The ∆cTG-AUC was significantly lower in males with low alcohol intake (3.0 ± 1.9 mmol·h/L) (n = 27) compared to males with no (7.0 ± 1.8 mmol·h/L) (n = 34), moderate (6.5 ± 1.8 mmol·h/L) (n = 54) or high alcohol intake (7.2 ± 2.2 mmol·h/L) (n = 24), when adjusted for age, smoking and body mass index . In males, low alcohol intake was associated with decreased diurnal triglyceridemia, whereas moderate and high alcohol intake was associated with increased triglycerides after dinner and at bed time.Fasting and postprandial triglyceride concentrations largely depend on dietary and lifestyle factors. Alcohol intake is associated with triglycerides, but the effect of alcohol on diurnal triglyceridemia in a free living situation is unknown. During three days, 139 men (range: 18–80 years) measured their own capillary triglyceride (cTG) concentrations daily on six fixed time-points before and after meals, and the total daily alcohol intake was recorded. The impact of daily alcohol intake  on diurnal triglyceridemia was analyzed by the incremental area under the cTG curve (∆cTG-AUC) reflecting the mean of the six different time-points. Fasting cTG were similar between the alcohol groups, but a trend of increased cTG was observed in men with moderate and high alcohol intake after dinner and at bedtime ( Many factors determine fasting and postprandial plasma triglycerides (TG)  like genThe effect of alcohol consumption on diurnal triglyceridemia has not yet been studied and data on the impact of alcohol on both fasting and non-fasting TG in an uncontrolled real life situation are limited. In this observational study we investigated the impact of alcohol consumption on capillary TG concentrations (cTG) during the day determined in free living male subjects, using a hand-held point-of-care testing device ,23,24,25Subjects were healthy male volunteers or patients, aged from 18 to 80 years, with hyperlipidemia, a known medical history of cardiovascular disease (CVD) or type 2 diabetes mellitus (T2DM) participating in several clinical studies with the same protocol, aimed at investigating factors influencing postprandial lipemia ,23,24,252), were determined using a Vitros 250 analyzer . Plasma apolipoprotein (apo) B was measured by nephelometry using apo B monoclonal antibodies . Plasma apo AI was measured by nephelometry using apo AI monoclonal antibodies . Plasma glucose was measured by glucose oxidase dry chemistry  and colorimetry, and insulin was measured by competitive radio immunoassay with polyclonal antibodies. Low density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula.Fasting blood samples were collected at inclusion after a period of 12 h fasting for measurement of lipids, apolipoproteins, insulin and glucose from plasma. Cholesterol, plasma triglycerides (TG) and high density lipoprotein (HDL) cholesterol . Subjects were instructed to carefully wash and dry their hands thoroughly before each measurement. With a lancing device, a drop of blood (30 μL) from the finger was obtained, which was applied to the TG test strip in the TG analyzer. The subjects recorded the diurnal TG profile in a diary. They were instructed to measure their cTG for three days  on the following fixed time-points: fasting, before lunch, 3 h after lunch, before dinner, 3 h after dinner and at bedtime. Subjects were requested to avoid heavy physical activity, although normal daily activities were allowed. The measurement range for cTG was 0.80–6.86 mmol/L. The TG-analyser is known to measure TG reliably, regardless of the nature of the TG-carrying lipoprotein species. The correlation coefficient between cTG measurements with the TG-analyser and plasma measurements according to enzymatic methods is 0.94 [Self-measurements of cTG were performed with a hand-held point-of-care testing device  and smoking. Diurnal triglyceridemia was defined as the incremental area under the curve (∆cTG-AUC). Dietary intake and diurnal triglyceridemia were calculated using the average over two or three days. Linear regression analysis was used to test for significant differences in diurnal triglyceridemia between the different alcohol groups with and without adjustment for age, BMI and smoking, since BMI closely correlates with TG and alcohol intake interacts with both smoking and TG [p < 0.05 (two-sided).Data were obtained from different studies using the same protocol. Data are given as mean ± SD unless stated otherwise. Alcohol intake was divided into: none, low (<10 g/day), moderate (10–30 g/day) and high (>30 g/day). Differences between groups were tested by ANOVA with Bonferroni as g and TG . Logaritp for trend 0.07). No differences were found in fasting plasma TG between males with different amounts of alcohol consumption. Total energy intake was similar between alcohol groups  and at bedtime (p for trend <0.001), which persisted after adjustment for age, BMI and smoking   . Low alcp < 0.05)  .p = 0.006), but not in men (p = 0.23), although men showed a similar trend [Our study suggests that low alcohol intake is associated with a decrease in diurnal triglyceridemia in males after adjustment for age, BMI and smoking. Our results are in line with a recent meta-analysis and other studies on alcohol consumption, suggesting that small to moderate alcohol consumption is associated with slightly lowered plasma TG ,8. A recar trend . The assper se is less important than the amount of alcohol consumed [We also observed a trend that moderate and high alcohol intake was associated with increased postprandial TG after dinner and at bedtime. The lack of differences before dinner most likely reflects an efficient TG clearance mechanism in this relatively healthy population. Moreover, since alcohol consumption occurred mostly around dinner as is usually the case in a healthy population , the mosconsumed . In contconsumed . IncreasClear mechanistic metabolic explanations are present to explain the increase in TG upon high alcohol intake. Alcohol has been shown to stimulate the secretion of very low density lipoproteins, to reduce lipoprotein lipase activity, to stimulate adipose tissue lipolysis resulting in elevated hepatic delivery of free fatty acids and to impair the oxidation of fatty acids in the mitochondria ,32,33,34Theoretically, it is possible that a bias was introduced and that some of the subjects altered their drinking habits during the study despite the fact that we asked all participants to adhere to their regular diet and drinking habits. Another limitation of the present study is the possibility of inaccuracy in the self-reported amount of alcohol intake, due to the fact that we used questionnaires and diaries . HoweverIn males, low alcohol intake was associated with lower diurnal triglyceridemia when compared to no, moderate or high alcohol intake, which was most pronounced during the evening. High alcohol intake seems to contribute to increased TG during the evening.


