When cholesterol is not cholesterol: a note on the enzymatic determination of its concentration in model systems containing vegetable extracts

Background: Experimental evidences demonstrate that vegetable derived extracts inhibit cholesterol absorption in the gastrointestinal tract. To further explore the mechanisms behind, we modeled duodenal contents with several vegetable extracts. Results: By employing a widely used cholesterol quantification method based on a cholesterol oxidase-peroxidase coupled reaction we analyzed the effects on cholesterol partition. Evidenced interferences were analyzed by studying specific and unspecific inhibitors of cholesterol oxidase-peroxidase coupled reaction. Cholesterol was also quantified by LC/MS. We found a significant interference of diverse (cocoa and tea-derived) extracts over this method. The interference was strongly dependent on model matrix: while as in phosphate buffered saline, the development of unspecific fluorescence was inhibitable by catalase (but not by heat denaturation), suggesting vegetable extract derived H2O2 production, in bile-containing model systems, this interference also comprised cholesterol-oxidase inhibition. Several strategies, such as cholesterol standard addition and use of suitable blanks containing vegetable extracts were tested. When those failed, the use of a mass-spectrometry based chromatographic assay allowed quantification of cholesterol in models of duodenal contents in the presence of vegetable extracts. Conclusions: We propose that the use of cholesterol-oxidase and/or peroxidase based systems for cholesterol analyses in foodstuffs should be accurately monitored, as important interferences in all the components of the enzymatic chain were evident. The use of adequate controls, standard addition and finally, chromatographic analyses solve these issues.

Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-Induced oxidative stress and apoptosis.

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Metflx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Metflx/flx and Met−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met-/- oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Metflx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Metflx/flx oval cells, whereas no effect was observed in Met−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.

Natural or naturalized? phylogeography suggests that the abundant sea urchin arbacia lixula Is a recent Colonizer of the Mediterranean

We present the global phylogeography of the black sea urchin Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94-205 kya in the Mediterranean, and 31-67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.

TGF-β dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells

Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-β. We analyzed the oxidative stress induced by TGF-β and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs. Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-β administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-β/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense. Conclusion: The data provide evidence that TGF-β induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

Dietary resveratrol prevents alzheimer's markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer's disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Dietary resveratrol prevents alzheimer's markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer's disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Effect of cocoa powder in the prevention of cardiovascular disease: biological, consumption and inflammatory biomarkers. A metabolomic approach

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have been partly attributed to its polyphenol content, in particular of flavanols. Bioavailability is a key issue for cocoa polyphenols in order to be able to exert their biological activities. In the case of flavanols, bioavailability is strongly influenced by several factors, such as their degree of polymerization and the food matrix in which the polyphenols are delivered. Furthermore, gut has become an active site for the metabolism of procyanidins (oligomeric and polymeric flavanols). Estimation of polyphenol consumption or exposure is also a very challenging task in Food and Nutrition Science in order to correlate the intake of phytochemicals with in vivo health effects. In the area of nutrition, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in Metabolomics or global metabolite analysis. In this chapter we have summarized the most relevant results of our recent research on the bioavailability of cocoa polyphenols in humans and the effect of the matrix in which cocoa polyphenols are delivered considering both targeted analysis and a metabolomic approach. Furthermore, we have also summarized the effect of long-term consumption of cocoa powder in patients at high risk of cardiovascular disease (CVD) on the inflammatory biomarkers of atherosclerosis.

