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Personalized medicine represents one of the major clinical outcomes of omics applications. Genomics and post-DNA omics allow an unprecedented characterization of the molecular landscape of each individual, endowing modern medicine with new predictive, preventive, personalized, and precision capabilities. Multiple omics techniques are available for all molecular families and over time a huge amount of molecular data has been produced in all the different biological and medical contexts. Their actionability requires a clinically meaningful rationalization, which with the use of bioinformatics has found a synthesis in the holistic approach of systems biology, where big data from omics can be represented as molecular networks of pathophysiological interest. Genomics, epigenomics, transcriptomics, proteomics, and metabolomics represent the major molecular layers of trans-omic networks that could allow Integrated Personal Omics Profiling (iPOP). However, given the intrinsic intercellular variability, bulk analysis can only provide average molecular signals from a multitude of different cells, each following its own fate and with its own epiphenomenal environment, thus limiting the potential of clinical applications of omics data in personalized medicine. Single-cell multiomics, thanks to its rapid development, is emerging as a strategy that can faithfully provide a real picture of the trans-omic relationships in multimolecular networks, highlighting pathogenetic cues that can be utilized in clinical practice. Single-cell multiomics applications are being developed, mostly in cancer, and some are proposing the technology for personalized approaches in other medical fields. Single-cell multiomics could also be exploited to overcome the cellular variability issue limiting the application of post-DNA omics, by applications to undifferentiated pluripotent stem cells, where the individual biological supersystem has yet to undergo the multitude of epiphenomenal events and the molecular pattern is closer to its original state. Dedifferentiation from somatic cells could provide induced pluripotent stem cells for improving the iPOP model in personalized medicine.

Despite significant advances in the diagnosis and treatment of cancer, the development of drug resistance still remains one of the principal causes that hampers the effectiveness of the therapy. Emerging evidences support the idea that the dysregulated metabolism could be related to drug resistance. The major goal of this study was to target cancer metabolic pathways using new pharmacological approaches coming from natural sources in order to possibly prevent or overcome this phenomenon. Firstly, the metabolic profile of human colorectal adenocarcinoma cells sensitive (LoVo WT) and resistant to doxorubicin (LoVo DOX) was delineated demonstrating that resistant cells remodel their metabolism toward a glycolytic phenotype. In particular it was observed that doxorubicin-resistant cancer cells exhibit an increased dependency from glucose for their survival, associated with overexpression of the glycolytic pathway. Moreover, both GLUT1 mRNA and protein expression significantly increased in LoVo DOX cells. Given the results about the metabolic profile, silybin, modulator of GLUTs, was selected as potential candidate to overcome doxorubicin resistance and, intriguingly, data revealed not only that silybin is more active in resistant cells than in wild type cells, but also that the combined treatment with doxorubicin and silybin presents a synergistic effect in LoVo DOX cells. Although many unanswered questions still remain about the molecular mechanism of silybin, these data suggest that targeting GLUTs may be a good strategy to restore doxorubicin sensitivity and elude drug resistance.

Conflicting results have been obtained by clinical studies investigating the effect of liver cirrhosis on enzyme induction. Because ethical concerns do not give consent for methodologically rigorous studies in humans, we addressed this question by examining the effect of the prototypical inducer dexamethasone (DEX) on the pregnane X receptor (PXR)-mediated induction of CYP3A1 and 3A2 in a validated animal model of liver cirrhosis obtained by exposure of rats to carbon tetrachloride. For this purpose, we assessed mRNA levels, protein expressions, and enzymatic activities of both CYP3A enzymes, as well as mRNA and protein expressions of PXR in rat populations rigorously stratified according to the severity of liver insufficiency. Constitutive mRNA and protein expressions of CYP3A1 and CYP3A2 and their basal enzyme activities were not affected by liver dysfunction. DEX treatment markedly increased steady-state mRNA level, protein content, and enzymatic activity of CYP3A1 in healthy and cirrhotic rats, irrespective of the degree of liver dysfunction. On the contrary, the inducing effect of DEX on gene and protein expressions and enzyme activity of CYP3A2 was preserved in moderate liver insufficiency, whereas it was greatly curtailed when liver insufficiency became severe. mRNA and protein expressions of PXR were neither reduced by liver dysfunction nor increased by DEX treatment. These results indicate that even the inducibility of cytochrome P450 isoforms under the transcriptional control of the same nuclear receptor may be differentially affected by cirrhosis and may partly explain why conflicting results were obtained by human studies.