Consumption of tomato fruits, like those of many other plant species that are part of the human diet, is considered to be associated with several positive effects on health. Indeed, tomato fruits are an important source of bioactive compounds with known beneficial effects including vitamins, antioxidants, and anticancer substances. In particular, antioxidant metabolites are a group of vitamins, carotenoids, phenolic compounds, and phenolic acid that can provide effective protection by neutralizing free radicals, which are unstable molecules linked to the development of a number of degenerative diseases and conditions. In this review, we will summarize the recent progress on tomatoes nutritional importance and mechanisms of action of different phytochemicals against inflammation processes and prevention of chronic noncommunicable diseases . In addition, we will summarize the significant progress recently made to improve the nutritional quality of tomato fruits through metabolic engineering and/or breeding. Lycopersicon genus Solanum consists of 13 species or subspecies: the cultivated tomato, Solanum lycopersicum, which is the only domesticated species, and 12 wild species . Tomato fruits are an important source of nourishment for the whole world's population. Its world production is estimated to be around 159 million tons, while the average annual fresh tomato consumption is 18 kg per European and 8 kg per capita in the US [The tomato belongs to the Solanaceae family that includes more than 3,000 species. In particular, the section of then the US . Many fan the US . In thisRecently, significant progresses have been made in order to improve the levels of human health promoting compounds in tomato fruits through metabolic engineering and/or breeding , 8. HoweTherefore, in this paper, we will summarize the recent progress made to improve the nutritional quality of tomato fruits according to current state of the art.Regular consumption of tomato fruit and its products is associated with lower risk of CNCD and several types of cancer and inflammation because of interaction of phytochemicals with metabolic pathways that are related to inflammatory response and oxidative stress.Some evidences support a role for tomato products in the prevention of lipid peroxidation that is a risk factor of atherosclerosis and cardiovascular disease .High dietary intake of tomato products can reduce LDL cholesterol levels and increase LDL resistance to oxidation . In addiβ-carotene and β-cryptoxanthin, and non-provitamin A carotenoids, such as lutein and lycopene. They contribute to the photosynthetic machinery in plants and protect them against photo damage [Carotenoids are tetraterpenes belonging to fat-soluble pigments. They include provitamin A carotenoids, such as o damage . More tho damage . Geograpo damage .Several studies have reported the health benefits of carotenoids related to their antioxidant power, such as immune system stimulation and antitumor activity , 45, 70.μg per gram fresh weight of lycopene, about 80% of total dietary intake of this carotenoid [CrtL), a gene conserved through evolution from the time of cyanogenic bacteria [ trans- and cis-configurations [ trans-conformation, while thermal treatments, light, acids, oxygen, and digestion can cause transformation into the more bioactive cis-form [ in situ or from external sources . Radical accumulation in the body generates a phenomenon called oxidative stress that results from an imbalance between formation and neutralization of ROS/RNS in cells [Tomatoes contain 8–40 rotenoid , 74. Fierotenoid . Accumulbacteria . Lycopenurations . In frescis-form . Lycopencis-form . Furtherin cells . One of in cells . These pα), IL-6, and IL-8 increased the inflammatory response. Lycopene stimulates the first one and inhibits the latter in macrophages and adipocytes [μmol/L) are needed to achieve significant response in humans [β-carotene and lycopene supplementation interact with the metabolism of linoleic acid, resulting in either an increase (β-carotene) or decrease (lycopene) in plasma concentration [μm/L) in comparison with the lowest lycopene group (0.18 μm/L) [ROS can activate the transcription nuclear factor kappa B (NFkB) that gives rise to the expression of genes of pro- and anti-inflammatory cytokines and their subsequent production . It is dipocytes . Doses oipocytes , interfeipocytes . In addiipocytes . Neverthn humans –88. In pn humans . Lycopenntration . An 83% 18 μm/L) .2), which started in the 1980s. Adolescent populations are currently the group mainly exposed to risk of obesity [α), and interleukin (IL)-6 are significantly correlated with weight, body mass index (BMI), and waist circumference [α play an important role in crosstalk between adipose tissue and skeletal muscle [Many chronic diseases and increasing of mortality are directly related to the global obesity epidemic . Following lycopene treatment, a significant elevation of plasma carotenoids, specifically lycopene, occurred in the treatment versus the placebo group. Kuopio Ischemic Heart Disease Risk Factor Study examined the relation between serum antioxidant and intima-mediated thickness of the common carotid artery, a marker related to the risk of having an acute coronary event. Lower levels of plasma lycopene were showed in men who had a coronary event compared with men who did not [In particular, lycopene reduces transcript levels of proinflammatory cytokines with downregulation of IL-6 expression , 17. Mar did not , 95.β-Carotene is considered a provitamin because it can be converted into retinol, a compound essential for vision. It is known as a strong antioxidant and the best quencher of singlet oxygen. At low partial pressures of oxygen, such as those found in most tissues under physiological conditions, β-carotene was found to inhibit oxidation. In tomatoes grown in a field located in the S. Marzano area, reported values were between 0.28 and 1 mg/100 g FW [β-carotene amount reached 1.2 mg/100 gr FW [β-carotene from raw tomatoes was reported to be about 0.1% [100 g FW . These l100 g FW , 97. In 00 gr FW . Bioacceout 0.1% .β-carotene and lycopene  totally abolished TNF-α-induced ROS levels  in TNF-α-treated human umbilical vein endothelial cells. This was due to the redox balance protection and to the maintenance of nitric oxide (NO) bioavailability [β-carotene prevents photooxidative damage and sunburn showing photoprotection in humans. Erythema formation was significantly diminished when β-carotene was applied on human skin or with a dietary intervention alone or in combination with α-tocopherol for 12 weeks [trans-β-carotene inhibit atherosclerosis in hypercholesterolemic rabbits. This could be due to stereospecific interactions with retinoic acid receptors in the artery wall [A study found that both lability . Several12 weeks . Metabolery wall .