Phylogeography of competing sexual and parthenogenetic forms of a freshwater flatworm: patterns and explanations

Background: Models of the maintenance of sex predict that one reproductive strategy, sexual or parthenogenetic, should outcompete the other. Distribution patterns may reflect the outcome of this competition as well as the effect of chance and historical events. We review the distribution data of sexual and parthenogenetic biotypes of the planarian Schmidtea polychroa. Results: S. polychroa lives in allopatry or sympatry across Europe except for Central and North-Western Europe, where sexual individuals have never been reported. A phylogenetic relationship between 36 populations based on a 385 bp fragment of the mitochondrial cytochrome oxidase I gene revealed that haplotypes were often similar over large geographic distances. In North Italian lakes, however, diversity was extreme, with sequence differences of up to 5% within the same lake in both sexuals and parthenogens. Mixed populations showed "endemic" parthenogenetic lineages that presumably originated from coexisting sexuals, and distantly related ones that probably result from colonization by parthenogens independent from sexuals. Conclusions: Parthenogens originated repeatedly from sexuals, mainly in Italy, but the same may apply to other Mediterranean regions (Spain, Greece). The degree of divergence between populations suggests that S. polychroa survived the ice ages in separate ice-free areas in Central, Eastern and Southern Europe and re-colonised Europe after the retreat of the major glaciers. Combining these results with those based on nuclear markers, the data suggest that repeated hybridisation between sexuals and parthenogenetic lineages in mixed populations maintains high levels of genetic diversity in parthenogens. This can explain why parthenogens persist in populations that were originally sexual. Exclusive parthenogenesis in central and western populations suggests better colonisation capacity, possibly because of inbreeding costs as well

The whereabouts of an ancient wonderer: Global phylogeography of the solitary ascidian Styela plicata

Genetic tools have greatly aided in tracing the sources and colonization history of introduced species. However, recurrent introductions and repeated shuffling of populations may have blurred some of the genetic signals left by ancient introductions. Styela plicata is a solitary ascidian distributed worldwide. Although its origin remains unclear, this species is believed to have spread worldwide by travelling on ship's hulls. The goals of this study were to infer the genetic structure and global phylogeography of S. plicata and to look for present-day and historical genetic patterns. Two genetic markers were used: a fragment of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and a fragment of the nuclear gene Adenine Nucleotide Transporter/ADP-ATP Translocase (ANT). A total of 368 individuals for COI and 315 for ANT were sequenced from 17 locations worldwide. The levels of gene diversity were moderate for COI to high for ANT. The Mediterranean populations showed the least diversity and allelic richness for both markers, while the Indian, Atlantic and Pacific Oceans had the highest gene and nucleotide diversities. Network and phylogenetic analyses with COI and ANT revealed two groups of alleles separated by 15 and 4 mutational steps, respectively. The existence of different lineages suggested an ancient population split. However, the geographic distributions of these groups did not show any consistent pattern, indicating different phylogeographic histories for each gene. Genetic divergence was significant for many population-pairs irrespective of the geographic distance among them. Stochastic introduction events are reflected in the uneven distribution of COI and ANT allele frequencies and groups among many populations. Our results confirmed that S. plicata has been present in all studied oceans for a long time, and that recurrent colonization events and occasional shuffling among populations have determined the actual genetic structure of this species.

Wine, alcohol, polyphenols and cardiovascular disease

Abstract. Excessive alcohol consumption is associated with increased morbidity and mortality as well as with labour and traffic accidents. However, current evidence suggests beneficial effects of moderate drinking on cardiovascular events including coronary heart disease, ischaemic stroke, peripheral arterial disease and congestive heart failure. The underlying mechanisms to explain these protective effects against coronary heart disease include an increase in high-density lipoprotein cholesterol and an increase in insulin sensitivity, and a decrease in platelet aggregation and circulating concentrations of fibrinogen. However, there are discrepancies regarding the specific effects of different types of beverages on the cardiovascular system, and also whether the possible protective effects of alcoholic beverages are due to their alcohol component (ethanol) or non-alcoholic products containing, mainly polyphenols. Recent randomised clinical trials have shown that wine, a polyphenol-rich alcoholic beverage, provides higher antioxidant and anti-inflammatory effects than some spirits such as gin, a polyphenol-free alcoholic beverage. In addition, dealcoholized red wine decreases blood pressure through a nitric oxide mediated mechanism, suggesting a protective effect of polyphenols on vascular function. Other studies performed in women have observed that daily doses of 1520 g of alcohol as red wine are sufficient to elicit protective effects similar to those observed in men who consumed higher doses of wine. In conclusion, moderate consumption of wine exerts a protective effect on biomarkers related to the progression and development of atherosclerosis due to its alcoholic (ethanol) and non-alcoholic (polyphenols) content. Women are more sensitive to the beneficial effects of wine.

