Caratterizzazione di popolazioni segreganti di vite per lo sviluppo di marcatori utili alla selezione di varietà resistenti alla Peronospora (Plasmopara viticola)

Il lavoro sperimentale svolto nell’ambito della presente tesi è parte del progetto PBA (Pedigree-Based Analysis) sviluppato presso il Laboratorio di Genomica Strutturale della Fondazione Edmund Mach di San Michele all’Adige (TN). Lo scopo del lavoro è la caratterizzazione genotipica e fenotipica di sei popolazioni segreganti di vite per la resistenza alla peronospora (Plasmopara viticola), mediante marcatori SSR (Simple Sequence Repeats) e fenotipizzazione di dischetti fogliari inoculati con spore fungine. Prima della fase di genotipizzazione, è stato messo a punto un protocollo di multiplex PCR per l’amplificazione di 9 SSR universali (This et al., 2004; Di Vecchi-Staraz et al., 2007) utili alla successiva identificazione varietale e verifica degli incroci. Successivamente, tutte le popolazioni oggetto di studio sono state genotipizzate impiegando 81 SSR situati su cromosomi noti, in letteratura, per portare elementi genetici di resistenza alla peronospora. La fenotipizzazione è stata effettuata inoculando dischetti fogliari prelevati dalle piante oggetto di studio con una sospensione di spore fungine a concentrazione nota. Dopo 4 e 6 giorni dall’inoculo, ai singoli dischetti è stata attribuita una classe fenotipica basandosi sulla densità di sporulazione e sull’estensione dei sintomi. Mediante analisi di immagine, effettuata sulle foto dei dischetti acquisite tra 4 e 7 dpi (days post-infection), è stato possibile ottenere un dato quantitativo (% sporulazione) impiegato per l’elaborazione di curve di progressione della malattia e nel calcolo di un parametro derivato (AUDPC: Area Under the Disease Progress Curve), utile nella caratterizzazione di genotipi il cui livello di resistenza alla peronospora non è noto. L’impiego dei 9 SSR universali ha assicurato la corretta identità dei genitori, ma ha rivelato l’erroneo scambio del parentale Cabernet Sauvignon (CS) con il Cabernet Franc (CF) nelle popolazioni O (LU1xCS) ed N (LU2xCS), e l’autofecondazione del 92% della popolazione M (PNxIV35), che è stata scartata. La genotipizzazione con 81 SSR ha evidenziato come l’impiego di 9 SSR universali possa non essere sufficiente per discriminare due varietà strettamente imparentate tra loro, come la Schiava Grossa e la Schiava Gentile. L’analisi della distribuzione delle popolazioni secondo le classi fenotipiche a 6 dpi permette di avanzare ipotesi sulla presenza di un major gene nelle popolazioni L (PNx54-2), N ed O, responsabile in maggior parte della resistenza alla peronospora. Le curve di progressione della malattia evidenziano notevoli differenze tra genotipi suscettibili e resistenti; individui fenotipicamente suscettibili mostrano un valore di AUDPC più elevato rispetto a genotipi resistenti e una maggiore pendenza della curva. I dati fenotipici e genotipici ottenuti sono stati impiegati nell’elaborazione di un unico file di input che verrà utilizzato in futuro nell’individuazione di regioni coinvolte nella resistenza alla peronospora mediante il software specifico per l’analisi PBA (Flex QTL: Quantitative Trait Loci). Gli SSR ad esse associati potranno essere impiegati per la selezione di individui interessanti mediante MAS (Marker Assisted Selection) ai fini della creazione di varietà di vite resistenti alla malattia. L’ottenimento di varietà contenenti più elementi genetici di resistenza (piramidizzazione) a diverse malattie fungine è un obiettivo fondamentale in un’ottica di salvaguardia ambientale e di riduzione dell’impiego di fungicidi di sintesi

Generic Substitution of Orphan Drugs for the Treatment of Rare Diseases: Exploring the Potential Challenges

Generic drugs are important components of measures introduced by healthcare regulatory authorities to reduce treatment costs. In most patients and conditions the switch from a branded drug to its generic counterpart is performed with no major complications. However, evidence from complex diseases suggests that generic substitution requires careful evaluation in some settings and that current bioequivalence criteria may not always be adequate for establishing the interchangeability of branded and generic products. Rare diseases, also called orphan diseases, are a group of heterogeneous diseases that share important characteristics: in addition to their scarcity, most are severe, chronic, highly debilitating, and often present in early childhood. Finding a treatment for a rare disease is challenging. Thanks to incentives that encourage research and development programs in rare diseases, several orphan drugs are currently available. The elevated cost of orphan drugs is a highly debated issue and a cause of limited access to treatment for many patients. As patent protection and the exclusivity period of several orphan drugs will expire soon, generic versions of orphan drugs should reach the market shortly, with great expectations about their impact on the economic burden of rare diseases. However, consistent with other complex diseases, generic substitution may require thoughtful considerations and may be even contraindicated in some rare conditions. This article provides an overview of rare disease characteristics, reviews reports of problematic generic substitution, and discusses why generic substitution of orphan drugs may be challenging and should be undertaken carefully in rare disease patients

Inhibitors of the Cdc34 acidic loop:a computational investigation integrating molecular dynamics, virtual screening and docking approaches

AbstractAmong the different classes of enzymes involved in the ubiquitin pathway, E2 ubiquitin-conjugating enzymes occupy a central role in the ubiquitination cascade. Cdc34-like E2 enzymes are characterized by a 12–14 residue insertion in the proximity of the catalytic site, known as the acidic loop. Cdc34 ubiquitin-charging activity is regulated by CK2-dependent phosphorylation and the regulatory mechanism involves the acidic loop. Indeed, the phosphorylation stabilizes the loop in an open conformation that is competent for ubiquitin charging.Cdc34 is associated with a variety of diseases, such as hepatocellular carcinomas and prostatic adenocarcinomas. In light of its role, the discovery of potential inhibitory compounds would provide the mean to effectively modulate its activity.Here, we carried out a computational study based on molecular dynamics, virtual screening and docking to identify potential inhibitory compounds of Cdc34, modulating the acidic loop conformation. The molecules identified in this study have been designed to act as molecular hinges that can bind the acidic loop in its closed conformation, thus inhibiting the Cdc34-mediated ubiquitination cascade at the ubiquitin-charging step. In particular, we proposed a pharmacophore model featuring two amino groups in the central part of the model and two lateral aromatic chains, which respectively establish electrostatic interactions with the acidic loop (Asp 108 and Glu 109) and a hydrogen bond with Ser 139, which is one of the key residues for Cdc34 activity

Sunitinib in metastatic renal cell carcinoma: The pharmacological basis of the alternative 2/1 schedule

No abstract availabl

Precision medicine in lymphoma by innovative instrumental platforms

Since the last years, many efforts have been addressed to the growing field of precision medicine in order to offer individual treatments to every patient on the basis of his/her genetic background. Formerly adopted to achieve new disease classifications as it is still done, innovative platforms, such as microarrays, genome-wide association studies (GWAS) and next generation sequencing (NGS), have made the progress in pharmacogenetics faster and cheaper than previously expected. Several studies in lymphoma patients have demonstrated that these platforms can be used to identify biomarkers predictive of drug efficacy and tolerability, discovering new possible druggable proteins. Indeed, GWAS and NGS allow the investigation of the human genome, finding interesting associations with putative or unexpected targets, which in turns may represent new therapeutic possibilities. Importantly, some objective difficulties have initially hampered the translation of findings in clinical routines, such as the poor quantity/quality of genetic material or the paucity of targets that could be investigated at the same time. At present, some of these technical issues have been partially solved. Furthermore, these analyses are growing in parallel with the development of bioinformatics and its capabilities to manage and analyze big data. Because of pharmacogenetic markers may become important during drug development, regulatory authorities (i.e., EMA, FDA) are preparing ad hoc guidelines and recommendations to include the evaluation of genetic markers in clinical trials. Concerns and difficulties for the adoption of genetic testing in routine are still present, as well as affordability, reliability and the poor confidence of some patients for these tests. However, genetic testing based on predictive markers may offers many advantages to caregivers and patients and their introduction in clinical routine is justified

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9

AbstractBone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9. rhBMP-15, in particular, is an excellent substrate for G-CK. In each protein a single residue is phosphorylated by G-CK, corresponding to the serine residue at the sixth position of the mature region of both rhBMP-15 and rhGDF-9, whose phosphorylation is required for biological activity

A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds

Abstract Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0–24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤2.0%) up to a maximum of 100% of herds with a within-herd AP ≥8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination

Effectiveness of combination of Mini-and Microsatellite loci to sub-type Mycobacterium avium subsp. paratuberculosis Italian type C isolates

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(Map) is the etiological agent of paratuberculosis. The aim of our study was to combine Mini-and Microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of Map isolates. For this purpose, 84 Italian Type C Map isolates, each from a different cattle herd, were submitted to MIRU-Variable-Number Tandem-Repeats (VNTRs) typing and Short Sequence repeats (SSRs) sequencing. Moreover, the method was used to analyse the variability inside 10 herds (from three to 50 isolates per herd).</p> <p>Results</p> <p>The molecular sub-typing, carried out using three SSR and 10 MIRU-VNTR loci, differentiated the 84 isolates into 33 clusters, reaching a Simpson's Discriminatory Index (SID) value of 0.952 (0.933 to 0.972, 95% confidence intervals). Among all considered loci, six (SSR2, MIRU2, SSR1, SSR8, VNTR3527 and VNTR1067) showed relevant allelic variability. Thirty-eight% of the isolates were clustered into four genotypes, differing from each other for the SSR2 locus. The other isolates, characterised by differences in two or more loci, were spread among the rest of the clusters. The intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters.</p> <p>Conclusions</p> <p>Our results revealed the advantage of using both Mini-and Microsatellite approaches for successfully discriminating among Map Type C isolates from the same geographic area, host species and herd. These data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies.</p

Implications of KRAS mutations in acquired resistance to treatment in NSCLC

Rationale: KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies. The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients. Materials and Methods: A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance

Concise Review: Chronic Myeloid Leukemia: Stem Cell Niche and Response to Pharmacologic Treatment

Nowadays, more than 90% of patients affected by chronic myeloid leukemia (CML) survive with a good quality of life, thanks to the clinical efficacy of tyrosine kinase inhibitors (TKIs). Nevertheless, point mutations of the ABL1 pocket occurring during treatment may reduce binding of TKIs, being responsible of about 20% of cases of resistance among CML patients. In addition, the presence of leukemic stem cells (LSCs) represents the most important event in leukemia progression related to TKI resistance. LSCs express stem cell markers, including active efflux pumps and genetic and epigenetic alterations together with deregulated cell signaling pathways involved in self-renewal, such as Wnt/β-catenin, Notch, and Hedgehog. Moreover, the interaction with the bone marrow microenvironment, also known as hematopoietic niche, may influence the phenotype of surrounding cells, which evade mechanisms controlling cell proliferation and are less sensitive or frankly resistant to TKIs. This Review focuses on the role of LSCs and stem cell niche in relation to response to pharmacological treatments. A literature search from PubMed database was performed until April 30, 2017, and it has been analyzed according to keywords such as chronic myeloid leukemia, stem cell, leukemic stem cells, hematopoietic niche, tyrosine kinase inhibitors, and drug resistance. Stem Cells Translational Medicine 2018