β-carotene may be associated with an increased risk of CHF (congestive heart failure) and cardiac death in men.Recently, Karppi et al.  found thβ-carotene assumption. The initial antioxidant activity of β-carotene is followed by a prooxidant action at high oxygen tension. This mechanism may be related to adverse effects observed under the supplementation of high doses of β-carotene. A study reported that supplements in doses of 20 mg daily for 5–8 years had no protective effect on macrovascular outcomes or total mortality of diabetic male smokers [β-carotene during radiation therapy compared with that of placebo. Other trials about β-carotene supplementation will be required in order to clarify the protection role associated with β-carotene.However, some studies do not support the absolute favorable role of  smokers . Margali smokers  reportedμg/100 g FW [μg/100 g FW in cherry variety. Increasing interest around this compound was due to its important role in preserving eye health in association with zeaxanthin [Lutein is a yellow carotenoid synthetized in chloroplasts and chromoplasts and found in high quantities in leaves whose main function is to contribute to photosynthesis , 103. It100 g FW , while G100 g FW  reportedaxanthin . The impaxanthin , 23. Nevaxanthin . in vitro and in mouse model demonstrated that increased dietary intake of lutein is protective against the development of early atherosclerosis in human and animals through the reduction of inflammation and oxidative stress in the artery wall [κB signaling may be one of the main mechanisms employed by lutein and lycopene to reduce inflammatory leukocyte adhesion to endothelium.Epidemiological studiesery wall . Kabagamery wall  and Riccery wall  reportedery wall . Armoza ery wall  studied Herrero-Barbudo et al.  reportedTomato protective action is commonly attributed to the tomato antioxidant lycopene. However, tomato products are also sources of other compounds such as vitamins A, B, and E. Also these compounds have important effects on human health that will be described below.α-, β-, γ-, and δ-tocopherol; and α-, β-, γ-, and δ-tocotrienol. This classification is based on the nature of the isoprenoid chain; tocopherols contain a phytyl chain while tocotrienols contain a geranylgeranyl chain. These molecules are potent liposoluble antioxidant. Tocopherols are the most abundant vitamins in tomato plant and contribute to maintain optimal plant photosynthesis rate under high levels of stress [α-tocopherol-lycopene mixtures, and also for lycopene-β-carotene, lycopene-lutein, and lutein-β-carotene mixtures. This effect could be due to the fact that electrons can transfer from the carotenoid to the α-tocopherolxyl radical to regenerate α-tocopherol. An analogous mechanism has been suggested for the interactions between vitamin C, carotenoids, and α-tocopherol [α-tocopherol during the process of lipid peroxidation is well known [Vitamin E family includes eight molecules: f stress . Frusciaf stress  reportedf stress  studied copherol . A synerll known . This syll known .Decreasing risk of advanced prostate cancer was associated with increasing dose of supplemental vitamin E uptake among men in the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial .In a study carried out on Finnish men and women, those who ingested higher intake of dietary vitamin E showed a decreased incidence of type 2 diabetes . On the L-ascorbic acid and dehydroascorbic acid are the main dietary forms of vitamin C, a labile molecule with reducing property. It is a water-soluble compound easily absorbed but it is not stored in the body. Abushita et al.  reportedAscorbic acid (AsA) content in fresh tomatoes depends on genotype, climatic conditions, fruit development, maturation, senescence, and time of storage. AsA content in tomato fruit increases reaching a maximum and then began to decline with ripening . Yahia eMost of the known functions of ascorbic acid are correlated with its ability as electron donor and potent antioxidant in humans. In fact, vitamin C protects against oxidation of LDL by different types of oxidative stresses and inhibits LDL oxidation by vascular endothelial cells . YogeetaVitamin C in tomato is highly bioavailable, so a regular intake of small amounts of tomato products can increase cell protection from DNA damage induced by oxidant species. This effect may originate from the synergism of vitamin C with lycopene . Some stJacob et al.  discoverμg/100 g FW.Folates represent all forms of vitamin B found in biological systems, while folic acid is the synthetic form found in dietary supplements and fortified foods . Iniestaμg/day) is recommended for pregnant women to reduce pregnancy-induced folate deficiency, which can lead to megaloblastic anemia [Folate metabolism is involved in several physiological mechanisms in the field of andrology and gynecology . In partc anemia .Homocysteine metabolism is regulated by the nutritional status of folate, vitamin B-12, and vitamin B-6 . Some re para-hydroxyl group. Phenolics may modulate cellular signaling processes during inflammation or may serve as signaling agents themselves [Phenolic compounds are widespread phytochemicals composed of an aromatic ring and one or more hydroxyl substituents. They can be constituted by simple phenolic molecules or polymerised compounds , 134. Inemselves , 136. Themselves . In partemselves  demonstrPolyphenolic compounds are associated with therapeutic tools in inflammatory diseases including cardiovascular diseases, obesity and type II diabetes, neurodegenerative diseases, cancer, and aging.κB); (c) the activation of peroxisome proliferators-activated receptor gamma (PPAR γ); and (d) the activation of mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and the modulation of several cell survival/cell-cycle genes [These effects are due to the phenolic ability to interact with a wide spectrum of molecular targets central to the cell-signaling machinery. Main molecular mechanisms include (a) the inhibition of proinflammatory enzymes, such as cyclooxygenase (COX-2), lipoxygenase (LOX), and inducible nitric oxide synthase (iNOS); (b) the inhibition of phosphoinositide 3-kinase (PI 3-kinase), tyrosine kinases, and nuclear factor-kappa B , nuclear respiratory factor 1 (NRF-1), and transcription mitochondrial factor A (TFAM). Potential beneficial effects of polyphenols have also been obtained for quercetin, able to reduce FFA levels, total cholesterol, and body weight in rats [Studies reported that resveratrol increases the survival of mice fed with a high caloric diet producing changes including improvement in insulin sensitivity and decreased fat accumulation and body weight. These changes are associated with induction of mitochondrial-related transcription factors such as estrogen-related receptor  in rats .Finally, in 2007 Shen et al.  conducteμg quercetin/g FW [Flavonoids constitute the largest group of naturally occurring phenolics in tomatoes and contribute to the determination of aroma, fragrance, and colour , 142. Matin/g FW . Detectetin/g FW .Some flavonoids such as rutin (a flavonol glycoside), quercetin, resveratrol, and catechin are active in rheumatoid arthritis. The mechanisms of action include inhibition of osteoclast/macrophage differentiation and function and estrogen modulation , 53.