INDUCED PLURIPOTENT STEM CELLS: AN INNOVATIVE TOOL TO DISSECT OVARIAN CANCER PATHOGENESIS

Ovarian cancer (OC) has one of the highest death-to-incidence ratios among all tumor types, which points to the need for novel therapeutic and prognostic strategies. Indeed, the absence of relevant tumor cell lines that can recapitulate disease histopathology highlights an acute need for new model systems to study this pathology. In particular, it is still unclear whether the most common and aggressive form of this disease, high grade serous ovarian cancer (HGSOC), could arise from in the ovarian surface epithelium (OSE), as initially thought, or might be arising from the fimbrial epithelium. Here I addressed these issues in two complementary ways based on induced pluripotent stem cells: i) the modeling of Ovarian Cancer by somatic cell reprogramming to pluripotency of tumor cells; ii) the molecular characterization of HGSOC and its putative cells of origin. Somatic cell reprogramming, by erasing tumor-associated epigenetic marks while preserving the underlying genetic mutations, would allow for the first time the precise dissection of genetic and epigenetic contribution to this disease, through the differentiation of OC-iPSC into disease-relevant cell types. I demonstrated the feasibility of OC reprogramming through a non-integrative platform, showing that OC-derived iPSC are closely similar to human ESC, and proving their tumoral origin by whole exome sequencing. Moreover, I showed that independent iPSC clones derived from the same tumor upon trilineage differentiation in vivo show differential tumorigenic potential. For a more precise dissection of this phenotype, I set up a differentiation protocol that allows differentiation of pluripotent cells into mesodermal progenitors, that are precursors of both fimbria and OSE. To isolate a pure population of these cells, I resorted to CRISPR/Cas9 to integrate a selection cassette in the MIXL1 locus. By this approach, I was able to show correct gene targeting at the intended site, allowing also for selection of mesodermal progenitors upon differentiation of normal iPSC. The same approach translated to OC-derived iPSC would allow to study the effects of genetic mutations deprived of tumor-associated epigenetic marks during differentiation, both at the stage of mesodermal progenitors and in cells directed towards the female reproductive epithelium in vivo. The second approach relies on the identification of specific molecular features of fimbria and ovarian surface epithelium, the two putative cells of origin of HGSOC. On this side, I offer a first glimpse on molecular features of HGSOC cancer and normal gynecological tissues. I could show that specific DNA methylation signatures of fimbrial epithelial cells and ovarian surface epithelium cells are partially retained in tumor samples and stratify HGSOC samples according to the putative cell of origin of this tumor. Moreover, I show for the first time a description of histone modifications in primary HGSOC, concentrating on marks of activation/repression sitting on promoter regions (H3K4me3 and H3K27me3, respectively) and marks that characterize active/closed-poised enhancers (H3K4me1, H3K27ac and H3K27me3)

Food ingredients and supplements : is this the future?

The concept of adequate nutrition has been changing in the last years in light of the increased knowledge about the relationship between diet and health and the new evidence on the protective role of numerous bioactive compounds introduced with specific categories of foods. In particular, the concept of adequate nutrition has been substituted with that of optimal nutrition, in other terms, attention has been focused on nutritional needs to optimize physiological functions and promote health, minimizing the risk of development of degenerative diseases. Moreover, it is always more clear that nutritional needs can vary depending on the sub-groups of population considered and also on factors related to individual genetic characteristics, as supported by several nutrigenetic and nutrigenomic studies

Anthocyanins and metabolites resolve TNF-α-mediated production of E-selectin and adhesion of monocytes to endothelial cells

This study investigated the capacity of an anthocyanin-rich fraction (ACN-RF) from blueberry, single anthocyanins (cyanidin, delphinidin and malvidin-3-glucoside; Cy, Dp and Mv-3-glc) and related metabolites (protocatechuic, gallic and syringic acid; PrA, GA and SA) to resolve an inflammation-driven adhesion of monocytes (THP-1) on endothelial cell (HUVECs) and secretion of cell adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). The adhesion of THP-1 to HUVECs was induced by tumour necrosis factor \u3b1 (TNF-\u3b1, 100\u202fng\u202fmL-1). Subsequently, ACN-RF, single ACNs and metabolites (from 0.01 to 10\u202f\u3bcg\u202fmL-1) were incubated for 24\u202fh. The adhesion was measured in a fluorescence spectrophotometer. E-selectin and VCAM-1 were quantified by ELISA. No toxicological effects were observed for the compounds and the doses tested. ACN-RF and Mv-3-glc reducedTHP-1 adhesion at all the concentrations with the maximum effect at 10 \u3bcg/ml (-60.2% for ACNs and-33.9% for Mv-3-glc). Cy-3-glc decreased the adhesion by about 41.8% at 10\u202f\u3bcg\u202fmL-1, while PrA and GA reduced the adhesion of THP-1 to HUVECs both at 1 and at 10\u202f\u3bcg\u202fmL-1 (-29.5% and -44.3% for PrA, respectively, and -18.0%and -59.3% for GA, respectively). At the same concentrations a significant reduction of E-selectin, but notVCAM-1 levels, was documented. No effect was observed following Dp-3-glc and SA supplementation. Overall, ACNs and metabolites seem to resolve, in a dose-dependent manner, the inflammation-driven adhesion of THP-1 to HUVECs by decreasing E-selectin concentrations. Interestingly, Mv-3-glc was active at physiologically relevant concentrations

