Analisi della variabilità genetica in una popolazione ovina di razza massese e studio di associazione con parametri di qualità del latte

RIASSUNTO Il presente lavoro ha come obiettivo la ricerca di associazioni tra marcatori microsatellite e la composizione chimica del latte. È stato condotto uno studio su 68 pecore di razza Massese provenienti da un allevamento toscano. A partire da latte fresco, sono stati valutati: composizione chimica standard, contenuto in caseina e frazioni caseiniche, proteine seriche, pH e parametri reologici. I capi sono stati sottoposti a genotipizzazione impiegando 17 marcatori microsatellite; sono, quindi, stati calcolati i valori di similarità genetica tra individui ed alcuni parametri genetici classici. Il numero medio di alleli per locus è risultato pari a 7,18 e l’eterozigosità osservata presentava variazioni tra 0,403 e 0,867 (media 0,677). La similarità genetica tra individui era 0,460. Cinque marcatori (BM8124, CSN3, BM1258, BMS468 e TGLA387) hanno mostrato una deviazione significativa dalle proporzioni di Hardy- Weinberg. Lo studio ha messo in evidenza alcuni microsatelliti con alleli significativamente associati a caratteri di composizione del latte (P<0,01). In particolare, la significatività più alta (P<0,001) è stata osservata per l’associazione dell’allele 2 del marcatore OIFNG con la a-lattoalbumina, dell’allele 10 del marcatore BL4 con il livello di immunoglobuline, dell’allele 2 del marcatore BMC1009 con il contenuto di grasso e dell’allele 9 del marcatore ILSTS42 con il parametro a30. Sono necessari ulteriori approfondimenti al fine di convalidare i risultati preliminari ottenuti nel presente lavoro e si rende necessaria l’estensione dello studio ad un numero maggiore di soggetti e la tipizzazione di un numero maggiore di loci, principalmente sui cromosomi 3 e 20 dove mappano i marcatori risultati più interessanti nella presente analisi. SUMMARY Current research aims to establish statistical associations between DNA microsatellites and milk chemical composition. Atrial was carried out on 68 Massese ewes reared in a farm of the Tuscany. The following parameters were evaluated on fresh milk: standard chemical composition, casein and its fractions, whey proteins, pH and rheological parameters. Animals were genotyped at 17 microsatellite loci. Genetic similarities among individuals and classical genetic parameters were evaluated. For each locus, average values of considered parameters were calculated in all the subjects carrying a given allele; these were compared statistically with the average values of subjects not carrying the allele, and the significance of the difference was estimated. The average number of alleles per locus resulted 7.18 and the observed heterozygosity ranged from 0.403 to 0.867 (0.677 medium value). The genetic similarity among individuals was 0.460. Five markers pointed out a significant deviation from the Hardy-Weinberg proportions (BM8124, CSN3, BM1258, BMS468 and TGLA387). The study revealed several microsatellites with alleles significantly linked to milk composition traits (P<0.01). In particular the highest significance (P<0.001) has been found for the allele 2 of OIFNG marker with a-lactoalbumin, for the allele 10 of the BL4 marker with immunoglobulins, for the allele 2 of BMC1009 with fat, and for allele 9 of ILSTS42 with a30. Further analyses are needed to validate these preliminary results, in particular increasing the number of subjects and of typed loci above all on the chromosomes 3 and 20 where the more interesting markers map

Liposomes as a potential ocular delivery system of distamycin A

Liposomes containing Distamycin A (DA) may be clinically useful in the treatment of ocular HSV infections, especially in acyclovir-resistant HSV keratitis. This study evaluated the in vitro and in vivo performance of a topical controlled release liposomal formulation containing DA (DA-Lipo) aimed at reducing the toxicity of the encapsulated active agent and improving drug uptake by ocular tissues. The bioavailability of DA in the tear fluid and the DA uptake into the cornea were increased after instillation of DA-Lipo in rabbits, reaching the DA corneal concentration corresponding to IC50 values against HSV without any sign of transcorneal permeation of drug. DA-Lipo was definitely less cytotoxic then plain DA in rabbit corneal epithelial cells. These results provide new insights into the correlation between the in vitro data and the drug kinetics following ocular applications of liposomal vesicle

Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells

Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells

Fundamentals of neurogastroenterology: Basic science

This review examines the fundamentals of neurogastroenterology that may underlie the pathophysiology of functional GI disorders (FGIDs). It was prepared by an invited committee of international experts and represents an abbreviated version of their consensus document that will be published in its entirety in the forthcoming book and online version entitled Rome IV. It emphasizes recent advances in our understanding of the enteric nervous system, sensory physiology underlying pain, and stress signaling pathways. There is also a focus on neuroimmmune signaling and intestinal barrier function, given the recent evidence implicating the microbiome, diet, and mucosal immune activation in FGIDs. Together, these advances provide a host of exciting new targets to identify and treat FGIDs, and new areas for future research into their pathophysiology

The Silent Epidemic of Diabetic Ketoacidosis at Diagnosis of Type 1 Diabetes in Children and Adolescents in Italy During the COVID-19 Pandemic in 2020

To compare the frequency of diabetic ketoacidosis (DKA) at diagnosis of type 1 diabetes in Italy during the COVID-19 pandemic in 2020 with the frequency of DKA during 2017-2019

“Modulazione dell'espressione genica in cellule linfoidi ed epitelioidi infettate con il Virus Erpetico Umano 8 (HHV8) ed analisi dell’attività chemioterapica/antivirale della Distamicina A (DA)”

Purpose of this PhD study have been the analysis of the modulation of cellular gene by Human Herpes virus 8 (HHV8) infection and of the potential antiviral effect of Distamycin A (DA), an oligopeptidic antibiotic. This study describes a comprehensive picture of global gene changes soon after HHV-8 active infection of two susceptible cell types. We found that host cell expression changes after only 24 hours of infection, and it is quite different from previous gene induction studies that were carried out at later time points of virus infection. In fact, Poole et al. have used a different primary effusion lymphoma cell line (JSC1) to produce the virus that was used to infect primary endothelial cells for a period of 3–5 weeks, and gene array experiments were done when almost all of the cells changed from typical cobblestone to spindle-shaped morphology and were positive for LANA. Several features were apparent from these analysis: a) there was virtually non-overlapping list of genes in all of two viral subtypes analyzed; b) HHV-8-induced transcriptional profiles in the epithelial and PBMC were closely different. This is probably because the criteria of > 4-fold gene induction as significant is a threshold filter very tight. Nevertheless, the genes that emerge from such comparison will be of greater interest for studying their role in the unique biology of HHV-8. Furthermore, the replicative capacity of the two different strains in the two cell types and the modulation of cell involved in the inflammatory process would seem to indicate that the genotypic difference could be translated into a different virus-host relationship, also attributable to a different pathogenetic mechanism. In addition to identifying specific host response genes within each functional pathway that are already known to be important for HHV-8 infection, our findings identify novel candidate genes that are not yet known in HHV-8 biology. Obtained results show a lack of cytotoxicity of DAlipo on cultured HEK293, RCE and PBMC cells. It might be noted that the CC50 value found in the present study falls at the low extreme of the range of CC50 values reported in the literature. In fact, available data show CC50 values ranging from 35 μM in a Hep-2 cell line to 154 μM in a HeLa cell line and 385 μM in rabbit primary kidney cells. It has been reported that DA inhibits the activity of all DNA polymerases. This mechanism of action could explain its antiviral activity. Because of the lack of information concerning the relevant genetic definition of HHV8 A1 and C3 subtypes from BCBL1 and BC3 cell lines, whether the observed behavior be the result of the expression of specific mutated genes or a non specific consequence of viral strain virulence has to be established. Clearly, further studies are needed to explain the different degree of sensitivity of HHV8 subtypes to DA and Dalipo. The significant antiviral activity of DAlipo and the lack of citotoxicity in epithelial and lymphoid cell lines shown in the present study indicates the compound as an antiherpetic drug potentially useful in both topic and systemic treatments. It could be used either alone or in association with other antiviral treatment, the latter to be employed at lower dosages and for shorter periods of time, thus limiting side effects. The antiviral activity of DAlipo formulation also in lymphoid cell lines may be due to the hydrophobic or amphiphilic drug penetration efficacy increased through inclusion of the drug in liposomes. DAlipo showed, when compared with the free drug, higher antiviral activity against HHV8 A1 and C3 subtypes in PBMCs and might therefore represent a promising DA formulation that could be used also for systemic treatments

Tolleranza all'insulto ischemico e invecchiamento mitocondriale: ruolo dei canali mito-KATP.