Interactions of bacteria and fungi on decomposing litter: differential extracellular enzyme activities

Fungi and bacteria are key agents in plant litter decomposition in freshwater ecosystems. However, the specific roles of these two groups and their interactions during the decomposition process are unclear. We compared the growth and patterns of degradativeenzymes expressed by communities of bacteria and fungi grown separately and in coexistence on Phragmites leaves. The two groups displayed both synergistic and antagonistic interactions. Bacteria grew better together with fungi than alone. In addition, there was a negative effect of bacteria on fungi, which appeared to be caused by suppression of fungal growth and biomass accrual rather than specifically affecting enzyme activity. Fungi growing alone had a high capacity for the decomposition of plant polymers such as lignin, cellulose, and hemicellulose. In contrast, enzyme activities were in general low when bacteria grew alone, and the activity of key enzymes in the degradation of lignin and cellulose (phenol oxidase and cellobiohydrolase) was undetectable in the bacteria-only treatment. Still, biomass-specific activities of most enzymes were higher in bacteria than in fungi. The low total activity and growth of bacteria in the absence of fungi in spite of apparent high enzymatic efficiency during the degradation of many substrates suggest that fungi provide the bacteria with resources that the bacteria were not able to acquire on their own, most probably intermediate decomposition products released by fungi that could be used by bacteria

Genetic diversity and species richness patterns in Baetidae (Ephemeroptera) in the Montseny Mountain range (North-East Iberian Peninsula)

This study aimed to describe patterns of diversity of Baetidae (Ephemeroptera) at the ommunity and population levels within the Montseny Mountain range (North-East Iberian Peninsula). We studied both the distribution of 4 species of baetids in 20 sites among three catchments along the altitudinal gradient (350-1700 masl); and the genetic diversity of the mtDNA cytochrome c oxidase subunit I (cox1) gene of the two common species Baetis alpinus and Baetis rhodani. We found a gradual replacement of the dominant species along the altitudinal gradient. Baetis alpinus inhabited sites at high-altitudes, and this species was replaced by B. rhodani when the altitude decreased. Baetis melanonyx and Alainites muticus attained low abundance at all river sections, and no clear altitudinal trend appeared. Our hypothesis at the population level was that genetic structuring is associated with geographic distance and limited by drainage boundaries among the three studied catchments because of the short-time dispersion of adults. Unexpectedly, analyses of molecular variance (AMOVA) and isolation-bydistance (IBD) showed genetic diversity was unstructured by distance for both species, which may be explained by the relatively short spatial scale studied and small topographic barriers among the three catchments. The Generalized Mixed Yule-Coalescent (GMYC) model showed that B. rhodani had two differentiated genetic lineages that co-occurred in all sites. Overall, diversity of baetids was structured at the community level along the altitudinal gradient, whereas it was unstructured at the population level within the Montseny Mountain range.

