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Fish embryos may be vulnerable to seawater acidification resulting from anthropogenic carbon dioxide (CO2) emissions or from excessive biological CO2 production in aquaculture systems. This study investigated CO2 effects on embryos of the European eel (Anguilla anguilla), a catadromous fish that is considered at risk from climate change and that is targeted for hatchery production to sustain aquaculture of the species. Eel embryos were reared in three independent recirculation systems with different pH/CO2 levels representing "control" (pH 8.1, 300 μatm CO2), end-of-century climate change ("intermediate", pH 7.6, 900 μatm CO2) and "extreme" aquaculture conditions (pH 7.1, 3000 μatm CO2). Sensitivity analyses were conducted at 4, 24, and 48 hours post-fertilization (hpf) by focusing on development, survival, and expression of genes related to acute stress response (crhr1, crfr2), stress/repair response (hsp70, hsp90), water and solute transport (aqp1, aqp3), acid-base regulation (nkcc1a, ncc, car15), and inhibitory neurotransmission (GABAAα6b, Gabra1). Results revealed that embryos developing at intermediate pH showed similar survival rates to the control, but egg swelling was impaired, resulting in a reduction in egg size with decreasing pH. Embryos exposed to extreme pH had 0.6-fold decrease in survival at 24 hpf and a 0.3-fold change at 48 compared to the control. These observed effects of acidification were not reflected by changes in expression of any of the here studied genes. On the contrary, differential expression was observed along embryonic development independent of treatment, indicating that the underlying regulating systems are under development and that embryos are limited in their ability to regulate molecular responses to acidification. In conclusion, exposure to predicted end-of-century ocean pCO2 conditions may affect normal development of this species in nature during sensitive early life history stages with limited physiological response capacities, while extreme acidification will negatively influence embryonic survival and development under hatchery conditions.

More than 300 genetic risk loci have been identified for male pattern baldness (MPB) but little is known about the exact molecular mechanisms through which the associated variants exert their effects on MPB pathophysiology. Here, we aimed at further elucidating the regulatory architecture of the MPB risk locus on chromosome (chr.) 2q35, where we have previously reported a regulatory effect of the MPB lead variant on the expression of WNT10A. A HaploReg database research for regulatory annotations revealed that the association signal at 2q35 maps to a binding site for the transcription factor EBF1, whose gene is located at a second MPB risk locus on chr. 5q33.3. To investigate a potential interaction between EBF1 and WNT10A during MPB development, we performed in vitro luciferase reporter assays as well as expression analyses and immunofluorescence co-stainings in microdissected human hair follicles. Our experiments confirm that EBF1 activates the WNT10A promoter and that the WNT10A/EBF1 interaction is impacted by the allelic expression of the MPB risk allele at 2q35. Expression analyses across different hair cycle phases and immunhistochemical (co)stainings against WNT10A and EBF1 suggest a predominant relevance of EBF1/WNT10A interaction for hair shaft formation during anagen. Based on these findings we suggest a functional mechanism at the 2q35 risk locus for MPB, where an MPB-risk allele associated reduction in WNT10A promoter activation via EBF1 results in a decrease in WNT10A expression that eventually results in anagen shortening, that is frequently observed in MPB affected hair follicles. To our knowledge, this study is the first follow-up study on MPB that proves functional interaction between two MPB risk loci and sheds light on the underlying pathophysiological mechanism at these loci.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.

Background Rituximab in combination with chemotherapy is now widely accepted as standard of care for AIDS-related lymphomas (ARLs) of B-cell origin. However, the clinical impact of rituximab in resource limited settings remains unknown. Different settings and patient heterogeneity may affect the effect of any given treatment. The study objectives were to determine if rituximab use was associated with improved 18-month overall survival (OS) of patients with ARLs and to identify correlates of 18-month OS. Methods A retrospective review of medical records of adult HIV infected patients treated for high-grade large cell non-Hodgkin’s lymphoma with chemotherapy +/- rituximab between 2015–2017 was conducted. Vital status and disease progression/relapse at 18 months were determined. Survival functions were estimated using Kaplan-Meier methodology. Equality of survival functions were assessed using Log-rank tests and Cox regression analysis to identify risk factors for mortality. Results One hundred and twenty-four eligible medical records were identified. This was a cohort of black Africans with a median age of 42 (IQR: 33–47) and a 57% male gender distribution. Overall survival at 6, 12 and 18 months for the population was 75.9%, 44.0% and 30.6% respectively. Over the study period, 72.6% of patients were diagnosed with disease progression/ relapse. There was a higher rate of rituximab use in patients who were treated at a private institution and those with medical insurance. Rituximab use was not associated with a reduction in 18-month mortality [adjusted hazard ratio (aHR)1.28, (95% CI 0.63–2.60)]. Risk factors for 18-month mortality were male gender [aHR 1.89, (95% CI 1.04–3.43)], age 40+ years [aHR 2.49, (1.33–4.67)], receipt of