β activated human astrocytes, reducing cytokines and chemokines and oxidative stress [Rutin, quercetin, glycosides of quercetin, and resveratrol have been shown to exert intestinal anti-inflammatory activity , 38. In e stress .Resveratrol acts on blood lipid levels and also at the atherosclerotic plaque and reduces cellular infiltration, fibrosis, and expression of inflammatory cytokines in a model of autoimmune myocarditis , 55.Rossi et al.  conducteα-induced inflammation through monocyte chemoattractant protein-1 [Anthocyanins are one of the flavonoid phytopigments; they may act on adipocytes and modulate the expression levels of adipocytokines. In particular, C3G (cyanidin 3-glucoside) was reported to upregulate the expression of adiponectin that can increase insulin sensitivity at the level of human adipocytes controlling obesity , 57. In rotein-1 . In animrotein-1 , 147. p-coumaric, and sinapic acids belong to hydroxycinnamic acids [ p-coumaric acid between values under limit of detection (in Liso and Remate varieties) and 5.77 mg/kg (in Daniella variety), and ferulic acid between 1.60 (in Riso variety) and 5.38 (in Durina variety) mg/kg FW.Phenolic acids are responsible for the astringent taste of vegetables . They inic acids . Chlorogic acids  analyzed p-coumaric acid between 3.5 and 5.5 mg/100 g FW, while the content of ferulic acid was between 0.9 and 1.5 mg/100 g FW.Luthria et al.  identifiOlthof et al. , 151 rep in vitro [ in vitro. They found that these compounds protected at low concentration in vitro against DNA oxidation induced by iron. Caffeic acid can block the biosynthesis of leukotrienes that are involved in immunoregulation. In addition, it was shown that caffeic acid possesses antitumor activity in humans [However, phenolic acids are studied mainly for their role as antioxidants , 63. Somin vitro . Lodovicin vitro  investign humans . Other sn humans .Cinnamic acids exhibit strong antioxidant properties. 1,5-Dicaffeoylquinic is a hepatoprotector when challenged by carbon tetrachloride. This mechanism involves, among others, radical scavenging .Tannins include compounds of hydrolysable tannins that are polymers of ellagic acid, or gallic and ellagic acids, with glucose and condensed tannins (proanthocyanidins), which derive from the condensation of monomers of flavanol units , 153. ThTomatoes are consumed fresh or are used to manufacture a wide range of processed products that can show a very different composition compared to fresh fruit. For example, significant losses of ascorbic acid can occur during postharvest storage period and during preparation and cooking of foods. This is due to oxidation and leaching into the water used for cooking , 154. in vitro bioaccessibility of lycopene [For the production of tomato paste from fresh tomatoes, both homogenization and heat treatment are used. Heat treatment and/or homogenization can disrupt the cellular matrix of tomatoes determining the bioavailability of different nutrients . For exalycopene , 160. Holycopene .D88°C value  of 276 min. In addition, they found an antioxidant activity of raw tomatoes equal to 4.13 ± 0.36 μmol of vitamin C equiv/g of tomato. After heat treatment at 88°C for 2, 15, and 30 min, the total antioxidant activity significantly increased to 5.29 ± 0.26, 5.53 ± 0.24, and 6.70 ± 0.25 μmol of vitamin C equiv/g of tomato, respectively (P < 0.01). This effect could be explained by the increased amount of lycopene, the major phytochemical in tomatoes.Veronica et al.  observedGahler et al.  investigβ-Carotene amount decreased or was stable while α-tocopherol content significantly rose during short-term heating.Seybold et al.  investigChang et al.  studied Patras et al.  reviewedAs a whole, these evidences could have a significant impact on consumer's food selection, increasing their awareness of the health benefits of processed tomatoes in the prevention of chronic diseases and inflammation.The ability of various tomato phytonutrients to be positively correlated to prevent or ameliorate chronic disease is appealing. The studies carried out on the effects of phytochemicals on human health prompted researchers to find novel ways to get biofortified tomato genotypes with enhanced levels of phytonutrients such as anthocyanins, lycopene, ascorbic acid, and folate. In the last few years, crop biofortification gave an enormous contribution to understand the relationship between diet and health, reduce the risk of chronic disease, and better understand the regulatory systems in plant species. As described below, attempts to create novel tomato plant lines that carry genes for the accumulation of essential nutrients in tomato fruit can be achieved both through metabolic engineering and conventional breeding , 166 Ta. Mdhar (monodehydroascorbate reductase) and Dhar (dehydroascorbate reductase) genes were overexpressed in tomato var. Micro-Tom. In the DHAR overexpressing lines, there was a 1.6-fold increase of AsA in fruits of plants grown in low light condition, while MDHAR transformants showed a reduced level of AsA by 0.7-fold in mature green fruits [ phytoene desaturase (crtI) of Erwinia uredovora and the yeast-derived GDP mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO) [ hydroxymethyl dihydropteridine (HMDHP), synthetized in the cytosol by GTP, and p-aminobenzoate (PABA), synthetized in chloroplasts from chorismate [ Arabidopsis (AtADCS), aimed at increasing the supply of PABA, did not result in any change of folate levels in tomato fruit. On the contrary, the expression of GCHI and AtADCS resulted in 20-fold higher levels of folate compared to the control [ lycopene β-cyclase gene from the Eubacterium Erwinia herbicola and the higher plant Narcissus pseudonarcissus into the tomato plastid genome and obtained a 50% increase in total carotenoid accumulation in genotypes expressing the plant enzyme. Other studies involve the accumulation of novel metabolites in tomato, such as resveratrol, using the expression of a gene encoding for a grape stilbene synthase [ DET1 (De-etiolated 1), using fruit-specific promoters combined with the RNA interference (RNAi) technology. The transformed plants showed an increase of flavonoid levels up to 3.5-fold. The last approach to increase levels of phytonutrients involves an enhancing expression of transcription factors that regulate specific biosynthetic pathways. This system does not work well in a complex branched pathway like those of folate biosynthesis [ Delia (Del) and Rosea1 (Ros1) from snapdragon Antirrhinum majus. The fruit of transformed plants accumulated high level of anthocyanins with concentrations similar to those found in blackberries and blueberries [AtMYB12), which regulates the caffeoylquinic acid and flavonol synthesis, proved to be a good candidate for the production of tomato fruits with high levels of polyphenolic antioxidants compounds [Plant metabolic engineering adopted different biochemical approaches to improve the amount of various phytonutrients in tomato fruits. One strategy is based on modification of key-rate limiting steps in metabolic pathways. For example, the expression in tomato of two isoforms of the GDP-mannose-3,5′-epimerase, a key enzyme of the ascorbic acid biosynthesis pathway, resulted in a modest increase of ascorbate (1.6-fold) in the fruits of transformed plant . In anotn fruits . Anotherse (ALO) , 170. Thorismate . The firorismate , 173. Th control . Using a control  introducsynthase . Anothersynthase  enhancedynthesis  but seemynthesis  and flavynthesis . The anteberries . Anotherompounds . Drawbacompounds , 185. S. pimpinellifolium, S. neorickii, S. habrochaites, S. chmielewskii, and S. pennellii) and heirlooms tomatoes have a rich genetic variability; therefore, one goal of plant breeding has become to screen wild genetic resources for valuable traits that could be introduced into modern varieties to improve specific traits [ Solanum pennellii were replaced with homologous intervals of the cultivated variety M82. In our laboratory, two introgression lines of Solanum pennellii were identified harboring QTLs that increase the content of ascorbic acid, phenols, and soluble solids [http://solgenomics.net/organism/solanum_lycopersicum/genome) by an international consortium [One way to produce biofortified plants is to take advantage of the natural variation of plant genomes. For example, recently a new genetic combination was obtained through conventional breeding, which produced a phenotype with deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peels. Such a genotype was named Sun Black . Tomato c traits , 187. Ine solids , 182. Ine solids . Althouge solids . In addinsortium . Using tnsortium . In TablThere are many evidences supporting the anti-inflammatory and anticancer action of tomato fruit bioactive compounds. Molecular mechanisms controlling these effects have been extensively studied and described.The present review reports a brief description, even though not exhaustive, of bioactive compounds available in tomato fruit and of their beneficial properties on human health.The content of these compounds might be increased to obtain biofortified food taking into account, among other things, the great influence of processing transformation that is required for some derived tomato foods. Various strategies might be used to increase these compounds in tomato fruit. Therefore, the final destination of biofortified tomatoes needs to be considered when selecting new genotypes for fresh market or processing.It is necessary to dissect tomatoes complex genetic control to identify key genes of biochemical pathways underlying their biosynthesis.The best strategy to transfer them into improved genotypes might be chosen between metabolic engineering via genetic transformation and precision breeding by the aid of molecular markers.In both cases, the richness of genetic and genomic resources today available for tomato might highly enhance the success of tomato breeding aimed to obtain new biofortified genotypes.


The aim of this study was to measure umbilical blood androgen concentrations in a birth cohort using a highly specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay and assesses the effects of sex, labor, and gestational age on fetal androgen levels at birth. We performed a prospective cohort study of androgen concentrations in mixed arterial and venous umbilical cord serum from 803 unselected singleton pregnancies from a general obstetric population in Western Australia. Total testosterone (TT), Δ4-androstenedione, and dehydroepiandrosterone were extracted from archived cord serum samples and measured using LC-MS/MS. SHBG was measured by ELISA; free testosterone (FT) and bioavailable testosterone (BioT) values were also calculated. Median values for all three androgens were generally lower than previously published values. Levels of TT, FT, BioT, and SHBG were significantly higher in male verses female neonates (P<0.0001), while dehydroepiandrosterone levels were higher in females (P<0.0001). Labor was associated with a significant (∼15–26%) decrease in median cord blood TT and FT levels (both sexes combined), but a modest (∼16–31%) increase in SHBG, Δ4-androstenedione, and dehydroepiandrosterone concentrations. TT and FT were significantly negatively correlated with gestational age at delivery, while SHBG, Δ4-androstenedione, and dehydroepiandrosterone were positively correlated. Antenatal glucocorticoid administration also had a significant effect in the multiple regression models. This is the first study to report umbilical cord androgen levels in a large unselected population of neonates using LC-MS/MS. Our findings suggest that previous studies have over-estimated cord androgen levels, and that fetal, maternal, and obstetric factors influence cord androgen levels differentially. Caution should be exercised when interpreting previously-published data that have not taken all of these factors into account. Fetal androgen concentrations have been the subject of considerable interest over several decades due to their association with maternal metabolic disorders 4-androstenedione (A4) and dehydroepiandrosterone (DHEA) Increasing awareness of the limitations of RIA for the measurement of low concentrations of sex steroids To address the weaknesses of previous studies and provide robust data on the relationships between obstetric variables, fetal sex and cord androgens, we have (as far as we are aware) undertaken the largest study to date of umbilical cord serum androgen concentrations determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The size of the cohort, combined with the accuracy and validity of the LC-MS/MS method, provides for the first time a robust and accurate assessment of the relationships between fetal sex, obstetric variables, gestational age at delivery and fetal androgen levels at birth.www.rainestudy.org.au) consists of 2868 unselected pregnancies recruited and sampled from the public antenatal clinic of King Edward Memorial Hospital in Perth, Western Australia between 1989–91 to study the effects of repeated ultrasound on fetal and post natal growth & development and pregnancy outcome. Study participants completed a questionnaire at 18 and 34 weeks gestation, which provided information on a wide range of variables including ethnicity, social and economic circumstances, lifestyle, medical history and environmental exposures. Pregnancy and neonatal outcome data were taken from hospital notes. The study was approved by the King Edward Memorial Hospital/University of Western Australia ethics committee and participants were re-consented in writing at 18 years of age. Umbilical cord blood was collected from 870 deliveries from the intensive ultrasound arm of the cohort, which consisted of 1415 singleton pregnancies in total. Immediately after delivery mixed umbilical arterial-venous (UA∶UV) cord blood was collected, allowed to clot and the resulting serum was frozen at −80°C and stored without thawing until the present study was performed. Detailed sequence analysis of DNA obtained from ten maternal-child pairs confirmed that the cord blood samples were not contaminated by maternal blood. Eight hundred and three cord blood samples (92.3%) representing 407 male and 396 female infants had sufficient serum  for steroid analysis. The demographic and obstetric characteristics of the sample set are shown in The Western Australian Pregnancy Cohort (known as the Raine Study: 4-androstenedione (A4), dehydroepiandrosterone (DHEA), and internal standards (d3-testosterone [IS-T] and 19-d3-androstenedione [IS-A4]) were extracted from 20 µL aliquots of cord serum with hexane∶dicholoromethane (3∶2). Steroid extracts were separated by HPLC on a Kinetex C18 column with a run time of 10 minutes, and the eluates monitored by an