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Nutrigenetics concerns individual differences in the reaction to food based on the genetic factors. Nutrigenomics analyses direct influences of nutrients on gene expression. Both terms are explained in our review article. Nutrigenetics and nutrigenomics require a deep understanding of nutrition, genetics and biochemistry and of new ‘omic’ technologies. It is often difficult to appreciate their relevance in the practice of preventive approaches for optimizing health, delaying onset of disease and diminishing its severity. This paper represents a review about the current research on Nutrigenomics and Nutrigenetics, and provide scientific data for a new integrated clinical approach.

Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.

The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (1). Mitochondria, beyond being the site of aerobic respiration, are at the crossroads of a variety of metabolic and signaling pathways resulting key regulatory organelles in cell life and death decision. Thus, it is no surprise that genomic, functional, and structural mitochondrial alterations have been associated with cancer and that mitochondria have become a pharmacological target in cancer therapy (2). Proliferating tumor cells show increased glycolysis and convert the majority of glucose to l-lactate, even in normoxic conditions. This is known as the Warburg effect. Actually, in many tumors, mitochondria are not defective in oxidative phosphorylation, and in the last decade, the molecular basis of Warburg effect has been reconsidered in the context of a set of concerted changes in energy metabolism and mitochondrial function that support tumorigenesis. This process, referred to as reprogramming of energy metabolism, is an emerging hallmark of cancer development (3, 4). This Research Topic presents one review, five mini-reviews, and an opinion article on the achievements and perspectives of studies on important aspects of cancer cell metabolic reprogramming whose mechanisms and regulation are still largely elusive. It also sheds light on certain novel functional components, which rewires cell metabolism in tumor transformation.

"Positive-strand RNA [(+)RNA] viruses include pathogens responsible for a number of diseases in humans, animals and plants. (+)RNA virus replication invariably occurs in association with specific host cell membranes, which are extensively rearranged to form partially enclosed vesicular enclaves, to scaffold and protect genome replication from the host plant defense reaction. The association of (+)RNA virus replication with the limiting membrane of peroxisomes and mitochondria has been studied with members of the genus Tombusvirus (family Tombusviridae). The tombusvirus carnation Italian ringspot virus (CIRV) genomic (+)RNA replication occurs on the mitochondrial outer membrane to produce numerous vesicles between the inner and outer membrane. CIRV p36 protein is required for targeting and anchoring the virus replication complex to the mitochondrial outer membrane in plant and when ectopically expressed in Saccharomyces cerevisiae. Interaction of CIRV p36 with yeast mitochondria is associated with increase in necrotic cell death and concomitant decrease in regulated cell death in response to acetic acid. Thus, in order to investigate p36-mitochondria interaction, we analyzed yeast cells expressing CIRV p36 under the control of the inducible GAL1 promoter, and measured several parameters of mitochondrial function. Concomitantly we also expressed the p33 replicase protein of another tombusvirus (cymbidium ringspot virus, CymRSV), which is known to be targeted to the endoplasmic reticulum in yeast cells. Endogenous and CCCP-stimulated respiration was dramatically reduced in p36-expressing yeast cells as compared with control cells. Similar results were obtained with isolated mitochondria using succinate as a substrate. A significant reduction of the activity of complexes II + III and IV was observed in yeast spheroplasts. No significant changes in mitochondrial respiration were observed in yeast cells expressing p33. Immunoblot analysis of either whole cell lysates or cell membrane-enriched fractions from p36- and p33-expressing or control cells showed that the level of marker proteins of mitochondrial matrix, inner and outer membrane was not changed by p36/p33 expression. These data suggest that p36 specifically alters mitochondrial function, without affecting mitochondrial biogenesis in yeast."

"Positive-strand RNA [(+)RNA] viruses, the largest class of viruses infecting humans, animals and plants, share common replication mechanisms. A feature conserved among these viruses is the association of the viral replication complex with specific intracellular membranes, which are rearranged and are induced to proliferate to form partially closed vesicles in which virus replication takes place. The (+)RNA Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae) replication in plants occurs in membranous structures derived from modified mitochondria. The determinants targeting CIRV replication complex to the mitochondrial outer membrane are contained in the non structural protein p36, which is indispensable for virus replication. However, the function and biological role of p36 are still elusive. Many DNA and RNA virus proteins behave as proapoptotic or antiapoptotic viral factors, either by direct or indirect interaction with the mitochondrial outer membrane. The yeast Saccharomyces cerevisiae has been widely used as a model host to study the replication of (+)RNA viruses, as well as cell death and survival processes. In view of the interaction of CIRV p36 with the mitochondrial outer membrane and the known role of mitochondria in cell fate decisions, some biological effects of p36 expression in yeast cells were investigated along with the possible effect of CIRV p36 on cell survival and death. The viability of yeast cells expressing p36 cloned under the inducible GAL1 promoter, in the absence or presence of 160 mM acetic acid (AA), was analyzed as a function of time by counting colony forming units (cfu) on YPD plates after 48 h growth at 30°C. Plasma membrane integrity was measured by propidium iodide (PI) staining. Phosphatidylserine (PS) exposure and membrane integrity were detected by a FITCcoupled Annexin V reaction. Cells were observed in an Axioplan 2 microscope (Zeiss). Protein analyses were performed either on whole cell lysates or after spheroplasting and separation into a membrane-enriched pellet and a supernatant fractions. It was shown that p36 expression was stable over time and affected cell growth rate, but did not promote cell death. In addition, p36 could change the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. Acetic acid treatment did not impair the strict association of p36 with mitochondrial membranes, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria. Yeast proved to be an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses, for fundamental studies and for the development of antiviral strategies.