Hypomethylating agents are a classical frontline low-intensity therapy for older patients with acute myeloid leukemia. Recently, TP53 gene mutations have been described as a potential predictive biomarker of better outcome in patients treated with a ten-day decitabine regimen., However, functional characteristics of TP53 mutant are heterogeneous, as reflected in multiple functional TP53 classifications and their impact in patients treated with azacitidine is less clear. We analyzed the therapeutic course and outcome of 279 patients treated with azacitidine between 2007 and 2016, prospectively enrolled in our regional healthcare network. By screening 224 of them, we detected TP53 mutations in 55 patients (24.6%), including 53 patients (96.4%) harboring high-risk cytogenetics. The identification of any TP53 mutation was associated with worse overall survival but not with response to azacitidine in the whole cohort and in the subgroup of patients with adverse karyotype. Stratification of patients according to three recent validated functional classifications did not allow the identification of TP53 mutated patients who could benefit from azacitidine. Systematic TP53 mutant classification will deserve further exploration in the setting of patients treated with conventional therapy and in the emerging field of therapies targeting TP53 pathway.

BACKGROUND:Hepatitis B virus (HBV) and hepatitis C virus (HCV) are a common cause of complications in liver disease and immunological impairment among human immunodeficiency virus (HIV)-infected patients. The aim of this study was to assess the seroprevalence of HBV and HCV and their correlation with CD4+ T-cells among HIV-infected patients in an HBV endemic area. METHODS:A cross-sectional observational and retrospective study was carried out in a reference center in Southern Brazil between January 2005 and December 2016. Socio-demographic data were collected by using a structured questionnaire. Serological tests and analysis of CD4+ T-cell count levels were performed using standard procedures. RESULTS:The seroprevalence of HIV-HBV, HIV-HCV, and HIV-HBV-HCV coinfections was 3.10%, 3.10%, and 0.16%, respectively. At baseline, anti-hepatitis B surface and anti-hepatitis B core antigens were detected in 46.27% and 16.74% of HIV-monoinfected patients and in 31.25% and 21.86% of the HIV-HCV coinfected patients, respectively. The median CD4+ T-cell count at baseline in the HIV-monoinfected group was higher than that in the HIV-coinfected groups, but without statistical significance. The median CD4+ T-cell count and the CD4/CD8 ratio were significantly higher in HIV-HBV and HIV-HCV groups after 24 months of combination antiretroviral therapy (cART) compared to the pre-cART values. When comparing patients with HIV-HBV and HIV-HCV on cART, CD4+ T-cell recovery was more rapid for HIV-HBV patients. CONCLUSION:Although the analyzed region was endemic for HBV, the prevalence of HIV-HBV and HIV-HCV coinfection was lower than the rate found in the general population of Brazil. HBV and HCV had no significant impact on CD4+ T-cell counts among HIV-infected patients at baseline.

BACKGROUND:For many years, long-acting intramuscular (LAI) antipsychotics have been prescribed predominantly to chronic and severe patients, as a last resort when other treatments failed. Recently, a broader and earlier use of LAIs, particularly second-generation LAIs, has been emphasized. To date, few studies attempted to frame how this change in prescribing took place in real-world practice. Therefore, this study aimed to describe the clinical features of patients prescribed with LAIs, and to explore possible prescribing differences between first- and second-generations LAIs under ordinary clinical practice in Italy. METHODS:The STAR Network "Depot" Study is an observational, longitudinal, multicenter study involving 35 centers in Italy. In the cross-sectional phase, patients prescribed with LAIs were consecutively recruited and assessed over a period of 12 months. Descriptive statistics and multivariable logistic regression analyses were employed. RESULTS:Of the 451 recruited patients, 61% were males. The level of social and working functioning was heterogeneous, as was the severity of disease. Seventy-two per cent of the patients had a diagnosis of the schizophrenia spectrum. Seventy per cent were prescribed with second-generation antipsychotic (SGA) LAIs (mostly paliperidone, aripiprazole and risperidone). Compared to first-generation antipsychotic (FGA) LAIs, patients prescribed with SGA LAIs were more often younger; employed; with a diagnosis of the schizophrenia spectrum or bipolar disorder; with higher levels of affective symptoms; with fewer LAI prescriptions in the past. DISCUSSION:LAIs' prescribing practices appear to be more flexible as compared to the past, although this change is mostly restricted to SGA LAIs.