API5500 Q-TRAP MS/MS detector in positive MRM mode. All three steroids were well resolved chromatographically; the single charged Q1/Q3 transitions for TT, A4, and DHEA were 289.3/96.9, 287.1/97.0, and 289.2/253.2 atomic mass units (amu), respectively, and 292.2/97.0, and 290.2/100.0 amu for IS-T and IS-A4, respectively. The data were acquired and processed by the data acquisition system Analyst® linked directly to the API5500 Q-TRAP MS/MS detector. Calibration curves  were prepared in stripped human serum and were run with each assay batch; calibration slope regression coefficients were ≥0.996 (n = 12). Recovery was calculated using deuterated internal standards added to the samples prior to extraction. The limits of quantitation for TT, A4 and DHEA were 0.025, 0.05 and 0.05 ng/ml, respectively . Extensive optimization and validation was carried out using fresh cord serum to ensure that the assays were specific and accurate. Quality controls (stripped serum) at low, medium and high values demonstrated inter-assay imprecision  of 5–8%, 5–14% and 8–11% for TT, A4 and DHEA, respectively (n = 24). Pooled male and female cord serum QC samples were also run with each batch and performed with comparable precision: CVs for TT, A4 and DHEA were 6–11%, 7–8% and 8–18%, respectively (n = 24). Recovery of target analytes from stripped serum across three concentrations ranged from 93–98% (TT), 104–111% (A4) and 93–96% (DHEA). Assay performance was determined to be unaffected by up to three freeze-thaw cycles or 24 h at room temperature. Steroid analysis was performed blind to sample identity or characteristics.Total testosterone (TT), ΔSex hormone binding globulin (SHBG) was measured by ELISA using a commercial kit  according to the manufacturer's instructions. All samples were measured in duplicate by a single operator using assay kits from the same batch. Samples with an initial replicate CV of >10% were reanalyzed. The inter-assay imprecision (CV) was <4.5% (n = 25); intra-assay CV was 5.2% (n = 861).http://www.issam.ch/freetesto.htm) vs. the empirical method carried out in n = 25 samples selected across a range of TT and SHBG values revealed that the empirical method generated values that were on average 32% lower than those derived by the Vermeulen method, consistent with expectations alb equal to 36,000 L/mol The mass action equation method of Vermeulen et al P value<0.05 was considered significant. Linear correlations were performed on Instat3 to explore the associations between androgen levels, gestational age at delivery, birth weight and placental weight after removal of extreme outliers ; there were no significant departures from linearity. Multiple regression analyses were then performed (using Instat3) to determine the independent effects of a range of maternal and obstetric variables  on cord blood androgen concentrations and their associations.Descriptive statistics were prepared on Microsoft Excel. All sets of continuous data failed the normality test of Kolmogrov and Smirnov so group medians were compared by Mann-Whitney U-test using Instat3 . Categorical or discrete variables were compared by Fisher's Exact Test. A The demographic details of the entire cohort, segregated according to fetal sex, are shown in P<0.0001), as were FT and BioT levels ; median SHBG concentrations were also modestly elevated in males . In contrast, DHEA concentrations were significantly higher (19.5%) in females verses males (P<0.0001). There were no significant differences in A4 concentrations. The distribution of all three androgens was skewed to the right of the median due to the presence of a number of values with elevated values  in 17 and 8 samples, respectively. These samples were assigned values at the LOQ for statistical analysis. Two samples were not assayed for SHBG. The mean, SD, median and range for all four analytes including the calculated FT and BioT values in male and female neonates are shown in d values Figure 12 = 0.125, P<0.0001), DHEA  and SHBG   and more strongly between A4 and DHEA . All correlations remained highly significant (P<0.0001) after controlling for ethnicity, socioeconomic status, birth weight, placental weight, gestational age at delivery, parity, tobacco smoking status , labor onset, labor induction, labor duration, oxytocin administration, tocolytic administration, antenatal steroid administration, APH, PTB, and PROM in multiple regression analysis. Maternal age showed a weak but significant negative correlation with cord blood A4 concentrations , but not with any of the other endocrine measurements; this association remained significant in the multiple regression analysis.Linear regression and multiple regression analyses were performed to assess the inter-relationship between androgen and SHBG levels, controlling for the maternal and obstetric variables listed in <0.0005) Figure 2P<0.0001), with weaker effects between SHBG and TT, fT and BioT (P = 0.0002). Linear correlations demonstrating a positive correlation between gestational age and SHBG, A4 and DHEA concentrations (P≤0.0001)), and a weak negative correlation with TT and FT concentrations , are shown in 2 = 0.014, P = 0.0019) but this did not remain significant after adjusting for other obstetric variables. On the other hand, delivery following the onset of labor compared to delivery by elective Cesarean section prior to labor had a significant effect on hormone levels in the multiple regression models. Group comparisons revealed that median SHBG, A4 and DHEA concentrations were increased with labor by 16.3%, 24.4% and 30.8%, respectively (P<0.0005) . Administration of antenatal glucocorticoids was also shown to have a significant effect on the associations between SHBG, TT, FT, BioT, A4 and DHEA levels in multiple regression analysis after adjusting for gestational age at delivery; however, a comparison of androgen levels in samples delivered with and without glucocorticoid administration did not find any significant differences between the groups, although mean levels of TT, FT, BioT and DHEA were somewhat higher in the steroid group .Multiple regression analysis also revealed a significant effect of gestational age at delivery on the association between SHBG levels and A4 and DHEA . The major concern is that degradation may have occurred in storage or with thawing, resulting in reduced concentrations of intact steroids. However, we believe such concerns are unfounded for several reasons. Firstly, studies of steroid stability (including testosterone) during long term storage confirm that serum samples are able to be stored for at least four decades at −80°C without loss or appreciable deterioration of steroid hormones An additional potential limitation of our study is the use of mixed UA∶UV blood samples. While we have confirmed that our samples are not contaminated by maternal blood, the relative proportion of umbilical arterial and venous blood is unknown and, in theory, may be altered by factors such as mode of delivery, gestational age, placental architecture and fetal hemodynamics. Studies which have compared UA∶UV differences in cord blood steroids suggest that UV concentrations of TT, A4 and DHEA are approximately 20%, 40% and 50–75% lower than UA levels et