Positive-strand RNA [(+)RNA] viruses, the largest class of viruses, include many important pathogens of humans, animals and plants, sharing common replication mechanisms. A highly conserved feature of (+)RNA virus replication is the association of the viral replication complex with specific intracellular membranes, which are induced to proliferate and are extensively rearranged to form vesicles (or spherules). These partially closed vesicular enclaves constitute the confined environment in which virus and host factors concentrate to allow for a productive viral RNA synthesis, under conditions protected from host defense reactions. Virus-encoded proteins are responsible for the intracellular localization of the replication complex and for the formation of spherules. The association of viral replicase proteins with the outer membrane of mitochondria has been studied in details with Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae), a virus with a (+)RNA genome 4.8 kb in size, containing five ORFs. In infected plants, CIRV replication takes place in membranous structures originating from vesiculation of the mitochondrial outer membrane. The signals targeting and anchoring CIRV replication complex to the mitochondrial membrane are contained in the 36-kDa product of ORF1 (p36). Most traits of CIRV replication can be reconstituted in Saccharomyces cerevisiae cells, thus representing a good model for virus-host interaction studies. Heterologous expression of p36 protein fused or not to GFP localizes to mitochondria in yeast cells and causes organelle and membrane proliferation. To gain insights into the interaction between p36 and mitochondria, the effects were studied of p36 heterologous expression on yeast cell viability as well as on programmed cell death induced by acetic acid. It was shown that p36 affects cell viability and seems to exert an inhibitory effect on the nature of acetic acid-induced cell death. Due to the conservation of replication mechanisms between (+)RNA viruses, data obtained with simple model viruses, like CIRV, in a simple eukaryotic host, could be extended to pathogens of higher eukaryotes.

The replication of positive-strand RNA [(+)RNA] viruses is always associated with specific cell membranes. The association of viral replicase proteins with mitochondria has been studied with Carnation Italian ringspot virus (CIRV), a (+)RNA plant virus replicating in membranous structures originating from vesiculation of the mitochondrial outer membrane. The replication-associated protein p36 is responsible for targeting and anchoring CIRV replication complex to the mitochondrial membrane. Yet, the mechanisms by which (+)RNA viruses control host cell fate are still unknown.Programmed cell death (PCD) is a primordial response of virus-infected cells, whose mechanism is conserved from yeast to mammals.Taking advantage of the successful CIRV replication in the yeast Saccharomyces cerevisiae,we investigated whether and how the heterologous expression of the mitochondria-targeted CIRV p36 alone or in combination with the replicase protein p95 and DI RNA could affect yeast cell viability or stress-induced PCD. S. cerevisiae YPH499 cells were transformed with plasmids containing the CIRV p36 sequence, fused or not to GFP, cloned under the control of the inducible GAL1 promoter. Acetic acid-induced PCD (AA-PCD) was triggered and cell viability measured as in Giannattasio et al. (2005).The type of yeast cell death was analyzed by measuring plasma membrane integrity and PS externalization by propidium iodide and FITC-annexin V staining, respectively, and fluorescence microscopy. Heterologous expression of p36/p95 did not affect yeast cell viability up to 72 h whereas p36 alone slowed down cell growth after 24 h expression compared to control cells, with virtually no loss in plasma membrane integrity. Interestingly,after AA-PCD induction, p36-expressing cells lost viability at the same rate as the controls but showed a change in the type of cell death judged by apoptotic and necrotic marker analysis. S. cerevisiae is a powerful model organism for virus-host interaction studies. Due to the conservation of replication mechanisms among (+)RNA viruses, the data obtained with plain model viruses in this simple eukaryotic host can potentially be transferred to pathogens of higher eukaryotes. In addition, this system can be extended to investigate cellular pathways such as PCD.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable.

Some experimental studies have been conducted upon the marine snail Nassarius mutabilis (Gastropoda, Prosobranchia) at Pesaro's Marine Department in the Adriatic Sea, from 2002-2005.

