Suppression of beta1,3galactosyltransferase beta3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly-N-acetyllactosamines and sialyl-Lewis x on O-glycans

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors

Effects of long term application of compost and poultry manure on soil quality of citrus orchards in Southern Italy.

A six-year study was carried out in an organically managed orange orchard located in Sicily (Southern Italy) to assess the effect of compost and organic fertilizers utilisation on soil quality. Adopting a randomized-block experimental design with three replicates, four treatments were carried out. In treatments 1 and 2, two different composts (C1 from distillery by products and C2 from livestock wastes) were applied. The plots of treatment 3 were fertilized using dried poultry manure. The control treatment was fertilized with mineral/synthetic fertilizers. In order to verify the hypothesis that composts and organic fertilizers improve soil fertility, soil quality was evaluated by selecting dynamic soil parameters, as indicators linked to C and N cycles. Total organic C, total N, C/N ratio, humified fraction, isoelectric focusing (IEF) of extracted organic matter, microbial biomass C, potentially mineralisable N under anaerobic conditions, potenzially mineralizable C, C mineralization quotient and metabolic quotient were determined for each sample. Furthermore, the Comunity level Physiological Profile (by Biolog tecnique) was defined, calculating derived functional biodiversity and versatility indexes. Parameters related to IEF and potentially mineralizable C showed significant differences among the treatments. Moreover, total C, total N and humification parameters tended to increase, while no differences were observed in biodiversity indexes. On these findings, it was concluded that composts and poultry manure only weakly affected soil properties, though they increased soil nutritive elements potentially available to crops

Expression of carbohydrate-antigen sialyl-Lewis a on colon cancer cells promotes xenograft growth and angiogenesis in nude mice

We investigated the role of carbohydrate antigen sialyl-Lewis a (sLea), an E-selectin ligand and epitope of tumor marker CA19.9, in the development of xenografts in nude mice. To this end, animals were inoculated with the human colon cancer cell line HCT-15, expressing no Lewis antigens, or with a clone expressing sLea (HCT-15-T5). The size of HCT-15-T5 xenografts appeared larger than those of HCT-15 and their average weight was over twice bigger. In both xenografts the mitotic index was found elevated, as determined by Ki-67 assay, and no apoptosis was detected in the tumor cells by both caspase 8 or TUNEL assays. Some apoptotic signals were instead detected in the vessels. Conversely, microvessel density, determined through CD-31 immunohistochemistry, was found 3.2-folds bigger in HCT-15-T5 xenografts (p < 0.012). Only the membranes of HCT-15-T5 cells grown as xenografts reacted intensively with the anti CA19.9 antibody 1116-NS-19-9 by immunofluorescence, but not by immunohistochemistry. Unknown structures were instead stained by such technique in both xenografts, as were in mouse tissues not expressing the antigen and in human colon adenocarcinoma. We conclude that expression of sLea on the surface of colon cancer cells improves xenograft growth and is associated with enhanced angiogenesis, while immunohistochemistry with 1116-NS-19-9 antibody appears not suitable to determine CA19.9 expression

Anthropic pressures on Nature 2000 Sites: recommendations and monitoring criteria for the pollution emergency response activities within the Orbetello lagoon

L’elevato valore naturalistico e socioeconomico di un Sito Natura 2000 richiede che tutte le attività antropiche che si svolgono al suo interno (es. pesca, turismo, trasporti, attività industriali, etc.) siano gestite in maniera tale da non pregiudicare le specie e gli habitat per i quali l’area è stata designata. Molti di questi Siti sono ambienti di transizione, ovvero zone che costituiscono il passaggio naturale tra terra e mare. La loro posizione di interfaccia tra questi due ambienti li rende ecosistemi unici e biologicamente molto produttivi, sede di meccanismi di regolazione dei processi interattivi della biosfera nelle due fasi, terrestre e marina. In alcuni Siti la presenza di attività antropiche diffuse e prolungate nel tempo ha portato al riscontro di stati di contaminazione elevata, fino all’inclusione di queste zone, o parti di esse, tra i Siti di bonifica di Interesse Nazionale (SIN). Il presente lavoro descrive le linee di indirizzo e le attività di monitoraggio da attuare per la salvaguardia della salute pubblica e dell’ambiente nel corso degli interventi di messa in sicurezza di emergenza predisposti nell’area lagunare antistante l’area industriale Ex Sitoco, all’interno della perimetrazione del SIN di Orbetello, incluso in un Sito di Importanza Comunitaria. Le matrici ambientali potenzialmente a rischio a causa dell’esecuzione di tali interventi sono: acqua, sedimento, biocenosi acquatiche, avifauna, uomo. È altresì importante valutare gli effetti che le ipotetiche modifiche su microscala, apportate a livello di ogni matrice, potrebbero causare nel lungo periodo su macroscala.The high naturalistic and socio-economic value of Natura 2000 sites requires that all human activities performed within their borders (e.g. fishing, tourism, transports, industrial activities) are regulated. Indeed, the site management should assure the effective safeguard of all species and habitats of European interest included in the protected area. A lot of such sites are located in transitional environments, that are areas characterised by a natural progression from the terrestrial to the water environments. Such environments include unique and very productive habitats, and they represent the regulation mechanisms of the interactive processes of the terrestrial and marine biosphere. In some sites, the presence of human activities that are distributed both in space and time has led to high levels of contamination, that in some cases even required their inclusion in Reclamation Sites of National Interest (SIN). The present study describes the planning and monitoring activities to be performed in order to safeguard human and environment health during the actions of MISE in the lagoonal area in front of the industrial area Ex Sitoco, within the borders of the Orbetello SIN, included in a SCI. The environmental parameters that are potentially at risk due to such activities are: water, sediment, water biocenosis, birds, humans. Furthermore, it is important to evaluate the effects that potential variations at the microscale level may cause at the macroscale level

Effect of Peat-Reduced and Peat-Free Substrates on Rosemary Growth

The objective of this work was to study the use of four composts, obtained by agro-industrial, urban and green wastes, as growing media components on Rosmarinus officinalis L. Substrates were obtained by mixing each compost with peat in different proportions. Main physical and chemical characteristics of prepared substrates have been compared and, at the end of growing cycle, the biometric survey on main growing parameters and plant nutritional status was performed. The obtained results showed that substrates with 30% compost have main physical and chemical parameters comparable with those of the control. Best quality plants have been obtained substituting peat with 30% of compost, except with the olive mill compost. At the end, the green pruning compost can be recommended as growing media component (up to 50%) for the growth of Rosmarinus officinalis L., being able to determine high quality plants, together with an implemented plant nutrient efficiency

Levers and Obstacles of Effective Research and Innovation for Organic Food and Farming in Italy

The objectives of this paper are to present the dynamic of organic food and farming (OFF) research and innovation, to outline challenges in deploying programs and accessing funding, and to define key actions to foster the development of tailored quality research on organic farming in Italy. The baseline starts from the main outcomes that emerged during the World Caf\ue9 held in the frame of the Salone Internazionale del biologico e del naturale (SANA Expo) in 2018, where the Italian OFF research community met to build a convergence on scope and modus operandi in the research endeavor. These outcomes were examined in the light of the key features of the research and innovation projects funded in Italy in the last 10 years, respectively by the Italian Ministry of Agriculture and the regional administrations through the innovation support instruments in the Rural Development Plan programming periods. In the period 2009\u20132018, 70 research projects for a total funding of 21.081 million \ubf (&lt;0.1% of the value of the sector) were launched, addressing nine dierent topic areas. Over a similar period (2007\u20132019), 53 regional innovation projects addressing organic farming were activated for a total budget of 14.299 million \ubf (&lt;10% of the entire available funding). The implementation of interventions in the research and the innovation areas were often scattered in terms of the topics, disciplines, and types of supply chain/network addressed. The relatively high share of multi/interdisciplinary research and innovation projects aswell as the acknowledgement of the multi-actor approach as a fundamental step toward co-research and co-innovation were upshots that emerged from our analysis. The outcomes of this study can be used by competent national and the regional authorities to design their future research and innovation policies and interventions

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in&nbsp;vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%-80% lower. In&nbsp;vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis

Realization of the farad from the dc quantum Hall effect with digitally-assisted impedance bridges

A new traceability chain for the derivation of the farad from dc quantum Hall effect has been implemented at INRIM. Main components of the chain are two new coaxial transformer bridges: a resistance ratio bridge, and a quadrature bridge, both operating at 1541 Hz. The bridges are energized and controlled with a polyphase direct-digital-synthesizer, which permits to achieve both main and auxiliary equilibria in an automated way; the bridges and do not include any variable inductive divider or variable impedance box. The relative uncertainty in the realization of the farad, at the level of 1000 pF, is estimated to be 64E-9. A first verification of the realization is given by a comparison with the maintained national capacitance standard, where an agreement between measurements within their relative combined uncertainty of 420E-9 is obtained.Comment: 15 pages, 11 figures, 3 table

Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥ 0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction