaluated in a continuous stirred membrane bioreactor. AaP-1-4 appears to be a novel non-expensive source of peroxidases suitable for biotechnological applications in the environmental field for the removal of aqueous (poly)phenols produced from several industrial processes.

Pioppino mushroom in southern Italy: an undervalued source of nutrients and bioactive compounds

BACKGROUND Agrocybe aegerita (V. Brig.) Singer, commonly known as Pioppino, is a popular edible mushroom, known in the Campania Region (Italy). Despite its habitual consumption, little nutritional and biochemical information is available. Thus, nutritional values, anti-radical properties and chemical composition of the wild Pioppino were compared to those of the cultivated Agaricus bisporus (J.E. Lange) Imbach (known as Champignon), equally analysed. RESULTS Macronutrient components (proteins, carbohydrates and lipids), free and protein amino acids and fatty acid content of poplar mushroom were achieved. Total phenol content of a defatted Pioppino alcoholic extract (PM) was determined, whereas DPPH and ABTS methods were applied to determine the radical scavenging capabilities of the extract. Ferricyanide and ORAC-fluorescein methods were also performed. Finally, LC-HRMS was used to identify and quantify the main metabolites in the extract. PM was mainly constituted of disaccharides, hexitol derivatives and malic acid. Coumaric acid isomers and C6 C1 compounds were also detected. CONCLUSION All data revealed that wild Pioppino is an excellent functional food, by far exceeding that of the Champignon. Therefore, these data are useful to promote the consumption of this mushroom encouraging thus its biological cultivation, due to wild availability is strongly compromised by the extensive use of fungicides.

Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the Basidiomycete mushroom Agrocybe aegerita.

BACKGROUND Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants. RESULTS The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway. CONCLUSIONS Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed. GENERAL SIGNIFICANCE Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita. Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies.

Nutritional profiling of Eurasian woodcock meat: chemical composition and myoglobin characterization: Nutritional profiling of Eurasian woodcock meat

BACKGROUND Meat from birds is a rich source of proteins for the human diet. In this framework, Eurasian woodcock (Scolopax rusticola L.), a medium-small wading bird hunted as game in many Eurasian countries, is considered one of the best meats for culinary purposes. Since the nutritional composition of Eurasian woodcock meat has not yet been reported, we decided to determine the nutritional profile of S. rusticola meat. RESULTS Macronutrient components (proteins, lipids and fatty acids) were determined, as well as free and total amino acids, and compared with those of the common pheasant. Eurasian woodcock meat contains high levels of proteins and essential amino acids. The levels of unsaturated fatty acids represent a great contribution to the total lipid amount. Among polyunsaturated fatty acids, linoleic acid (C18:2, n-6) is the major essential fatty acid. Finally, we report the characterization of myoglobin (Mb) from Eurasian woodcock. CONCLUSION The data revealed that meat from this bird could be a good source of quality raw proteins because of its amino acid composition, and it had a low lipid content. On the other hand, Mb characterization might be of benefit to the meat industry, by providing useful information for the determination of species-specific differences in meat from birds.

Structural and enzymatic properties of Ageritin, a novel metal-dependent ribotoxin-like protein with antitumor activity

Ageritin has been recently described as the first ribotoxin-like from Basidiomycota division (mushroom Agrocybe aegerita) with known antitumor activity (BBA 2017, 1861: 1113-1121). By investigating structural, catalytic and binding properties, we demonstrate that Ageritin is a unique ribotoxin-like protein. Indeed, typical of the ribotoxin family, it shows the specific ribonucleolytic activity against the ribosomal Sarcin-Ricin Loop in a rabbit reticulocytes assay. However, it displays several elements of novelty, as this activity is strongly metal-dependent and completely suppressed in the presence of EDTA, different from other representative members of the ribotoxin family. Consistently, we prove that Ageritin is able to bind magnesium ions with low micromolar affinity. We also show that Ageritin is significantly more stable than other ribotoxins in thermal and chemical denaturation experiments. These peculiar features make Ageritin the prototype of a new ribotoxin-like family present in basidiomycetes. Finally, given its high stability, this enzyme is a promising candidate as a new tool in immunoconjugates and nanoconstructs.

Free amino acid profile of Bubalus bubalis L. meat from the Campania region

In this study, we determined the amount of carnosine and anserine in water buffalo meat without hanging treatment and the free amino acid profile by using amino acid analyser with post-column ninhydrin derivatization procedure. The main free amino acids present in samples were glutamic acid (>60 mg/100 g), followed by alanine, glycine, and arginine. Other protein amino acids were detected in minor amounts (less than 2 mg/100 g). Among the non-protein amine-containing compounds, taurine and urea were the most abundant. The analysis showed that 50% of the total free amino acids was represented by dipeptides carnosine (average ~130.3 mg/100 g) and anserine (average ~17.9 mg/100 g). Thus, this study for the first time reports the free amino acids profile of water buffalo meat and the content of carnosine and anserine, potentially involved in the darkening meat process and their ratio, that could be used to estimate the water buffalo meat portion in mixed meat products.