Different effects of anthocyanins and phenolic acids from wild blueberry (Vaccinium angustifolium) on monocytes adhesion to endothelial cells in a TNF-alpha stimulated proinflammatory environment

SCOPE: Monocyte adhesion to the vascular endothelium is a crucial step in the early stages of atherogenesis. This study aims to investigate the capacity of an anthocyanin (ACN) and phenolic acid (PA)-rich fraction (RF) of a wild blueberry, single ACNs (cyanidin, malvidin, delphinidin) and related metabolites (protocatechuic, syringic and gallic acid) to counteract monocytes (THP-1) adhesion to endothelial cells (HUVECs) in a tumor necrosis \u3b1 (TNF-\u3b1) mediated pro-inflammatory environment. METHODS AND RESULTS: HUVECs were incubated with different concentrations (from 0.01 to 10 \u3bcg mL-1 ) of the compounds for 24 h. Labelled monocytic THP-1 cells were added to HUVECs and their adhesion was induced by TNF-\u3b1 (100 ng mL-1 ). ACN-RF reduced THP-1 adhesion to HUVECs with a maximum effect at 10 \u3bcg mL-1 (-33%). PA-RF counteracted THP-1 adhesion at 0.01, 0.1 and 1 \u3bcg mL-1 (-45%, -48.7% and -27.6%, respectively), but not at maximum concentration. Supplementation with gallic acid reduced THP-1 adhesion to HUVECs with a maximum effect at 1 \u3bcg mL-1 (-29.9%), while malvidin-3-glucoside and syringic acid increased the adhesion. No effect was observed for the other compounds. CONCLUSION: These results suggest that ACNs/PA-RF may prevent atherogenesis while the effects of the single ACNs and metabolites are controversial and merit further exploration

Role of polyphenols and polyphenol-rich foods in the modulation of PON1 activity and expression

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme involved in the protection of low-density lipoprotein and HDLs against lipid peroxidation. Several studies documented the capacity of polyphenols to stimulate PON1 transcription activation. The objective of the present review is to provide the main evidence about the role and the potential mechanism of action of polyphenols and polyphenol-rich foods in the modulation of PON1 gene expression and activity. A total of 76 in vitro and in vivo studies were included in the review. Overall, while evidence obtained in vitro is limited to quercetin and resveratrol, those deriving from animal models seem more convincing for a wide range of polyphenols but only at pharmacological doses. Evidence from human studies is promising but deserves more substantiation about the role of polyphenol-rich foods in the regulation of PON1 activity and expression. Research focused on the understanding of the structure\u2013activity relationship of polyphenols with PON1 and on the mechanisms at the base of PON1 modulation is warranted. Well-designed human intervention studies are encouraged to corroborate the findings of polyphenols also at physiological doses

The central role of iron in human nutrition : from folk to contemporary medicine

Iron is a fundamental element in human history, from the dawn of civilization to contemporary days. The ancients used the metal to shape tools, to forge weapons, and even as a dietary supplement. This last indication has been handed down until today, when martial therapy is considered fundamental to correct deficiency states of anemia. The improvement of the martial status is mainly targeted with dietary supplements that often couple diverse co-factors, but other methods are available, such as parenteral preparations, dietary interventions, or real-world approaches. The oral absorption of this metal occurs in the duodenum and is highly dependent upon its oxidation state, with many absorption influencers possibly interfering with the intestinal uptake. Bone marrow and spleen represent the initial and ultimate step of iron metabolism, respectively, and the most part of body iron circulates bound to specific proteins and mainly serves to synthesize hemoglobin for new red blood cells. Whatever the martial status is, today\u2019s knowledge about iron biochemistry allows us to embrace exceedingly personalized interventions, which however owe their success to the mythical and historical events that always accompanied this metal

Berries and oxidative stress markers: an overview of human intervention studies

Berries are an excellent source of bioactive compounds such as vitamins, minerals but above all polyphenols with anthocyanins as the most representative compounds. Several in vitro and in vivo studies documented the beneficial effects of berries and their bioactives in the modulation of numerous cell functions related to oxidative stress and/or antioxidant protection. The following review summarizes published results about the role of berries (either fresh, juice, freeze-dried or dehydrated) on total plasma and serum antioxidant status and on the modulation of biomarkers of oxidative stress in acute and chronic human intervention trials. The biomarkers considered include DNA, protein and lipid oxidation, and endogenous antioxidant enzymes. Though limited, there is indication that the consumption of berries may reduce oxidative stress by modulating protein and lipid oxidation, and by improving total antioxidant status. In particular, these effects are more evident following chronic dietary interventions with respect to postprandial studies. Benefits are observed in healthy subjects as well as in those with cardiovascular risk factors or other diseases. On the contrary, data regarding the effect of berries on DNA damage and endogenous antioxidant enzyme activities are still scarce and inconclusive. In conclusion, much remains to be elucidated before a comprehensive understanding of the effects of berries on the modulation of oxidative stress markers is achieved. Robust clinical evidence supporting the role of berries in counteracting oxidative stress in humans is encouraged

Effect of two different sublingual dosages of vitamin B12 on cobalamin nutritional status in vegans and vegetarians with a marginal deficiency : a randomized controlled trial

Background & Aims: Vegetarians and vegans are more vulnerable to vitamin B12 deficiency with severe risks of megaloblastic anemia, cognitive decline, neuropathy, and depression. An easy and simple method of supplementation consists of taking one weekly dosage of 2000 \ub5g. However, single large oral doses of vitamin B12 are poorly absorbed. The present research evaluates the ability of two different sublingual dosages of vitamin B12 (350 \ub5g/week vs. 2000 \ub5g/week) in improving cyanocobalamin (vitamin B12) nutritional status in vegans and vegetarians with a marginal deficiency. Methods: A 12-week randomized, double-blind, controlled, parallel intervention trial was performed. Forty subjects with marginal vitamin B12 deficiency were enrolled and randomly divided into two groups: test group Ld (low dose, 350 \ub5g/week) and control group Hd (high dose, 2000 \ub5g/week) vitamin B12 supplementation. Blood samples were collected at baseline and after 15, 30, 60, and 90 days from the intervention for the determination of vitamin B12, related metabolic markers, and blood cell counts. Results: Two-way analysis of variance showed a significant effect of time (P < 0.0001) and of time x treatment interaction (P = 0.012) on serum concentration of vitamin B12. In particular, 90 days of supplementation increased the levels of cyanocobalamin (+81.8% in the Ld group and +144.0% in the Hd group) compared to baseline. A significant increase was observed for the levels of holotranscobalamin (+64.5% in the Ld group and +165.2% in the Hd group), while a decrease occurred for the levels of methylmalonic acid (-72.3% in the Ld group and -69.4% in the Hd group), homocysteine (-56.8% in the Ld group and -53.6% in the Hd group), and folate (-22.8% in the Ld group and -17.7% in the Hd group) compared to baseline (time effect, P < 0.0001). No difference was observed between groups (Ld vs. Hd). No effect was detected for the other variables under study. Conclusions: In our experimental conditions, both supplements were able to restore adequate serum concentrations of vitamin B12 and to improve the levels of related metabolic blood markers in subjects with a marginal deficiency. The results support the use of a sublingual dosage of 50 \ub5g/day (350 \ub5g/week) of cobalamin, instead of 2000 \ub5g/week (provided as a single dose), to reach a state of nutritional adequacy of vitamin B12 in this target population

Effect of fiber and protein-enriched pasta formulations on satiety-related sensations and afternoon snacking in Italian healthy female subjects

The objective of the study was to investigate the effect of consuming different fiber and protein-enriched pasta formulations on satiety response and on mid-afternoon energy intake. Twenty Italian young healthy female subjects participated to a randomized repeated measure study design developed to evaluate the effect on satiety and energy intake of five different pasta formulations, i.e. high fiber, high fiber and high protein, high protein from soy, high protein from egg white, and standard commercial pasta consumed at lunch. The formulations together with a portion of fruit were consumed on five different occasions followed by an ad libitum snack meal proposed 2 h later. Before, immediately after the lunch consumption, and every 30 min until snack time, satiety sensations were assessed by visual analogue scales. In addition, mid-afternoon energy and macronutrient intake consumed with the snacks were calculated. Compared to the control pasta, all the formulations significantly affected satiety-related sensations. Palatability-related attributes of pasta were positively correlated to snack energy intake, whereas fullness sensation was negatively correlated. Among the formulations tested only the fiber and protein-enriched pasta significantly reduced energy intake following the ad libitum snack consumption (p < 0.05). Overall, these findings suggest that pasta enriched with a combination of fiber and protein, might be effective in the modulation of appetite sensations, thus suggesting a new concept-pasta formulation for the modulation of eating behavior. These results are interesting considering that pasta is a staple food in different target groups of the population

Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche.

While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies