ANALISI FUNZIONALE DI GENI REGOLATORI DELLO SVILUPPO EMBRIONALE DI PARACENTROTUS LIVIDUS

Parole chiave: TRIM, E3-ubiquitina ligasi, ectoderma orale, scheletogenesi, riccio di mare. La recente acquisizione di consistenti dati genomici ha confermato che organismi bilateri, semplici come i nematodi o complessi come l’uomo, fanno uso degli stessi strumenti di base, quali fattori di trascrizione e molecole segnale, per decodificare le informazioni necessarie allo sviluppo embrionale. Nel contesto di tali regolatori, la famiglia di proteine TRIM/RBCC (Tripartite motif/RING-Bbox-Coiled coil) rappresenta una delle principali classi di E3 ubiquitina ligasi putative, che svolgono ruoli essenziali nella regolazione di processi quali ciclo cellulare e sviluppo embrionale. Nel genoma di riccio di mare esistono copie multiple di geni contenenti trim box. In particolare, in Paracentrotus lividus, è stato identificato un locus che include due copie, probabilmente originatesi da un evento di duplicazione, di un gene codificante per il fattore con motivo TRIM, denominato strim1 (sea urchin trim1). Entrambi i geni consistono di un singolo esone e mostrano un’elevata identità di sequenza. Con particolare interesse è stata individuata, a livello del promotore putativo di entrambe le copie di strim1, la presenza di regioni di sequenza con elevata identità a segmenti dell’elemento trasponibile Helitron-N2. Ciò potrebbe suggerire meccanismi complessi di evoluzione della famiglia multigenica in esame. Durante l’embriogenesi, la trascrizione zigotica di strim1 ha inizio a stadi precoci e raggiunge il picco massimo allo stadio di gastrula. I livelli di trascritto si riducono, poi, progressivamente, e allo stadio di pluteo risultano difficilmente rilevabili. Inoltre, esperimenti di ibridazione in situ su embrioni interi hanno mostrato che i trascritti strim1 sono caratterizzati da una localizzazione asimmetrica nell’embrione, ristretta esclusivamente all’ectoderma orale. In particolare, allo stadio di gastrula si riscontra localizzazione ectodermica in corrispondenza dei siti di localizzazione ventro-laterale dei clusters di cellule del mesenchima primario, coinvolte nel processo di scheletogenesi. L’over-espressione di strim1 determina anomalie scheletriche, mentre la perdita di funzione, indotta mediante l’iniezione in zigoti di oligonucleoditi morfolino antisenso, altera il posizionamento delle PMCs e blocca la scheletogenesi. Tale processo risulta ripristinato da una sorgente clonale di strim1 in embrioni morfanti. Interessanti risultati mostrano che Otp e il fattore trascrizionale Pax2/5/8 costituiscono bersagli molecolari della segnalazione Strim1 dipendente in cellule ectodermiche. Inoltre, l’iniezione in zigoti dei relativi trascritti esogeni consente il recupero del corretto programma scheletogenico, bloccato dalla perdita di funzione di strim1. Ulteriori evidenze, suggeriscono, infine, che fgfA è parte del medesimo network di segnalazione di Strim1. strim1 svolge, quindi, un ruolo cruciale nel controllo del differenziamento e del posizionamento delle PMCs nell’ambito della morfogenesi dello scheletro embrionale

Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-specific, TRIM genes as well as multiple copies of known ones. We also retrieved a landscape of cDNA sequences from staged EST libraries, indicating that most of these genes are actively transcribed during development. Phylogenetic analysis of the deduced proteins, using set of TRIMs from various species, revealed a degree of genetic variation between species. Worth of mention, we predicted the occurrence of transposition events involving some of these genes, according with the documented rapid evolution of this family. Next, we adopted heuristic algorithms and post-processing steps to investigate the evolutionarily distant Paracentrotus lividus, Allocentrotus fragilis and Lytechinus variegatus genomes, whose sequencing projects are actually in progress. We assembled partial pools of TRIM genes and specifically associated them to EST-derived cDNA sequences. Such a collection of data should provide a framework for unravel gene regulatory networks involving TRIM genes from an evolutionary perspective. Indeed, in the Pl species, we have previously isolated and functionally characterized the cDNA sequence encoding the first echinoderm TRIM factor, Strim1. Here, we identified five strim1 genes, all sharing a intronless organization, and roughly located their cis-regulatory apparatus

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis

Yeast ecology of vineyards within Marsala wine area (western Sicily) in two consecutive vintages and selection of autochthonous Saccharomyces cerevisiae strains

In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards showed some differences in species composition and concentration levels among 2008 and 2009 vintages. Due to the enological relevance, all Saccharomyces cerevisiae isolates were differentiated applying two genotypic tools (interdelta analysis and microsatellite multiplex PCR of polymorphic microsatellite loci) that recognized 51 strains. Based on the low production of H2S, acetic acid and foam, ethanol resistance, growth in presence of high concentrations of potassium metabisulphite (KMBS) and CuSO4 and at low temperatures, 14 strains were selected and used as starter to ferment grape must at 13 C and 17 C in presence of 100 mg/L of KMBS. Three strains (CS160, CS165 and CS182) showed optimal technological aptitudes

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: application on a traditional raw ewes’ milk cheese.

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes’ milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the nonstarter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5- month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes

Development of new non-dairy beverages from Mediterranean fruit juices fermented with water kefir microorganisms

The aim of this work was to explore the use of several Mediterranean fruit juices as fermentable substrates to develop new non-dairy fermented beverages. Microbiological, chemical and sensory features of kefir-like beverages obtained after the fermentation of juices extracted from fruits cultivated in Sicily (southern Italy) with water kefir microorganisms were investigated. Results indicated that both lactic acid bacteria and yeasts were able to develop in the fruit juices tested, but the highest levels were registered with prickly pear fruit juice. All fruit juices underwent a lactic fermentation, since a lactic acid content was detected in the resulting kefir-like beverages. Except kiwifruit and quince based kefirs, total titratable acidity increased for the other experimental products. A general decrease of the soluble solid content and an increase of the number of volatile organic compounds (VOCs) was also observed after fermentation. As expected, the fermentation increased the concentration of alcohols. The main fermentation in KLBs resulted to be yeast-based. Kiwifruit and pomegranate juices possessed a high antioxidant activity. Esters compounds were present at higher amount after the fermentation, especially in grape, pomegranate and quince. Aldehydes showed an opposite trend. Changes in colour attributes were registered as noticeable at human perception scale. The overall quality evaluation indicated that, among the Mediterranean fruit juices tested, apple and grape beverages were the products mostly appreciated by the tasters. Therefore, these findings support the possibility to develop fruit-based kefirlike beverages with high added value and functional properties

A survey of the main technology, biochemical and microbiological features influencing the concentration of biogenic amines of twenty Apulian and Sicilian (Southern Italy) cheeses

Abstract Twenty Apulian and Sicilian cheeses were analysed for their concentrations of eight biogenic amines (BAs), free amino acids, pH, water activity, and subjected to microbiological characterisation. In addition, lactic acid bacteria isolated from cheeses were assayed for their capacity to generate BAs. Principal component analysis was performed to find the effect of different parameters on the distribution of the cheeses. Although short-ripened (≤30 d) cheeses did not show significant BA concentrations, the only BA showing high positive correlation with time of ripening was histamine. Concentration of histidine and, especially, percentage of histidine-decarboxylase bacteria presumably affected histamine concentration. High pH values were negatively correlated to the concentration of tyramine, putrescine, and cadaverine. Fifty percent of the cheeses contained at least one BA at potentially toxic concentrations. Unambiguous and ever-valid relations among parameters and BAs are difficult to determine, because BAs are the result of combined and varied factors

Identification, typing, and investigation of the dairy characteristics of lactic acid bacteria isolated from 'Vastedda della valle del Belìce' cheese

Traditional cheeses made without starter cultures can be characterised by the attribute of instability. The addition of autochthonous starter cultures can ensure stability without compromising the characteristics of the final product. This study aimed to characterise the autochthonous lactic acid bacteria (LAB) population in “Vastedda della valle del Belìce” cheeses, which have a protected designation of origin (PDO) status, in order to develop an ad hoc starter culture to be used in its future production. Winter and spring productions were analysed to ensure isolation of specific LAB that had adapted to perform fermentation at low temperatures. Plate counts revealed total microbial numbers nearing 109 CFU.g−1. All of the cheese samples were dominated by coccus-shaped LAB. When enterobacteria were present, their concentrations were at similar levels (3.3–5.6 Log CFU.g−1) in both seasons. All of the colonies that differed in morphological appearance were isolated and differentiated on the basis of phenotypic characteristics and genetic polymorphisms, as analysed by random amplification of polymorphic DNA-polymerase chain reaction. A total of 74 strains were identified and further genotyped by sequencing the 16S rRNA gene, resulting in the identification of 16 LAB species belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). The species most frequently present were Streptococcus gallolyticus subsp. macedonicus, Streptococcus thermophilus, Lactococcus lactis and Leuconostoc mesenteroides. The 74 strains were also investigated in vitro for general dairy parameters such as acidification capacity, diacetyl generation and antibacterial activity. Several strains of the most frequently represented species displayed traits relevant to the production of PDO “Vastedda della valle del Belìce”

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multistrain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality