Neuropilin 1mediates keratinocyte growth factor signaling in adipose-derived stem cells: potential involvement in adipogenesis

Adipogenesis is regulated by a complex network of molecules, including fibroblast growth factors. Keratinocyte growth factor (KGF) has been previously reported to promote proliferation on rat preadipocytes, although the expression of its specific receptor, FGFR2-IIIb/KGFR, is not actually detected in mesenchymal cells. Here, we demonstrate that human adipose-derived stem cells (ASCs) show increased expression of KGF during adipogenic differentiation, especially in the early steps. Moreover, KGF is able to induce transient activation of ERK and p38 MAPK pathways in these cells. Furthermore, KGF promotes ASC differentiation and supports the activation of differentiation pathways, such as those of PI3K/Akt and the retinoblastoma protein (Rb). Notably, we observed only a low amount of FGFR2-IIIb in ASCs, which seems not to be responsible for KGF activity. Here, we demonstrate for the first time that Neuropilin 1 (NRP1), a transmembrane glycoprotein expressed in ASCs acting as a coreceptor for some growth factors, is responsible for KGF-dependent pathway activation in these cells. Our study contributes to clarify the molecular bases of human adipogenesis, demonstrating a role of KGF in the early steps of this process, and points out a role of NRP1 as a previously unknown mediator of KGF action in ASCs

CloudSat-based assessment of GPM Microwave Imager snowfall observation capabilities

The sensitivity of Global Precipitation Measurement (GPM) Microwave Imager (GMI) high-frequency channels to snowfall at higher latitudes (around 60◦N/S) is investigated using coincident CloudSat observations. The 166 GHz channel is highlighted throughout the study due to its ice scattering sensitivity and polarization information. The analysis of three case studies evidences the important combined role of total precipitable water (TPW), supercooled cloud water,and background surface composition on the brightness temperature (TB) behavior for different snow-producing clouds. A regression tree statistical analysis applied to the entire GMI-CloudSat snowfall dataset indicates which variables influence the 166 GHz polarization difference (166∆TB)and its relation to snowfall. Critical thresholds of various parameters (sea ice concentration (SIC), TPW, ice water path (IWP)) are established for optimal snowfall detection capabilities. The 166∆TB can identify snowfall events over land and sea when critical thresholds are exceeded (TPW \u3e 3.6 kg·m−2, IWP \u3e 0.24 kg·m−2 over land, and SIC \u3e 57%, TPW \u3e 5.1 kg·m−2 over sea). The complex combined 166∆TB-TB relationship at higher latitudes and the impact of supercooled water vertical distribution are also investigated. The findings presented in this study can be exploited to improve passive microwave snowfall detection algorithms

Inquinamento atmosferico e ricoveri ospedalieri urgenti in 25 citt? italiane: risultati del progetto EpiAir2 Air pollution and urgent hospital admissions in 25 Italian cities: results from the EpiAir2 project

OBJECTIVE: to evaluate the relationship between air pollution and hospital admissions in 25 Italian cities that took part in the EpiAir (Epidemiological surveillance of air pollution effects among Italian cities) project. DESIGN: study of time series with case-crossover methodology, with adjustment for meteorological and time-dependent variables. The association air pollution hospitalisation was analyzed in each of the 25 cities involved in the study; the overall estimates of effect were obtained subsequently by means of a meta-analysis. The pollutants considered were PM10, PM2.5 (in 13 cities only), NO2 and ozone (O3); this last pollutant restricted to the summer season (April-September). SETTING AND PARTICIPANTS: the study has analyzed 2,246,448 urgent hospital admissions for non-accidental diseases in 25 Italian cities during the period 2006- 2010; 10 out of 25 cities took part also in the first phase of the project (2001-2005). MAIN OUTCOME MEASURES: urgent hospital admissions for cardiac, cerebrovascular and respiratory diseases, for all age groups, were considered. The respiratory hospital admissions were analysed also for the 0-14-year subgroup. Percentage increases risk of hospitalization associated with increments of 10 μg/m3 and interquartile range (IQR) of the concentration of each pollutant were calculated. RESULTS: reported results were related to an increment of 10 μg/m3 of air pollutant. The percent increase for PM10 for cardiac causes was 0.34% at lag 0 (95%CI 0.04-0.63), for respiratory causes 0.75%at lag 0-5 (95%CI 0.25-1.25). For PM2.5, the percent increase for respiratory causes was 1.23% at lag 0- 5 (95%CI 0.58-1.88). For NO2, the percent increase for cardiac causes was 0.57%at lag 0 (95%CI 0.13-1.02); 1.29% at lag 0-5 (95%CI 0.52-2.06) for respiratory causes. Ozone (O3) did not turned out to be positively associated neither with cardiac nor with respiratory causes as noted in the previous period (2001-2005). CONCLUSION: the results of the study confirm an association between PM10, PM2.5, and NO2 on hospital admissions among 25 Italian cities. No positive associations for ozone was noted in this period.OBIETTIVO: valutare la relazione tra inquinamento atmosferico e ricoveri ospedalieri nelle citt? italiane partecipanti alla seconda fase del progetto EpiAir (Sorveglianza epidemiologica dell\u27inquinamento atmosferico: valutazione dei rischi e degli impatti nelle citt? italiane). DISEGNO: studio di serie temporali con metodologia case-crossover, con aggiustamento per i fattori temporali e meteorologici rilevanti. L\u27associazione inquinamento atmosferico- ospedalizzazioni ? stata analizzata in ciascuna delle 25 citt? in studio, le stime complessive di effetto sono state ottenute successivamente mediante una metanalisi. Gli inquinanti considerati sono stati il particolato (PM10), il biossido di azoto (NO2) e l\u27ozono (O3), quest\u27ultimo limitatamente al semestre estivo (da aprile a settembre). In 13 citt? in cui i dati erano disponibili ? stata analizzata anche la frazione fine del particolato (PM2.5). SETTING E PARTECIPANTI: lo studio ha esaminato 2.246.448 ricoveri ospedalieri urgenti per cause naturali di pazienti residenti e ricoverati, nel periodo 2006-2010, in 25 citt? italiane, di cui 10 gi? partecipanti alla prima fase del progetto EpiAir (2001-2005). PRINCIPALIMISURE DI OUTCOME: sono stati considerati i ricoveri ospedalieri urgenti per malattie cardiache, cerebrovascolari e respiratorie per tutte le fasce di et?. I ricoveri per cause respiratorie sono stati analizzati separatamente anche per la fascia di et? 0-14 anni. L\u27esposizione ? stata valutata per incremento sia di 10 μg/m3 sia pari all\u27intervallo interquartile (IQR) della concentrazione di ciascun inquinante. RISULTATI: considerando un incremento di 10 μg/m3 per inquinante, per il PM10 ? stato osservato un incremento percentuale di rischio per patologie cardiache dello 0,34%a lag 0 (IC95% 0,04-0,63), e per patologie respiratorie dello 0,75% a lag 0-5 (IC95% 0,25-1,25). Per il PM2.5 l\u27incremento percentuale di rischio per patologie respiratorie ? risultato dell\u271,23%a lag 0-5 (IC95%0,58-1,88). Per l\u27NO2 la stima di effetto per patologie cardiache ? risultata dello 0,57% a lag 0 (IC95% 0,13-1,02), e per patologie respiratorie dell\u271,29% a lag 0-5 (IC95% 0,52-2,06). L\u27ozono non ? risultato positivamente associato n? alle patologie cardiache n? a quelle respiratorie (a differenza del periodo 2001-2005). CONCLUSIONE: i risultati dello studio confermano l\u27effetto a breve termine dell\u27inquinamento atmosferico da PM10, PM2.5 e NO2 sulla morbosit?, in particolare respiratoria, nelle citt? italiane. Non sono state rilevate associazioni positive per l\u27O3

Common Breast Cancer Susceptibility Alleles and the Risk of Breast Cancer for BRCA1 and BRCA2 Mutation Carriers: Implications for Risk Prediction

The known breast cancer (BC) susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1,LSP1 and 2q35 confer increased risks of BC for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of three additional SNPs, rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11 and rs10941679 at 5p12 and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased BC risk for BRCA2 carriers (per-allele Hazard Ratio (HR)=1.10, 95%CI:1.03-1.18, p=0.006 and HR=1.09, 95%CI:1.01-1.19, p=0.03, respectively). Neither SNP was associated with BC risk for BRCA1 carriers and rs6504950 was not associated with BC for either BRCA1 or BRCA2 carriers. Of the nine polymorphisms investigated, seven were associated with BC for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, p-values:7×10−11-0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (p=0.0049, 0.03 respectively). All risk associated polymorphisms appear to interact multiplicatively on BC risk for mutation carriers. Based on the joint genotype distribution of the seven risk associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e. between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing BC by age 80, compared with 42-50% for the 5% of carriers at lowest risk. Our findings indicated that these risk differences may be sufficient to influence the clinical management of mutation carriers

STRATEGIES FOR THE IDENTIFICATION OF ALLELES INVOLVED IN HEREDITARY BREAST CANCER

Breast cancer is the most common malignancy affecting women in the Western world. At present, several risk factors have been identified and, among them, the most significant is a positive family history for the disease due to the presence of inherited predisposing germline alterations. Despite the discovery of many susceptibility genes, among which the highly penetrant BRCA1 and BRCA2 are the most relevant, more than 70% of the genetic predisposition to breast cancer remains unexplained. During my three-years Ph.D., I used different analytical approaches to investigate DNA alterations and loci that could be relevantly implicated in the aetiology and progression of hereditary breast cancers. As first project on non informative BRCA1/2 families, I was involved in the assessment of the pathogenetic role of the BRCA1 p.Val1688del allele, which recurs in the Northeast Italy. Unclassified variants (UVs) provide a considerable challenge in the clinical management of high-risk families. Indeed, the absence of conclusive data on UVs pathogenicity, due to the lack of an evident deleterious effect on protein function, prevents their use in the identification of individuals predisposed to breast cancer development. To assess the pathogenetic role of BRCA1 p.Val1688del, we used the integrated multifactorial likelihood model described by Goldgar et al. (2004). Several independent evidences were derived from epidemiologic and linkage studies as well as histopathology, LOH and evolutionary conservation analyses, in 12 families carrying the p.Val1688del allele. All the evidences were than properly evaluated to obtain, from each, the probability of the observed data given that the variant is a deleterious mutation, against the probability of the observed data under the hypothesis that the variant is neutral. The likelihood ratios were integrated to obtain a combined odd of variant causality, resulting in a clear assessment of the BRCA1 p.Val1688del pathogenicity. Indeed, the final integrated odd of 349,000:1 in favor of disease causality largely exceeds the established cut off 1,000:1 needed to state the deleterious effect of the variant. The second project, described herein, focused on the identification of molecular alterations involved in non-BRCA1/BRCA2 breast cancers. To this purpose, I performed a comprehensive genomic profile of the breast cancer cell line HCC1500, which was established from a patient who probably harboured a predisposing germline mutation affecting genes other than BRCA1 and BRCA2. Indeed, despite the early age of disease development and a positive family history for breast and colon cancer, we did not detect any deleterious BRCA1, BRCA2 or TP53 alteration. The HCC1500 cell line was analysed by two complementary approaches: i) a novel combined strategy named GINI (Gene Identification by NMD Inhibition; Noesie and Dietz, 2001) for the identification of nonsense mutations at the transcriptome level, and ii) a copy number alteration (CNA) analysis that was achieved by array-based high-density SNP genotyping. This approach led to the identification of genes specifically mutated by either point mutations or major genomic rearrangements. In particular, we identified an intronic substitution in the PNLIPRP3 gene, which is predicted to create a PTC by altering pre-mRNA splicing. Moreover, high-resolution CNA analysis allowed the detection of small homozygously deleted regions as well as focal amplifications, involving single or few genes. A gene selection based on the specific biological function and/or type of mutation and/or previously reported involvement in cancer pathways, allowed the definition of a relatively small number of candidate breast cancer genes that will deserve further and more specific investigation. Finally, among the minor research projects that I will not discuss in my thesis, I was also directly involved in studies carried out by our research group as part of an international consortium, named CIMBA (Consortium of Investigators of Modifiers of BRCA1/2), whose aim is the identification of genetic factors implicated in the modification of BRCA1 and BRCA2 penetrance (Chenevix-Trench et al., 2007). Candidate single nucleotide polymorphisms (SNPs), most of which derived from genome-wide association studies, were genotyped in 213 BRCA1/2 mutation carriers from our cohort by the taqman allele discrimination technique, and subsequently statistically analyzed together with the data obtained from more than 30 study groups world-wide. The genotyping of SNP alleles in thousands of BRCA1/BRCA2 mutation carriers provide the necessary statistical power for the identification of significant association between common SNPs with typical low effects and specific subclasses of individuals at higher risk. These studies were carried out on more than 9400 BRCA1 and 5600 BRCA2 mutation carriers and allowed the identification of a significant association between rs3817198 in LPS1 and rs13387042 at 2q35 (already found to be associated with increased breast cancer risks in the general population) and increased breast cancer risk in BRCA2 and both BRCA1 and BRCA2 mutation carriers, respectively. The identification of genetic modifiers of breast cancer risk not only will favor a better understanding of breast cancer predisposition and pathogenesis but will also allow more precise cancer risk estimates and will represent a useful tool for the design of new therapeutic approaches.Il tumore della mammella e’ la neoplasia più frequente nelle donne dell’Occidente. Ad oggi sono stati identificati alcuni fattori di rischio per lo sviluppo della neoplasia mammaria, ma il maggiormente significativo è la presenza di storia familiare, che è associata alla presenza nei membri della famiglia