Le patologie cardiovascolari, in particolare l’infarto del miocardio, risultano incrementate con l’invecchiamento, in più si osserva una minore tolleranza all’insulto ischemico da parte di cuori senescenti, e una perdita di efficacia delle strategie cardioprotettive. Come conseguenza si osserva anche un’aumentata vulnerabilità cellulare, riconducibile a una disfunzione dei mitocondri. In più sappiamo che i canali mitocondriali del potassio ATP-sensibili (mito-KATP) svolgono un ruolo fondamentale nella cardioprotezione da danno ischemico, tanto che la ricerca si è indirizzata verso lo sviluppo di attivatori sempre più selettivi verso questo target. Recenti studi dimostrano che l’attivatore di riferimento dei canali mito-KATP, diazossido, perde la sua efficacia protettiva quando somministrato a cuori ottenuti da animali senescenti (Schulman et al., 2001), senza tuttavia fare alcuna speculazione sulle alterazioni fisiologiche responsabili di tale mancato effetto. Pertanto, lo scopo della mia ricerca, è stato quello di valutare gli effetti sui parametri funzionali di mitocondri cardiaci ottenuti da animali giovani e senescenti, da parte di due attivatori dei canali mito-KATP, diazossido e F163. Attraverso il primo protocollo sperimentale è stato valutato il potenziale di membrana mitocondriale (Δψm), in mitocondri isolati da ratto giovane e senescente in seguito all’aggiunta degli attivatori dei mito-KATP. L’analisi è stata condotta potenziometricamente con l’utilizzo di un mini-elettrodo sensibile al Tetrafenilfosfonio (TPP+), ione liposolubile che si distribuisce fra interno ed esterno del mitocondrio in relazione al potenziale di membrana, come descritto dall’equazione di Nernst. Nella seconda parte della ricerca sono stati valutati i movimenti del K+ utilizzando una sonda fluorescente tallio-sensibile (catione K+-mimetico). Mediante questo approccio sperimentale sono stati osservati, attraverso l’analisi spettrofluorimetrica, i flussi di ioni Tl+ indotti dagli attivatori dei mito-KATP, diazossido e F163, e indotti dalla valinomicina (ionoforo del K+, usato come riferimento) nel ratto giovane e nel ratto senescente. Nell’ultima parte della tesi, sono stati analizzati i movimenti del Ca++ attraverso la membrana mitocondriale nel ratto giovane e nel ratto senescente con l’utilizzo degli attivatori dei canali mito-KATP, diazossido e F163. L’analisi è stata condotta potenziometricamente con l’utilizzo di un mini-elettrodo sensibile al Ca++. Il lavoro ha permesso di identificare che la maggiore vulnerabilità al danno da ischemia/riperfusione tipica negli animali senescenti può risiedere in un cattivo funzionamento del mitocondrio, dovuto ad una alterata gestione dei flussi di K+ tra citosol e matrice mitocondriale. Tale alterazione sembra avere pesanti ripercussioni sul corretto funzionamento dei canali mito-KATP, che rappresentano un fondamentale meccanismo endogeno di protezione da danno da ischemia/riperfusione

Human herpesvirus 8 cellular gene modulation

P0663 Human herpesvirus 8 cellular gene modulation Barbara Matteoli* 1 , Rita Romano 2 ,FrancescaTabacchi 2 , Moreno Ferroni 1 1 LAMM,SRL,Lucca, Italy, 2 Department of Public Health and Infectious Diseases,Sapienza University of Rome, Rome, Italy Background:Sinceinflammation is the basis of viral pathogenesis and may beinduced either directly by thevirus or by thecell in responseto infection, theexpression pattern of cellular genes involved in this process was investigated in human epithelial and lymphoid cells infected with Human herpes virus 8 (HHV8) . Materials/methods:Theexpression of 297 human genes involved in 65 pathways associated with inflammation have been studied in HEK293 and PBMC cells 24h after infection with HHV8 (A1 and C3 strains) by DualChip Microarray.Signal quantification was performed by SilverQuant Image Viewer and analysis software(Eppendorf, Germany) and SPSS 16.0 for Windows (SPSS, USA) with two way ANOVA followed by Scheffecontrast. Results:Expression ratios of most of the genes werecloseto 1 or changed only <2-fold cutoff (in positive or negativevalue), suggesting that they were not significantly changed in consequenceto HHV8 infection. Overall,a higher number of genes resulted modulated in HHV8-infected HEK293 cells with respect to PBMCs. A robust increasein theexpression was found in genes involved in inflammation (i.e., AGT, CCR8, CXCR4, IL-6, IL-9, IL10RA, IL-17, IL-23A), signal transduction (i.e.,FGF5,FGF7, MAP3K1, MAPk9m TGFBR1,TRAF2),cellular cycle(IGFBP1), immuneresponse(i.e., IL-16,TLR4),apoptosis (TNFSF4,TNFSF11),and others genes belonging to different categories (i.e., BMP4, BMPR2,EPHA3, HGFBP1, RUNX1, RUNX2,SP1).With a >2-fold threshold filter gene modulation as significant,a high numbers of genes resulted significantly modulated both in HEK293 cell lineand PBMCs infected both with the A1 (169 and 104 genes, respectively) and C3 subtypes (131 and 93 genes, respectively). Next, to increasethesignificance, only those genes with >4-fold changes in their expression were considered. A total of 24 genes resulted strongly induced,especially after A1 subtypeinfection.Thecomparison of transcriptional profiling between both viral subtypes showed that only 4 genes arestrongly induced by different viral subtypes (i.e., BMP4, BMPR2, RUNX2 and TNFSF11). Conclusions: New human genes have been involved in HHV8 infection;commonly modulated genes may be used as target for antiviral/chemiotherapic treatmen