Honey bees are considered large-scale monitoring tools due to their environmental exploration and foraging activities. Traces of these activities can be recovered in the honey that also may reflect the hive ecological micro-conditions in which it has been produced. This study applied a next generation sequencing platform (Ion Torrent) for shotgun metagenomic analysis of honey environmental DNA (eDNA). The study tested a methodological framework to interpret DNA sequence information useful to describe the complex ecosystems of the honey bee colony superorganism, its pathosphere and the heterogeneity of the agroecological environments and environmental sources that left DNA marks in the honey. Analysis of two honeys reported sequence reads from five main organism groups (kingdoms or phyla): arthropods (that mainly included reads from Apis mellifera, several other members of the Hymenotpera, in addition to members of the Diptera, Coleoptera and Lepidoptera, as well as aphids and mites), plants (that clearly confirmed the botanical origin of the two honeys, i.e. orange tree blossom and eucalyptus tree blossom honeys), fungi and bacteria (including common hive and honey bee gut microorganisms, honey bee pathogens and plant pathogens), and viruses (which accounted for the largest number of reads in both honeys, mainly assigned to Apis mellifera filamentous virus). The shotgun metagenomic approach that was used in this study can be applied in large scale experiments that might have multiple objectives according to the multi-kingdom derived eDNA that is contained in the honey.

BACKGROUND:Improving the knowledge of angiomyolipoma physiopathology might help in refining its pharmacological treatment. We investigated if angiomyolipoma cells have migratory properties, how their growth and motility can be influenced by the hormonal milieu, and if this can be related to a specific gender. METHODS:Primary cells were isolated from angiomyolipomas surgically resected for therapeutical reasons in a female and in a male patient. The genetic control demonstrated no TSC2 deletion. Bi- (wound healing) and three-dimensional (transwell assay) migration were analyzed in vitro in basal conditions and under the influence of 17- β-estradiol and SDF-1α. RESULTS:Treatment up to 72 hours with 17-β-estradiol (0.1-100 nM), tamoxifen (0.2-20 μM) or with both, does not modify angiomyolipoma cells proliferation. On the other hand, SDF-1α and 17-β-estradiol treatment induce a significant motility increase (both bi- and three-dimensional) which becomes evident already after 2 hours of incubation. Angiomyolipoma cells express mRNA coding for SDF-1α and 17-β-estradiol receptors and secrete both the metalloproteases principally involved in malignant phenotype acquisition, i.e. MMP-2 and MMP-9. CONCLUSION:Angiomyolipoma cells behave similarly, despite their different source. Primary angiomyolipoma cells migrate in response to hormonal milieu and soluble factors, and produce active metalloproteases, both aspects being consistent with the theory claiming they can migrate to the lungs (and/or other organs) and colonizing them. No main feature, among the aspects we analyzed, seems to be referable to the gender of origin.

Canine Inflammatory Bowel Disease (IBD) is considered a multifactorial disease caused by complex interactions between the intestinal immune system, intestinal microbiota and environmental factors in genetically susceptible individuals. Although IBD can affect any breed, German shepherd dogs (GSD) in the UK are at increased risk of developing the disease. Based on previous evidence, the aim of the present study was to identify single nucleotide polymorphisms (SNPs), which may confer genetic susceptibility or resistance to IBD using a genome-wide association study (GWAS). Genomic DNA was extracted from EDTA blood or saliva samples of 96 cases and 98 controls. Genotyping of cases and controls was performed on the Canine Illumina HD SNP array and data generated was analyzed using PLINK. Several SNPs and regions on chromosomes 7,9,11 and 13 were detected to be associated with IBD using different SNP-by-SNP association methods and FST windows approach. Searching one Mb up-and down-stream of the most significant SNPs, as identified by single SNP analysis as well as 200Kb before and after the start and the end position of the associated regions identified by FST windows approach, we identified 63 genes. Using a combination of pathways analysis and a list of genes that have been reported to be involved in human IBD, we identified 16 candidate genes potentially associated with IBD in GSD.

Shot-gun next generation sequencing (NGS) on whole DNA extracted from specimens collected from mammals often produces reads that are not mapped (i.e. unmapped reads) on the host reference genome and that are usually discarded as by-products of the experiments. In this study, we mined Ion Torrent reads obtained by sequencing DNA isolated from archived blood samples collected from 100 performance tested Italian Large White pigs. Two reduced representation libraries were prepared from two DNA pools constructed each from 50 equimolar DNA samples. Bioinformatic analyses were carried out to mine unmapped reads on the reference pig genome that were obtained from the two NGS datasets. In silico analyses included read mapping and sequence assembly approaches for a viral metagenomic analysis using the NCBI Viral Genome Resource. Our approach identified sequences matching several viruses of the Parvoviridae family: porcine parvovirus 2 (PPV2), PPV4, PPV5 and PPV6 and porcine bocavirus 1-H18 isolate (PBoV1-H18). The presence of these viruses was confirmed by PCR and Sanger sequencing of individual DNA samples. PPV2, PPV4, PPV5, PPV6 and PBoV1-H18 were all identified in samples collected in 1998-2007, 1998-2000, 1997-2000, 1998-2004 and 2003, respectively. For most of these viruses (PPV4, PPV5, PPV6 and PBoV1-H18) previous studies reported their first occurrence much later (from 5 to more than 10 years) than our identification period and in different geographic areas. Our study provided a retrospective evaluation of apparently asymptomatic parvovirus infected pigs providing information that could be important to define occurrence and prevalence of different parvoviruses in South Europe. This study demonstrated the potential of mining NGS datasets non-originally derived by metagenomics experiments for viral metagenomics analyses in a livestock species.

Microbiota plays an important role in the homeostasis of the gastrointestinal tract. Understanding the variations of the commensal microbiota composition is crucial for a more efficient control of enteric infectious diseases and for the reduction of the use of antibiotics in animal production, which are the main points of interest for improved animal healthcare and welfare and for consumer health protection. Even though the intestinal microbiota has been extensively studied, little is known about the gastric microbiota. This pilot study was aimed at a descriptive analysis of the gastric microbiota in healthy pigs and at the identification of any differences among four potentially distinct microbial niches in the stomach. Gastric mucosal samples from the oxyntic area, the pylorus and the gastric groove, and a sample of gastric contents were collected from four healthy weaned pigs. Bacterial DNA was isolated and extracted from each sample and amplicons from the V6 region of the 16S rRNA gene were sequenced using Ion Torrent PGM. The data were analysed by an "unsupervised" and a "supervised" approach in the Ribosomal Database Project (RDP) pipeline. Proteobacteria was the dominant phylum in all the samples. Differences in bacterial community composition were found between mucosal and content samples (one-way ANOSIM pairwise post hoc test, p < 0.05); instead, the different mucosal regions did not show differences between them. The mucosal samples were characterised by Herbiconiux and Brevundimonas, two genera which include cellulolytic and xylanolytic strains. Nevertheless, additional larger trials are needed to support the data presented in this pilot study and to increase the knowledge regarding the resident microbiota of the stomach.

Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism.

Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources.

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Accumulating evidence suggests that nonalcoholic fatty liver disease (NAFLD) is associated with left ventricular diastolic dysfunction (LVDD) in nondiabetic individuals. To date, there are very limited data on this topic in patients with type 2 diabetes and it remains uncertain whether NAFLD is independently associated with the presence of LVDD in this patient population. We performed a liver ultrasonography and trans-thoracic echocardiography (with speckle-tracking strain analysis) in 222 (156 men and 66 women) consecutive type 2 diabetic outpatients with no previous history of ischemic heart disease, chronic heart failure, valvular diseases and known hepatic diseases. Binary logistic regression analysis was used to examine the association between NAFLD and the presence/severity of LVDD graded according to the current criteria of the American Society of Echocardiography, and to identify the variables that were independently associated with LVDD, which was included as the dependent variable. Patients with ultrasound-diagnosed NAFLD (n = 158; 71.2% of total) were more likely to be female, overweight/obese, and had longer diabetes duration, higher hemoglobin A1c and lower estimated glomerular filtration rate (eGFR) than those without NAFLD. Notably, they also had a remarkably greater prevalence of mild and/or moderate LVDD compared with those without NAFLD (71% vs. 33%; P

Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.

Impaired balance control is a hallmark symptom in Parkinson's disease (PD). Altered sensory-motor integration contributes to the deficiency. We aimed to determine whether impaired vestibular signal processing added to the disorder. We exposed patients (N = 11; 68±6y) and age-matched healthy subjects (hS: N = 19; 65±11y) on a motion platform in complete darkness to two consecutive forward tilt movements (12 series; N = 24; overall 288 trials) and asked them to indicate which tilt was perceived larger. By combing tilt movements with translations we manipulated vestibular sensory input in order to investigate whether putative impairment resulted from a deficiency of the sensory organs (semicircular canals in 'single-SCC-cue-condition', otoliths in 'single-OT-cue-condition') themselves or to a sensory integration failure ('multi-cue-condition').Tilt discrimination in the multi-cue-condition was inferior in patients compared to hS (p = 0.02). No significant differences between the two groups were found for both single-cue-conditions. Comparison of multi-cue-condition with a prediction resulting from the combination of both single-cue-conditions by optimal observer theory revealed that patients (p = 0.04), in contrast to hS, failed to efficiently combine SCC and OT information to improve tilt perception.We found that PD patients distinguished forward tilts less precise than hS, suggesting impaired vestibular perception. Tilt discrimination in patients, moreover, did not improve as much as in hS in conditions where both SCC and OT information was available compared to conditions where only SCC or OT cues were activated. The latter provides evidence that tilt misperception in PD most likely results from an integration failure of vestibular signals.

BACKGROUND: Recent epidemiological data suggest that non-alcoholic fatty liver disease (NAFLD) is closely associated with aortic valve sclerosis (AVS), an emerging risk factor for adverse cardiovascular outcomes, in nondiabetic and type 2 diabetic individuals. To date, nobody has investigated the association between NAFLD and AVS in people with type 2 diabetes, a group of individuals in which the prevalence of these two diseases is high. METHODS AND RESULTS: We recruited 180 consecutive type 2 diabetic patients without ischemic heart disease, valvular heart disease, hepatic diseases or excessive alcohol consumption. NAFLD was diagnosed by liver ultrasonography whereas AVS was determined by conventional echocardiography in all participants. In the whole sample, 120 (66.7%) patients had NAFLD and 53 (29.4%) had AVS. No patients had aortic stenosis. NAFLD was strongly associated with an increased risk of prevalent AVS (odds ratio [OR] 2.79, 95% CI 1.3-6.1, p

BACKGROUND:The endothelium is not a homogeneous organ. Endothelial cell heterogeneity has been described at the level of cell morphology, function, gene expression, and antigen composition. As a consequence of the genetic, transcriptome and surrounding environment diversity, endothelial cells from different vascular beds have differentiated functions and phenotype. Detection of circulating endothelial cells (CECs) by flow cytometry is an approach widely used in cancer patients, and their number, viability and kinetic is a promising tool to stratify patient receiving anti-angiogenic treatment. METHODOLOGY/PRINCIPAL FINDINGS:Currently CECs are identified as positive for a nuclear binding antigen (DNA+), negative for the pan leukocyte marker CD45, and positive for CD31 and CD146. Following an approach recently validated in our laboratory, we investigated the expression of CD109 on CECs from the peripheral blood of healthy subject and cancer patients. The endothelial nature of these cells was validated by RT-PCR for the presence of m-RNA level of CDH5 (Ve-Cadherin) and CLDN5 (Claudin5), two endothelial specific transcripts. Before treatment, significantly higher levels of CD109+ CECs and viable CD109+CECs were found in breast cancer patients and glioblastoma patients compared to healthy controls, and their number significantly decreased after treatment. Higher levels of endothelial specific transcripts expressed in developing endothelial cells CLEC14a, TMEM204, ARHGEF15, GPR116, were observed in sorted CD109+CECs when compared to sorted CD146+CECs, suggesting that these genes can play an important role not only during embryogenesis but also in adult angiogenesis. Interestingly, mRNA levels of TEM8 (identified as Antrax Toxin Receptor1, Antrax1) were expressed in CD109+CECs+ but not in CD146+CECs. CONCLUSION:Taken together our results suggest that CD109 represent a rare population of circulating tumor endothelial cells, that play a potentially useful prognostic role in patients with glioblastoma. The role of CD109 expression in cancer vessel-specific endothelial cells deserves to be further investigated by gene expression studies.

Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4-1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4-1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future management of AA.

BackgroundThe relationship between non-alcoholic fatty liver disease (NAFLD) and atrial fibrillation (AF) in type 2 diabetes is currently unknown. We examined the relationship between NAFLD and risk of incident AF in people with type 2 diabetes.Methods and resultsWe prospectively followed for 10 years a random sample of 400 patients with type 2 diabetes, who were free from AF at baseline. A standard 12-lead electrocardiogram was undertaken annually and a diagnosis of incident AF was confirmed in affected participants by a single cardiologist. At baseline, NAFLD was defined by ultrasonographic detection of hepatic steatosis in the absence of other liver diseases. During the 10 years of follow-up, there were 42 (10.5%) incident AF cases. NAFLD was associated with an increased risk of incident AF (odds ratio [OR] 4.49, 95% CI 1.6-12.9, pConclusionsOur results indicate that ultrasound-diagnosed NAFLD is strongly associated with an increased incidence of AF in patients with type 2 diabetes even after adjustment for important clinical risk factors for AF.

BackgroundBone metastasis represents an increasing clinical problem in advanced gastric cancer (GC) as disease-related survival improves. In literature, few data on the natural history of bone disease in GC are available.Patients and methodsData on clinicopathology, skeletal outcomes, skeletal-related events (SREs), and bone-directed therapies for 208 deceased GC patients with evidence of bone metastasis were statistically analyzed.ResultsMedian time to bone metastasis was 8 months (CI 95%, 6.125-9.875 months) considering all included patients. Median number of SREs/patient was one. Less than half of the patients (31%) experienced at least one and only 4 and 2% experienced at least two and three events, respectively. Median times to first and second SRE were 2 and 4 months, respectively. Median survival was 6 months after bone metastasis diagnosis and 3 months after first SRE. Median survival in patients who did not experience SREs was 5 months. Among patients who received zoledronic acid before the first SRE, the median time to appearance of first SRE was significantly prolonged compared to control (7 months vs 4 months for control; P: 0.0005).ConclusionsTo our knowledge, this retrospective analysis is the largest multicenter study to demonstrate that bone metastases from GC are not so rare, are commonly aggressive and result in relatively early onset of SREs in the majority of patients. Indeed, our large study, which included 90 patients treated with ZOL, showed, for the first time in literature, a significant extension of time to first SRE and increase in the median survival time after diagnosis of bone metastasis. Taken together, these data may support the beneficial effects of ZOL in GC patients.

OBJECTIVE: Acetylation or deacetylation of histone proteins may modulate cytokine gene transcription such as TNF alpha (TNF). We evaluated the balance between histone deacetytlase (HDAC) and histone acetyltransferase (HAT) in patients with rheumatoid arthritis (RA) or ankylosing spondylitis (AS) compared to healthy controls (HC) and determined the influence of HDAC inhibitors (trichostatin A -TSA- or Sirtinol -Sirt-) on these enzymatic activities and on the PBMC production of TNF. METHODS: 52 patients with RA, 21 with AS and 38 HC were evaluated. HAT and HDAC activities were measured on nuclear extracts from PBMC using colorimetric assays. Enzymatic activities were determined prior to and after ex vivo treatment of PBMC by TSA or Sirt. TNF levels were evaluated in PBMC culture supernatants in the absence or presence of TSA or Sirt. RESULTS: HAT and HDAC activities were significantly reduced in AS, while these activities reached similar levels in RA and HC. Ex vivo treatment of PBMC by HDACi tended to decrease HDAC expression in HC, but Sirt significantly reduced HAT in RA. TNF production by PBMC was significantly down-regulated by Sirt in HC and AS patients. CONCLUSION: HAT and HDAC were disturbed in AS while no major changes were found in RA. HDACi may modulate HDAC and HAT PBMC expression, especially Sirt in RA. Sirtinol was able to down regulate TNF production by PBMC in HC and AS. An imbalance between HAT and HDAC activities might provide the rationale for the development of HDACi in the therapeutic approach to inflammatory rheumatic diseases.

Eccentric gaze in darkness evokes minor centripetal eye drifts in healthy subjects, as cerebellar control sufficiently compensates for the inherent deficiencies of the brainstem gaze-holding network. This behavior is commonly described using a leaky integrator model, which assumes that eye velocity grows linearly with gaze eccentricity. Results from previous studies in patients and healthy subjects suggest caution when this assumption is applied to eye eccentricities larger than 20 degrees. To obtain a detailed characterization of the centripetal gaze-evoked drift, we recorded horizontal eye position in 20 healthy subjects. With their head fixed, they were asked to fixate a flashing dot (50 ms every 2 s)that was quasi-stationary displacing(0.5 deg/s) between ± 40 deg horizontally in otherwise complete darkness. Drift velocity was weak at all angles tested. Linearity was assessed by dividing the range of gaze eccentricity in four bins of 20 deg each, and comparing the slopes of a linear function fitted to the horizontal velocity in each bin. The slopes of single subjects for gaze eccentricities of ± 0-20 deg were, in median,0.41 times the slopes obtained for gaze eccentricities of ± 20-40 deg. By smoothing the individual subjects' eye velocity as a function of gaze eccentricity, we derived a population of position-velocity curves. We show that a tangent function provides a better fit to the mean of these curves when large eccentricities are considered. This implies that the quasi-linear behavior within the typical ocular motor range is the result of a tuning procedure, which is optimized in the most commonly used range of gaze. We hypothesize that the observed non-linearity at eccentric gaze results from a saturation of the input that each neuron in the integrating network receives from the others. As a consequence, gaze-holding performance declines more rapidly at large eccentricities.

Perceived direction of gravity, as assessed by the subjective visual vertical (SVV), shows roll-angle dependent errors that drift over time and a bias upon return to upright. According to Bayesian observer theory, the estimated direction of gravity is derived from the posterior probability distribution by combining sensory input and prior knowledge about earth-vertical in a statistically optimal fashion. Here we aimed to further characterize the stability of SVV during and after prolonged roll-tilts. Specifically we asked whether the post-tilt bias is related to the drift pattern while roll-tilted. Twenty-nine healthy human subjects (23-56 yo) repetitively adjusted a luminous arrow to the SVV over periods of 5 min while upright, roll-tilted (± 45°, ± 90°), and immediately after returning to upright. Significant (p

Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB).rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry.A baseline count of CD109+ CEC higher than 41.1/ml (1(st) quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45-) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacC(KO) strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa.

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca(+2)-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca(+2)-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Chronic hepatitis C is the main cause of death in patients with end-stage liver disease. Prognosis depends on the increase of fibrosis, whose progression is twice as rapid in men as in women. Aim of the study was to evaluate the effects of reproductive stage on fibrosis severity in women and to compare these findings with age-matched men.A retrospective study of 710 consecutive patients with biopsy-proven chronic hepatitis C was conducted, using data from a clinical database of two tertiary Italian care centers. Four age-matched groups of men served as controls. Data about demographics, biochemistry, liver biopsy and ultrasonography were analyzed. Contributing factors were assessed by multivariate logistic regression analysis.Liver fibrosis was more advanced in the early menopausal than in the fully reproductive (P

BACKGROUND: The rotational vestibulo-ocular reflex (rVOR) generates compensatory eye movements in response to rotational head accelerations. The velocity-storage mechanism (VSM), which is controlled by the vestibulo-cerebellar nodulus and uvula, determines the rVOR time constant. In healthy subjects, it has been suggested that self-motion perception in response to earth-vertical axis rotations depends on the VSM in a similar way as reflexive eye movements. We aimed at further investigating this hypothesis and speculated that if the rVOR and rotational self-motion perception share a common VSM, alteration in the latter, such as those occurring after a loss of the regulatory control by vestibulo-cerebellar structures, would result in similar reflexive and perceptual response changes. We therefore set out to explore both responses in patients with vestibulo-cerebellar degeneration. METHODOLOGY/PRINCIPAL FINDINGS: Reflexive eye movements and perceived rotational velocity were simultaneously recorded in 14 patients with chronic vestibulo-cerebellar degeneration (28-81 yrs) and 12 age-matched healthy subjects (30-72 yrs) after the sudden deceleration (90°/s2) from constant-velocity (90°/s) rotations about the earth-vertical yaw and pitch axes. rVOR and perceived rotational velocity data were analyzed using a two-exponential model with a direct pathway, representing semicircular canal activity, and an indirect pathway, implementing the VSM. We found that VSM time constants of rVOR and perceived rotational velocity co-varied in cerebellar patients and in healthy controls (Pearson correlation coefficient for yaw 0.95; for pitch 0.93, p0.8). CONCLUSIONS/SIGNIFICANCE: Our results confirm that self-motion perception in response to rotational velocity-steps may be controlled by the same velocity storage network that controls reflexive eye movements and that no additional, e.g. cortical, mechanisms are required to explain perceptual dynamics.

Prognostic models applied in medicine must be validated on independent samples, before their use can be recommended. The assessment of calibration, i.e., the model's ability to provide reliable predictions, is crucial in external validation studies. Besides having several shortcomings, statistical techniques such as the computation of the standardized mortality ratio (SMR) and its confidence intervals, the Hosmer-Lemeshow statistics, and the Cox calibration test, are all non-informative with respect to calibration across risk classes. Accordingly, calibration plots reporting expected versus observed outcomes across risk subsets have been used for many years. Erroneously, the points in the plot (frequently representing deciles of risk) have been connected with lines, generating false calibration curves. Here we propose a methodology to create a confidence band for the calibration curve based on a function that relates expected to observed probabilities across classes of risk. The calibration belt allows the ranges of risk to be spotted where there is a significant deviation from the ideal calibration, and the direction of the deviation to be indicated. This method thus offers a more analytical view in the assessment of quality of care, compared to other approaches.

Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

BackgroundGain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.Methodology/principal findingsWe describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.Conclusions/significanceOur data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.

Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size ( approximately 2-3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[(18)F]fluoroethyl-D-glucopyranose ([(18)F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [(18)F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells.Dynamic PET/CT imaging demonstrated rapid accumulation of [(18)F]FEDL in peritumoral pancreatic tissue (4.04+/-2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65+/-0.50 min and 14.14+/-3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [(18)F]FEDL in peritumoral pancreatic tissue was estimated as 3.57+/-0.60 and 0.94+/-0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [(18)F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2-4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm.We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [(18)F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [(18)F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

BACKGROUND: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. METHODOLOGY/PRINCIPAL FINDINGS: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. CONCLUSIONS: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.

Fish embryos may be vulnerable to seawater acidification resulting from anthropogenic carbon dioxide (CO2) emissions or from excessive biological CO2 production in aquaculture systems. This study investigated CO2 effects on embryos of the European eel (Anguilla anguilla), a catadromous fish that is considered at risk from climate change and that is targeted for hatchery production to sustain aquaculture of the species. Eel embryos were reared in three independent recirculation systems with different pH/CO2 levels representing "control" (pH 8.1, 300 μatm CO2), end-of-century climate change ("intermediate", pH 7.6, 900 μatm CO2) and "extreme" aquaculture conditions (pH 7.1, 3000 μatm CO2). Sensitivity analyses were conducted at 4, 24, and 48 hours post-fertilization (hpf) by focusing on development, survival, and expression of genes related to acute stress response (crhr1, crfr2), stress/repair response (hsp70, hsp90), water and solute transport (aqp1, aqp3), acid-base regulation (nkcc1a, ncc, car15), and inhibitory neurotransmission (GABAAα6b, Gabra1). Results revealed that embryos developing at intermediate pH showed similar survival rates to the control, but egg swelling was impaired, resulting in a reduction in egg size with decreasing pH. Embryos exposed to extreme pH had 0.6-fold decrease in survival at 24 hpf and a 0.3-fold change at 48 compared to the control. These observed effects of acidification were not reflected by changes in expression of any of the here studied genes. On the contrary, differential expression was observed along embryonic development independent of treatment, indicating that the underlying regulating systems are under development and that embryos are limited in their ability to regulate molecular responses to acidification. In conclusion, exposure to predicted end-of-century ocean pCO2 conditions may affect normal development of this species in nature during sensitive early life history stages with limited physiological response capacities, while extreme acidification will negatively influence embryonic survival and development under hatchery conditions. [ABSTRACT FROM AUTHOR]