Genetic diversity and species richness patterns in Baetidae (Ephemeroptera) in the Montseny Mountain range (North-East Iberian Peninsula)

This study aimed to describe patterns of diversity of Baetidae (Ephemeroptera) at the ommunity and population levels within the Montseny Mountain range (North-East Iberian Peninsula). We studied both the distribution of 4 species of baetids in 20 sites among three catchments along the altitudinal gradient (350-1700 masl); and the genetic diversity of the mtDNA cytochrome c oxidase subunit I (cox1) gene of the two common species Baetis alpinus and Baetis rhodani. We found a gradual replacement of the dominant species along the altitudinal gradient. Baetis alpinus inhabited sites at high-altitudes, and this species was replaced by B. rhodani when the altitude decreased. Baetis melanonyx and Alainites muticus attained low abundance at all river sections, and no clear altitudinal trend appeared. Our hypothesis at the population level was that genetic structuring is associated with geographic distance and limited by drainage boundaries among the three studied catchments because of the short-time dispersion of adults. Unexpectedly, analyses of molecular variance (AMOVA) and isolation-bydistance (IBD) showed genetic diversity was unstructured by distance for both species, which may be explained by the relatively short spatial scale studied and small topographic barriers among the three catchments. The Generalized Mixed Yule-Coalescent (GMYC) model showed that B. rhodani had two differentiated genetic lineages that co-occurred in all sites. Overall, diversity of baetids was structured at the community level along the altitudinal gradient, whereas it was unstructured at the population level within the Montseny Mountain range.

FFQ para el estudio del consumo de cacao (portugués)

Cocoa has been highlighted as a food with potential benefits to human health due to it being animportant source of polyphenols. However, few studies show the contribution of cocoa andchocolate products in polyphenol intake. The aim of this work was to develop and validate afood frequency questionnaire (FFQ) evaluating the intake of food products containing cocoa (CFFQ).A sample of university students was recruited to complete the 90-item questionnaire inSpain and therefore in Spanish (http://diposit.ub.edu/dspace/handle/2445/60475) and in Portugalin a Portuguese version, which is the present document. The developed FFQ questionnaire canbe considered as a valid option for assessing the consumption frequency of cocoa- andchocolate-derived products, thereby allowing the evaluation of cocoa polyphenol intake infurther studies.

Inappropriate prescribing to the oldest old patients admitted to hospital: prevalence, most frequently used medicines, and associated factors

Background: Scientific evidence on treatments of chronic diseases in patients 85 years old or older is very limited, as is available information on inappropriate prescription (IP) and its associated factors. The study aimed to describe medicine prescription, potentially inappropriate medicines (PIM) and potentially prescribing omissions (PPO) and their associated factors on this population. Methods: In the context of an observational, prospective and multicentric study carried out in elderly patients admitted to seven Spanish hospitals for a year, a sub-analysis of those aged 85 years and over was performed. To assess PIMs, the Beers and STOPP criteria were used, and to assess PPOs, the START and the ACOVE-3 criteria were used. To assess factors associated with IP, a multivariate logistic regression analysis was performed. Patients were selected randomly every week on consecutive days from the hospitalization lists. Results: A total of 336 patients were included in the sub-analysis with a median (Q1-Q3) age of 88 (8690) years. The median medicines taken during the month prior to admission was 10 (713). Forty-seven point two per cent of patients had at least one Beers-listed PIM, 63.3% at least one STOPP-listed PIM, 53.6% at least one START-listed PPO, and 59.4% at least one ACOVE-3-listed PPO. Use of benzodiazepines in patients who are prone to falls (18.3%) and omission of calcium and vitamin D supplements in patients with osteoporosis (13.3%) were the most common PIM and PPO, respectively. The main factor associated with the Beers-listed and the STOPP-listed PIM was consumption of 10 or more medicines (OR = 5.7, 95% CI 1.8-17.9 and OR = 13.4, 95% CI 4.0-44.0, respectively). The main factors associated with the START-listed PPO was a non-community dwelling origin (OR 2.3, 95% CI 1.0-5.0), and multimorbidity (OR1.8, 95% CI 1.0-3.1). Conclusions: Prescribed medicines and PIM and PPO prevalence were high among patients 85 years and over. Benzodiazepine use in those who are prone to falls and omission of calcium and vitamin D in those with osteoporosis were the most frequent PIM and PPO, respectively. Factors associated with PIM and PPO differed with polypharmacy being the most important factor associated with PIM.