Molecular analysis of 289 chromosomes has been performed in a cohort of phenylketonuria (PKU) patients whose ancestors lived in five Italian regions, Calabria, Campania, Piemonte, Puglia/Basilicata and Sicilia. Phenylalaninehydroxylase (PAH) gene mutations and minihaplotypes (combinations of PAH gene STR and VNTR systems) have been determined for 78.5 and 64%, respectively, of the chromosomes studied. 21 different minihaplotypes and 24 PKU mutations were found. Heterogeneity tests carried out for the frequencies of mutations and minihaplotypes show that the distribution of eight mutations and four minihaplotypes is statistically heterogeneous in the five Italian regions. Although the evolutionary rate of microsatellites or the age of these mutations is difficult to estimate with accuracy, our findings taken together show a genetic stratification of the Italian population. These results rule out allelic homogeneity of PKU at the molecular level between regions of Italy, yet minihaplotype data may be of practical use for a multistep approach to PAH gene genotyping.

Microorganisms face constant stressful conditions, such as weak acid stress, both in natural habitats and during their use for biotechnological applications. Microbes respond to stress by activating either cell adaptation or death pathways. Yeast Saccharomyces cerevisiae has been a valuable model to study the mechanisms of cell response to stressful environmental changes. This chapter summarizes current knowledge on molecular mechanisms of general weak acid stress response and programmed cell death in response to acetic acid as unraveled in S. cerevisiae. Future perspectives aimed at clarifying the complex intracellular signaling networks, integrating cell adaptation and death pathways in response to acetic acid stress are envisaged. Elucidation of finely regulated integration mechanisms of such pathways represents a challenge for understanding aspects of eukaryotic cell homeostasis as well as for improving the performance of a given yeast strain in industrial processes and applications.

Programmed cell death can occur through two separate pathways caused by treatment of Saccharomyces cerevisiae with acetic acid (AA-PCD), which differ from one another essentially with respect to their sensitivity to Nacetylcysteine (NAC) and to the role played by cytochrome c and metacaspase YCA1. Moreover, yeast can also undergo macroautophagy which occurs in NAC-insensitive manner. In order to gain some insight into the relationship between AA-PCD and macroautophagy use was made of WT and knock-out cells lacking YCA1 and/or cytochrome c. We show that i. macroautophagy is modulated by YCA1 and by cytochrome c in a negative and positive manner, respectively, ii. the NAC-insensitive AA-PCD and macroautophagy differ from one another and iii. NAC-insensitive AA-PCD pathway takes place essentially without macroautophagy, even if the shift of extracellular pH to acidic values required for AA-PCD to occur leads itself to increased or decreased macroautophagy in YCA1 or cytochrome c-lacking cells.

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Since cystic fibrosis (CF) is the most common lethal hereditary monogenic disease in Caucasian populations with an average prevalence of 1:2500 newborns, population screening by genetic testing for CF prenatal diagnosis and clinical phenotype determination seems worthwhile to implement in all countries as is the case for other common inherited disease screening protocols. Worldwide mutational analysis of the CF transmembrane regulator (CFTR) gene has revealed that p.F508del mutation accounts on the average for 70-80% of CF chromosomes from northern Europe and America. Yet, more than 1500 mutations of CFTR have been identified to cause CF in different populations. In addition, the phenotypic spectrum associated with mutations in the CFTR gene extends beyond the classically defined CF. Due to the high allelic and phenotypical heterogeneity of CF chromosomes, it is important to identify the CFTR mutation

In Saccharomyces cerevisiae, SAL1 encodes a Ca(2+)-binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca(2+)-binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/P(i) exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2(R252I) and Aac2(R253I) variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V(-)R(+) Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.

To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.

To gain further insight into the mechanism by which yeast programmed cell death (PCD) occurs, we investigated whether and how proteasome activity changes in Saccharomyces cerevisiae cells undergoing PCD as a result of treatment with acetic acid (AA-PCD). We show that proteasome activation starts 60 min after AA-PCD induction, with a maximum at 90 min, and decreases at 150 min. Moreover, cell survival measurements carried out in the absence or presence of MG132, which inhibits proteasome function, show that the inhibition of proteasome activity partially prevents AA-PCD, thus indicating that a transient proteasome activation is needed for AA-PCD to occur.

Yeast cells lacking the metacaspase-encoding gene YCA1 (Deltayca1) were compared with wild-type (WT) cells with respect to the occurrence, nature and time course of acetic-acid triggered death. We show that Deltayca1 cells undergo programmed cell death (PCD) with a rate lower than that of the WT and that PCD in WT cells is caused at least in part by the caspase activity of Yca1p. Since in Deltayca1 cells this effect is lost, but z-VAD-fmk does not prevent both WT and Deltayca1 cell death, PCD in WT cells occurs via a Yca1p caspase and a non-caspase route with similar characteristics.