Principal component analysis (PCA) multivariate analysis was applied to study the cytotoxic activity of essential oils from various species of the Pistacia genus on human tumor cell lines. In particular, the cytotoxic activity of essential oils obtained from P. lentiscus, P. lentiscus var. chia (mastic gum), P. terebinthus, P. vera, and P. integerrima, was screened on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). The results indicate that all the Pistacia phytocomplexes, with the exception of mastic gum oil, induce cytotoxic effects on one or more of the three cell lines. PCA highlighted the presence of different cooperating clusters of bioactive molecules. Cluster variability among species, and even within the same species, could explain some of the differences seen among samples suggesting the presence of both common and species-specific mechanisms. Single molecules from one of the most significant clusters were tested, but only bornyl-acetate presented cytotoxic activity, although at much higher concentrations (IC50 = 138.5 µg/mL) than those present in the essential oils, indicating that understanding of the full biological effect requires a holistic vision of the phytocomplexes with all its constituents.

The objective of this study was to compare RT-PCR, Western blot and determination of enzyme activity in the assessment of the induction of cytochromes P450 (CYPs) 1A1 and 1A2 by benzo[a]pyrene (BaP) in Sprague-Dawley and Wistar rats. Inhibition studies and kinetic analyses confirmed literature data indicating that methoxyresorufin is a specific CYP1A2 substrate in both uninduced and BaP-treated rats, whereas ethoxyresorufin is a specific CYP1A1 substrate only in BaP-treated rats. BaP treatment increased mRNA and protein expressions of both CYP1A enzymes to a greater extent in Wistar than Sprague-Dawley rats. It consistently caused a higher increase in mRNA and protein expression of the aryl hydrocarbon receptor in the former rats. By contrast, CYP1A2 enzyme activity was much more markedly increased in Sprague-Dawley than Wistar rats and CYP1A1 activity was induced to similar levels. A BaP-induced increase in the turnover number of CYP1A enzymes in Sprague-Dawley rats, relative to Wistar rats, may provide a plausible explanation for the differential effect of BaP on gene expression and enzyme activity. These results have methodological implications, since they show that RT-PCR and Western blot may not provide a quantitative measure of induction of CYP1A activity, which is the actual measure of the change in CYP1A-mediated metabolism.

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.

Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes.

Methods Selectivity ofthe relationships between hypoxia, HIF2 and SerpinB3 versus SerpinB4 polymorphic isoform expression has been investigated in control HepG2 cells or in HepG2 cells stably transfected to overexpress HIF1 or HIF2 by employing cell and molecular biology techniques. HIF2 and SerpinB3 transcript levels were evaluated (quantitative real-time PCR) on frozen HCC specimens obtained at the time of surgical resection from 67 patientswith cirrhosis of different aetiology. A series of 18 paraffinincluded liver specimens from cirrhotic HCV patients carrying HCC wasalso included in the study for immuno-histochemistry analysis. Results Selectivity of SerpinB3 up-regulation by hypoxia via HIF2 was established by the following experimental evidence: i) HIF2 (not HIF1 ), binds to the Serpin B3 promoter (ChIP assay); ii) only HepG2 overexpressing HIF2 show SerpinB3 upregulation; iii) hypoxic conditions do not affect expression of SerpinB4. Immuno-histochemistry and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation (Spearman r coefficient 0,3533, p

Fucus vesiculosus and Ascophyllum nodosum have been traditionally used for the treatment of obesity and several gastrointestinal diseases. We have recently demonstrated that the phytocomplex obtained from these algae (Gdue™) controls postprandial glucose levels in a mouse model of steatohepatitis, a condition often associated with obesity and type 2 diabetes mellitus. We analyzed the effect of Gdue™ on HOMA index, waist circumference, fasting blood glucose and insulin levels in overweight or obese subjects. Waist circumference decreased significantly after 6 months of treatment (112 ± 17 at t0 vs 105 ± 13 cm after 6 months of treatment; p™ (110 ± 15 at t0 vs 98 ± 15 mg/dL after 6 months for glucose; p0 vs 4.419 ± 2.382 after 6 months; p