di alterazioni germinali predisponenti. Fino ad oggi sono stati identificati alcuni geni responsabili dell’aumento di rischio per lo sviluppo del tumore della mammella, tra cui i più rilevanti sono i geni ad alta penetranza BRCA1 e BRCA2. Tuttavia più del 70% dell’eccesso di rischio che si riscontra in casi familiari di tumore della mammella rispetto la popolazione generale non trova ad oggi un riscontro in alterazioni genetiche predisponenti. Durante il mio dottorato mi sono occupata dell’identificazione di alterazioni genetiche coinvolte nella predisposizione e progressione del tumore eredo-familiare della mammella in cui sono state escluse mutazioni chiaramente patogenetiche dei geni BRCA1/BRCA2. In un primo progetto ho partecipato alla caratterizzazione del ruolo patogenetico della variante BRCA1 p.Val1688del, identificata come variante di incerto significato clinico (Unclassified Variant, UV) e riscontrata frequentemente in pazienti provenienti dalla regione Veneto. In assenza di un chiaro effetto deleterio sulla funzionalità della proteina, le UV non possono essere utilizzate per l’identificazione e la sorveglianza degli individui ad alto rischio per lo sviluppo della neoplasia mammaria. Allo scopo di chiarire il ruolo patogenetico della delezione p.Val1688del, abbiamo utilizzato un approccio multi fattoriale, descritto da Golgar et al. (2004), attraverso il quale evidenze di diversa natura vengono integrate per derivare un rapporto di probabilità in favore della patogenicità o neutralità della variante. A questo scopo, i membri di 12 famiglie portatrici della variante sono stati analizzati per ottenere dati quali la co-segregazione della variante con il fenotipo tumorale, la co-presenza di mutazioni patogenetiche nel gene BRCA1, l’istopatologia del tumore, la perdita di eterozigosi (LOH) e la conservazione filogenetica del residuo aminoacidico alterato. Assunta l’indipendenza delle evidenze raccolte, i rapporti di probabilità associati a ciascuna evidenza sperimentale e clinica sono stati combinati, ottenendo un valore di 349000:1 in favore del ruolo patogenetico della variante, che supera di molto il cut off di 1000:1 stabilito per poter classificare la variante come alterazione predisponente. Il secondo progetto di cui mi sono occupata riguarda la definizione del profilo genetico della linea cellulare di carcinoma mammario HCC1500, derivata da una paziente con probabile tumore eredo-familiare della mammella. Nonostante l’età precoce di insorgenza e la storia familiare di tumore della mammella e del colon, il coinvolgimento dei geni BRCA1, BRCA2 e TP53 è stato escluso mediante screening mutazionale. La linea HCC1500 è stata quindi analizzata mediante due approcci complementari: i) l’identificazione di mutazioni nonsenso mediante l’utilizzo di un’approccio di analisi recentemente descritto chiamato GINI (Gene Identification by NMD Inhibition; Noesie and Dietz, 2001), e ii) l’identificazione di alterazioni numeriche mediante l’utilizzo di array per la genotipizzazione di un elevato numero di SNP, una recente tecnologia che permette un’elevata risoluzione di analisi. Mediante questo approccio è stato possibile identificare singoli geni mutati con specifiche alterazioni puntiformi o a causa di più estesi riarrangiamenti genomici. In particolare, nel gene PNLIPRP3 abbiamo identificato una sostituzione intronica che, modificando lo splicing del pre-mRNA, potrebbe dare origine ad un trascritto con mutazione troncante. Inoltre, l’elevata risoluzione dell’analisi per lo studio delle alterazioni cromosomiche numeriche, ci ha permesso di identificare singoli o pochi geni coinvolti in delezioni in omozigosi, amplificazioni di piccole regioni e riarrangiamenti intragenici derivanti da eventi di amplificazione o delezione. Sulla base di criteri quali la funzione biologica e mutazioni ritenute rilevanti per la selettività con cui alterano specifiche regioni codificanti, abbiamo selezionato alcuni geni che, prioritariamente ad altri, andrebbero confermati e analizzati in maggior dettaglio con ulteriori studi.. Un terzo progetto che ho seguito direttamente, ma che comunque non tratterò nella mia tesi, riguarda l’identificazione di fattori genetici coinvolti nella modificazione del rischio per i soggetti portatori di mutazione BRCA1 e BRCA2. Tale progetto si inserisce nell'ambito di un consorzio internazionale, denominato CIMBA (Consortium of Investigators of Modifiers of BRCA1/2; (Chenevix-Trench et al., 2007). SNP identificati prevalentemente attraverso studi di associazione genome-wide sono stati genotipizzati in 213 carriers di mutazione BRCA1/2 della nostra casistica mediante discriminazione allelica con sonde TaqMan. L’analisi statistica è stata effettuata sui dati genotipici provenienti da più di 30 gruppi di lavoro, permettendo la raccolta di dati derivanti da migliaia di individui. In questo modo è possibile ottenere la potenza statistica necessaria per l’identificazione dell’associazione tra polimorfismi con debole effetto e un aumentato rischio di sviluppo del tumore della mammella in specifiche sottoclassi di soggetti già portatori di mutazione nei geni ad alta penetranza BRCA1/2. Effettuati su più di 9400 e 5600 carriers di mutazione rispettivamente di BRCA1 e BRCA2, tali studi hanno permesso di identificare un’associazione significativa tra gli SNP rs3817198 (LPS1) e rs13387042 (2q35) ed un aumentato rischio in soggetti portatori di mutazioni BRCA2, e, nel secondo caso BRCA1 o BRCA2. L’identificazione e studio di fattori genetici modificatori permetterà di acquisire una più approfondita conoscenza della predisposizione e patogenesi del tumore della ereditario mammella, ma anche di stimare con migliore precisione il rischio di sviluppo della neoplasia così come di fornire utili informazioni per l'identificazione di nuovi approcci terapeutici

BDE-47, -99, -209 and Their Ternary Mixture Disrupt Glucose and Lipid Metabolism of Hepg2 Cells at Dietary Relevant Concentrations: Mechanistic Insight through Integrated Transcriptomics and Proteomics Analysis

Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals implied as flame retardants. Humans are mainly exposed to BDE-47, -99, and -209 congeners by diet. PBDEs are metabolic disruptors with the liver as the main target organ. To investigate their mode of action at a human-relevant concentration, we exposed HepG2 cells to these congeners and their mixture at 1 nM, analyzing their transcriptomic and proteomic profiles. KEGG pathways and GSEA Hallmarks enrichment analyses evidenced that BDE-47 disrupted the glucose metabolism and hypoxia pathway; all the congeners and the MIX affected lipid metabolism and signaling Hallmarks regulating metabolism as mTORC1 and PI3K/AKT/MTOR. These results were confirmed by glucose secretion depletion and increased lipid accumulation, especially in BDE-47 and -209 treated cells. These congeners also affected the EGFR/MAPK signaling; further, BDE-47 enriched the estrogen pathway. Interestingly, BDE-209 and the MIX increased ERα gene expression, whereas all the congeners and the MIX induced ERβ and PPARα. We also found that PBDEs modulated several lncRNAs and that HNRNAP1 represented a central hub in all the four interaction networks. Overall, the PBDEs investigated affected glucose and lipid metabolism with different underlying modes of action, as highlighted by the integrated omics analysis, at a dietary relevant concentration. These results may support the mechanism-based risk assessment of these compounds in relation to liver metabolism disruption

Functional Interaction between Adenosine A_2A and mGlu₅ Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu₅R Binding in Mouse Hippocampus and Neuroblastoma Cell Line

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity