Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus - associated hepatocellular carcinoma.

Background: The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. Methods: We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR)and confocal immunofluorescence. Results: Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling Pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. Conclusions: Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets

Spectroscopic and in silico studies on the interaction of substituted pyrazolo[1,2-a]benzo[1,2,3,4]tetrazine-3-one derivatives with c-myc g4-dna

Herein we describe a combined experimental and in silico study of the interaction of a series of pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives (PBTs) with parallel G-quadruplex (GQ) DNA aimed at correlating their previously reported anticancer activities and the stabilizing effects observed by us on c-myc oncogene promoter GQ structure. Circular dichroism (CD) melting experiments were performed to characterize the effect of the studied PBTs on the GQ thermal stability. CD measurements indicate that two out of the eight compounds under investigation induced a slight stabilizing effect (2–4 °C) on GQ depending on the nature and position of the substituents. Molecular docking results allowed us to verify the modes of interaction of the ligands with the GQ and estimate the binding affinities. The highest binding affinity was observed for ligands with the experimental melting temperatures (Tms). However, both stabilizing and destabilizing ligands showed similar scores, whilst Molecular Dynamics (MD) simulations, performed across a wide range of temperatures on the GQ in water solution, either unliganded or complexed with two model PBT ligands with the opposite effect on the Tms, consistently confirmed their stabilizing or destabilizing ability ascertained by CD. Clues about a relation between the reported anticancer activity of some PBTs and their ability to stabilize the GQ structure of c-myc emerged from our study. Furthermore, Molecular Dynamics simulations at high temperatures are herein proposed for the first time as a means to verify the stabilizing or destabilizing effect of ligands on the GQ, also disclosing predictive potential in GQ-targeting drug discovery

The role of iron and copper on the oligomerization dynamics of DR_2577, the main S-layer protein of deinococcus radiodurans

Surface (S)-layers are cryptic structures that coat the external surface of the bacterial cell in many species. The paracrystalline regularity of the S-layer is due to the self-assembling of one or more protein units. The property of self-assembling seems to be mediated by specific topologies of the S-layer proteins as well as the presence of specific ions that provide support in building and stabilizing the bi-dimensional S-layer organization. In the present study, we have investigated the self-assembling mechanism of the main S-layer protein of Deinococcus radiodurans (DR_2577) finding an unusual role played by Fe3+ and Cu2+ in the oligomerization of this protein. These findings may trace a structural and functional metallo-mediated convergence between the role of these metals in the assembling of the S-layer and their well-known roles in protecting against oxidative stress in D. radiodurans

Contractile reserve in systemic sclerosis patients as a major predictor of global cardiac impairment and exercise tolerance

Several studies have evidenced high prevalence of myocardial systolic and diastolic dysfunction among patients with systemic sclerosis (SSc). Exercise echocardiography has shown a diagnostic and prognostic role in identifying early left ventricular (LV) dysfunction in several myocardial pathological settings. The aim of our study was to evaluate early signs of LV impairment under exercise and their correlation to patient's exercise tolerance. Forty-five patients (age 60.4 ± 10.3 years) with SSc and 20 age and sex comparable controls were enrolled in the study. All patients underwent clinical evaluation, 2D echocardiography associated with Tissue Doppler and speckle tracking to evaluate LV deformation indexes, and an exercise echocardiography to evaluate left ventricle contractile reserve (LVCR) and exercise pulmonary pressures. Finally, a 6-minute walking test (6MWT) to evaluate exercise tolerance was also performed. Compared to controls, SSc patients showed an impaired diastolic function (E/E' 10.9 ± 3.7 vs 8.36 ± 2.01; p < 0.01) associated with larger left atrial dimensions (LAVI 28.4 ± 8.7 vs 19.3 ± 4.6 mL/m2; p < 0.01). During exercise echocardiography, a reduced global longitudinal strain at peak exercise (S-GLS) was highlighted compared to controls (15.7 ± 3.6 vs 18.2 ± 2.2; p = 0.001). A S-GLS cutoff <18 %, identified by ROC analysis, identified SSc patients with a reduced diastolic function, exercise tolerance at the 6MWT and higher pulmonary pressures. Our data show that in SSc patients a reduced LVCR characterizes the patients with a more extensive cardiovascular impairment in terms of LV diastolic function, pulmonary pressures and exercise tolerance. These data underline the importance of exercise echocardiography for the preclinical screening of the LV impairment in this population

Association of chronic hepatitis C with major depressive disorders: irrespective of interferon-alpha therapy

Abstract Background Mood and anxiety symptoms in chronic hepatitis C (CHC) may be related to the patient awareness of the diagnosis and prognosis, to side effects induced by interferon (IFN)-alpha treatment, as well as to substance abuse. However, the observation of metabolic alterations in patients with CHC has led to hypothesize a direct effect of hepatitis C virus (HCV) on brain function. This study was aimed at elucidating whether CHC is associated with specific anxiety or mood disorders independently of confounding factors. Methods Patient cohort: consecutive patients, 135 with CHC and 76 with chronic hepatitis B (CHB). Exclusion criteria: previous treatment with IFN-alpha, co-infection with HCV and hepatitis B virus, infection with human immunodeficiency virus, drug or alcohol abuse, or malignancies. Controls: subjects without evidence of hepatitis randomly extracted from the database of a previous epidemiological study; they were divided into two groups of 540 (332 males) and 304 (220 males) as controls for patients with CHC and CHB, respectively. The psychiatric diagnosis was formulated by means of the Composite International Diagnostic Interview Simplified carried out by a physician according to DSM-IV criteria. Results A higher lifetime prevalence of major depressive disorder (MDD) was observed among CHC compared to CHB or controls. The risk of MDD was not statistically different between CHB and controls. Both the CHC and CHB groups showed a significantly higher frequency of panic disorder when compared to controls. No statistical differences were observed in the prevalence of general anxiety disorder and social phobia when CHC or CHB were compared to controls. Conclusion The present study provides the first evidence of an association between CHC and MDD, diagnosed on the basis of well-defined international criteria. This association is independent of treatment with IFN-alpha and is not influenced by substance or alcohol abuse. By contrast, anxiety disorders do not appear to be specifically associated with CHC

Characterization of antibody-mediated neutralization directed against the hypervariable region 1 of hepatitis C virus E2 glycoprotein

The hypervariable region 1 (HVR1) comprising the first 27 aa of E2 glycoprotein is a target for neutralizing antibodies against hepatitis C virus (HCV), but the mechanisms of this neutralization in the cell-culture-infectious genotype 2a strain JFH1 HCV virus (HCVcc) system are unknown. Two rabbit polyclonal sera, R1020 and R140, recognizing the HVR1 of the genotype 1a isolates H77c and Glasgow (Gla), respectively, and a Gla HVR1-specific mouse mAb AP213 have been described previously. However, attempts to generate of antibodies to the JFH1 HVR1 were unsuccessful. Therefore, this study produced chimeric JFH1 HCVcc viruses harbouring the H77c or Gla HVR1 to assess the reactivity of antibodies to this region and their effects on virus infectivity. The inter-genotypic HVR1 swap did not significantly affect virus infectivity. The genotype 1a HVR1-specific antibodies neutralized chimeric viruses in an isolate-dependent manner, underlining the role of HVR1 in HCV infection. The neutralizing antibodies reacted mainly with the C-terminal portion of HVR1, and detailed mapping identified A17, F20 and Q21 in the Gla HVR1 sequence and T21 (and possibly L20) in the corresponding H77c sequence as key epitope residues for AP213 and R140, and R1020, respectively. Importantly, none of the antibodies inhibited in vitro binding of viral envelope glycoproteins to the best-characterized HCV receptor, CD81, or to the glycosaminoglycan attachment factors. However, the HVR1 antibodies were capable of post-attachment neutralization. Overall, this study emphasizes the role of HVR1 in HCVcc entry and provides new tools to study this region further in the context of complete virions

An integrated care pathway for cancer patients with diabetes: A proposal from the Italian experience

Diabetes and cancer frequently coexist in the same subject, often with relevant clinical effects on the management and prognosis of the comorbid patient. The existing guidelines, however, do not appropriately address many clinical issues in this setting. Although collaboration between diabetologists and oncologists should play an important role in achieving appropriate levels of care, close coordination or agreement between these specialists is seldom offered. There is an urgent need for greater interdisciplinary integration between all specialists involved in this setting, for a shared approach ensuring that organisational silos are overcome. To this end, the Italian Associations of Medical Diabetologists (AMD) and the Italian Association of Medical Oncology (AIOM) recently established a dedicated Working Group on 'Diabetes and Cancer'. The working group outlined a diagnostic and therapeutic clinical pathway dedicated to hospitalised patients with diabetes and cancer. In this article, we describe the Italian proposal including some suggested measures to assess, monitor and improve blood glucose control in the hospital setting, to integrate different specialists from both areas, as well as to ensure discharge planning and continuity of care from the hospital to the territory

On farm agronomic and first environmental evaluation of oil crops for sustainable bioenergy chains.

Energy crops, and in particular oil crops, could be an important occasion for developing new non food production rows for a new multi-functional agriculture in Italy. In this view, the use of local biomass is a fundamental starting point for the development of a virtuous energy chain that should pursue not only agricultural profitability, but also chain sustainability and that is less dependent on the global market, characterized by instability in terms of biomass availability and price. From this perspective, particular attention must be paid to crop choice on the basis of its rusticity and of its adaptability to local growing conditions and to low input cropping systems. In this context, alike woody and herbaceous biomasses, oil crops such as sunflower and rapeseed should be able to support local agricultural bioenergy chain in Italy. In addition, in a local bioenergy chain, the role of the farmers should not be limited just to grain production; but also grain processing should be performed at farm or consortium level in oilseed extraction plants well proportioned to the cropped surface. In this way, by means of a simple power generator, farmer could thus produce its own thermal and electric energy from the oil, maximizing his profit. This objective could also be achieved through the exploitation of the total biomass, including crop residues and defatted seed meals, that may be considered as fundamental additional economic and/or environmental benefits of the chain. This paper reports some results of three-years on-farm experiments on oil crop chain carried out in the framework of "Bioenergie" project, that was focused to enhance farmers awareness of these criteria and to the feasibility at open field scale of low-input cultivation of rapeseed, sunflower and Brassica carinata in seven Italian regions. In several on-farm experiences, these crops produced more than 800 kg ha-1 of oil with good energy properties. Defatted seed meals could be interesting as organic fertilizers and, in the case of B. carinata, as a biofumigant amendment that could offer a total or partial alternative to some chemicals in agriculture. Furthermore, biomass soil incorporation could contribute to C sequestration, catching CO2 from atmosphere and sinking a part in soil as stable humus. Finally, four different open field experiences carried out again in the second year of the project, have been analysed in order to evaluate their energy and greenhouse gasses balance after cultivation phase