al. reported mean TT values of 0.5 nmol/L in seven male infants measured by LC-MS/MS, in keeping with our results, although half were below the limit of detection of 0.24 nmol/L et al., in a GC-MS study of the umbilical blood steroid metabolome et al.While we anticipated that our results would be marginally lower than many previous studies as a result of improved assay accuracy and precision, we were surprised that the median TT values for our cohort were markedly lower than published values, even some of those derived by mass chromatographic methods. For instance, Krogh and colleagues recently reported TT values in the range of 0.8–0.9 nmol/L in a LC-MS/MS study of 10-year-old cord blood samples from approximately 200 male infants from the Danish National Birth Cohort vs. females fetuses, whereas the converse is true with respect to relative androgen underexposure. This has important implications for studies investigating the relationships between the development of androgen-sensitive neurocognitive abnormalities or metabolic disorders and in-utero androgen exposure. It also emphasizes the need for developmental studies to be sufficiently powered to accommodate such ‘extremes’ in their analyses. We also described novel findings of higher DHEA concentrations in cord blood from female infants compared to males. Small differences have been observed in previous studies vs. female infants In agreement with several previous studies All three androgens measured in this study are substrates for estrogen biosynthesis via the actions of placental aromatase/CYP19 and are also inter-convertible via other placental enzymes SHBG was measured primarily to allow determination of FT and BioT values. Unexpectedly, we uncovered some interesting associations between SHBG levels and fetal and obstetric factors. A highly significant positive relationship was observed between cord SHBG concentrations and gestational age at delivery which remained after controlling for multiple variables; this was described previously in a small study Some studies suggest that smoking in pregnancy may increase circulating postnatal testosterone and cortical concentrations and reduce estriol concentrations In conclusion, we report that levels of androgens in umbilical cord blood samples from unselected male and female neonates are lower than previously described, and that umbilical cord androgen levels are influenced by fetal sex, onset of labor and gestational age at delivery. We suggest that most previous studies examining the relationships between cord blood androgens and later-life/developmental events are compromised by inaccurate steroid measurements or inadequate control for fetal or obstetric factors. As such, some of their conclusions may be questionable and considerable caution should be exercised when interpreting previously-published data that have not taken these factors into account.Figure S1Androgen and SHBG concentrations  in cord blood samples from pregnancies with or without (A) preeclampsia/severe hypertension, or (B) maternal antenatal glucocorticoid administration. Note: fT values were multiplied by 100 to allow representation on the same graph. No significant differences between groups were detected by Kruskal-Wallis test (P>0.05)(TIF)Click here for additional data file.


We wanted to investigate if BMI is related to clinico-pathological characteristics, hormone receptor status in primary tumour, and disease outcome in endometrial cancer.Endometrial cancer incidence is increasing in industrialised countries. High body mass index  and protein expression by immunohistochemistry . In contrast, oestrogen receptor (ERα) status was not associated with BMI. Overweight/obese women had significantly better disease-specific survival (DSS) than normal/underweight women in univariate analysis (P=0.035). In multivariate analysis of DSS adjusting for age, FIGO stage, histological subtype, and grade, BMI showed no independent prognostic impact.High BMI was significantly associated with low International Federation of Gynaecology and Obstetrics (FIGO) stage, endometrioid histology, low/intermediate grade, and high level of progesterone receptor (PR) mRNA by qPCR (High BMI was significantly associated with markers of non-aggressive disease and positive PR status in a large population-based study of endometrial carcinoma. Women with high BMI had significantly better prognosis in univariate analysis of DSS, an effect that disappeared in multivariate analysis adjusting for established prognostic markers. The role of PR in endometrial carcinogenesis needs to be further studied. In all, 91% of the women underwent hysterectomy with bilateral salpingo-oophorectomy as primary treatment and were classified according to the FIGO 1988 criteria . If surgFollow-up time was defined as the time interval between date of primary diagnosis and date of death or last follow-up. The median follow-up time was 4.9 years (range 0.01–23.2). In all, 223 patients (20%) died from endometrial carcinoma during the follow-up period, while 207 (18%) died from other causes. These data were cross-checked with information from the Cancer Registry of Norway and the Register of Statistics Norway. Last follow-up was 20 December 2009.2), both measured at the time of diagnosis. These data were available for 949 patients (84%). For the statistical analyses on BMI we used the quartiles for the data set as cut points, as well as the established WHO classification system; BMI under 18.5 (underweight), between 18.5 and 24.9 , between 25 and 29.9 (overweight), and >30 (obese). Height and weight of outliners  was double-checked. All analyses were also performed excluding these; this did not affect any of the conclusions.Body mass index was calculated as weight (kg) divided by squared height (mα and PR) and available for 437 and 433 patients for ERα and PR, respectively (38% of study population). The method for immunohistochemical staining was as previously described, using the lower quartile to define receptor loss  as previously described (α and PR were investigated by qPCR using the TaqMan Low Density Array technique (From a subset of 150 patients (13%), fresh frozen tumour tissue was collected prospectively and was available for mRNA analysis in parallel with the immunohistochemical staining. Total RNA was extracted using the RNeasy kit , with quality control and method for data processing as previously reported  and overall survival (OS) were performed using the Kaplan–Meier method (log-rank test). The Cox proportional hazard regression analysis was applied to evaluate the prognostic impact of BMI adjusted for the established prognostic markers in endometrial carcinoma. We compared the distribution of clinico-pathological variables and prognosis for patients with available data for BMI to patients where these data were missing (16%). Women lacking BMI data were older, with median age 69.3 years compared with 65.2 years for the group where BMI was registered, P=0.004 (Mann–Whitney U-test). No other significant differences were identified. The statistical software PASWStatistics18.0 was used for data analyses .Body mass index in WHO categories was applied to assess the distribution of various clinico-pathological variables, using the Pearson's The study was approved by the IRB .P=0.002 (P=0.99), but a tendency to more adjuvant therapy given to patients with BMI<25 (P=0.06).The median BMI at diagnosis was 26.4 (range 14.7–73.0), with significantly increasing BMI throughout the study period, P=0.002 . There wvs 26.9, respectively . We did not find any significant correlation between BMI and ERα status in tumours (P=0.08) . For ERα, no such association with BMI was observed for mRNA expression levels (P=0.21). Loss of ERα and PR (by IHC) was associated with postmenopausal status .When investigating biomarkers for receptor status in tumours related to BMI we found that patients with PR-negative tumours (by IHC) had lower median BMI compared with the patients who had PR-positive tumours, median 25.5 (P=0.08) . To furtvs normal/underweight as defined by the WHO had better DSS, with a 5-year survival of 82% for women with BMI⩾25 compared with 76% for BMI<25 . When BMI was applied as a continuous variable in the same Cox model, we found a similar insignificant HR for BMI of 1.01 (CI 0.98–1.04), and pattern for the other variables with independent impact for FIGO stage and age only. In contrast, when using OS as end point in the Cox model, we found that BMI had independent impact on prognosis when introduced as a continuous variable with an HR 1.02 . FIGO stage and age were also independent predictors of prognosis (P<0.0001 for both), while histology was of borderline significance (P=0.053) and grade was non-significant (P=0.166).The survival effect of BMI observed in univariate analysis for DSS disappeared when adjustment was made for age at diagnosis (continuous variable), FIGO stage, histological subtype, and grade in the Cox multivariate regression analysis as listed in To our knowledge, this is the most comprehensive study of clinico-pathological variables to date. It is also the largest study to date exploring the relationship between BMI and a large panel of markers for tumour phenotype in endometrial carcinoma. The large sample size with careful characterisation of FIGO stage, histological subtype, and grade confers more accuracy to the estimates for the independent prognostic impact of BMI compared with smaller previous studies. Also, the fact that the patient series studied was derived from a well-defined geographic region in Norway, previously shown to be representative for the total Norwegian population . We found that BMI was significantly linked to alterations in PR but not ERα status in tumours, confirmed by two different techniques estimating mRNA and protein levels for PR and ERα. The biological function of PR may be altered by genetic variations. Interestingly, recent studies have identified a single-nucleotide polymorphism in the gene coding for the PR, which has been associated with increased risk for endometrial carcinoma (The rise in endometrial carcinoma incidence has been associated with an epidemic of obesity and physical inactivity . The sig


Coronary artery bypass grafting (CABG) with cardioplegic cardiac arrest and cardiopulmonary bypass (CPB) is associated with myocardial injury. The aim of this study was to investigate whether a modified mechanical post-conditioning (MMPOC) technique has a myocardial protective effect by enhancing early metabolic recovery of the heart following revascularization.A prospective, randomized trial was conducted at a single-center university hospital performing adult cardiac surgery. Seventy-nine adult patients undergoing first-time elective isolated multivessel coronary artery bypass grafting were prospectively randomized to MMPOC or control group. Anesthetic, cardiopulmonary bypass, myocardial protection, and surgical techniques were standardized. The post reperfusion cardiac indices, inotrope use and biochemical-electrocardiographic evidence of myocardial injury were recorded. The incidence of postoperative complications was recorded prospectively.Operative characteristics, including CPB and aortic cross-clamp time, were similar between the two groups (p>0.05). The MMPOC group had lower troponin I and other cardiac biomarkers level post CPB and postoperatively, with greater improvement in cardiac indices (p<0.001). MMPOC shortened post surgery hospitalization from 9.1 ± 2.1 to 7.5 ± 1.6 days (p<0.001).MMPOC technique promotes early metabolic recovery of the heart during elective CABG, leading to better myocardial protection and functional recovery. Reperfusion has the potential to cause additional reversible and irreversible damage to the myocardium, which is called reperfusion injury,2. The eThe aim of our study was to determine the efficacy of a modified type of mechanical post-conditioning (MMPOC) in patients undergoing elective coronary revascularization, with specific attention to biochemical markers of ischemic injury and post-surgical recovery of the patients and to show whether there is room for protection by post-conditioning amongst all the other cardioprotective factors.This study was approved by the Institutional Review Board of the University of Ankara and consisted of 79 patients undergoing elective primary coronary revascularization with ≥99% stenosis of the left anterior descending (LAD) artery. So as to include patients with relatively large volumes of at-risk myocardium, we limited our analysis to those patients exhibiting proximal occlusion of the LAD. Patients undergoing valve replacement, combined valve replacement/coronary revascularization, or preoperative coronary revascularization was excluded from the study. Informed consent was obtained from all patients before enrollment.Patients were allocated to the MMPOC group or the study group using a computer-generated randomization code. Participants were randomly assigned in a 1:1 ratio. The study was open label and the primary investigator, who was not the treating physician and nurses informed the participants about their allocated treatment. Data for primary outcomes were assessed by use of a computer.Baseline, procedural, and follow-up data were stored prospectively in a database located at the University of Ankara. Patients’ preoperative risk factors were recorded and EuroSCOREs were calculated for each patient. Patients’ preoperative characteristics were recorded including age, sex, size, preexisting medical conditions, preoperative medications, preoperative ejection fractions. Intraoperative variables of which number of coronary bypass grafts, duration of cardiopulmonary bypass (CPB), duration of aortic cross-clamp, requirement for inotropic drugs, and/or intra-aortic balloon support, and blood product use were included. Postoperative data comprised myocardial infarction, cardiac tamponade, reoperation for occlusion or other causes, requirement of intra-aortic balloon pump support, neurologic complications, renal dysfunction, chest tube drainage during the first 24 postoperative hours, total chest tube drainage, the length of mechanical ventilator support, pneumonia, multiorgan failure, gastrointestinal complications, sepsis, coma, the length of intensive care unit (ICU) stay, and readmission within 90 days after surgery.Adverse events were defined as death, perioperative myocardial infarction, stroke, re-exploration due to bleeding, respiratory insufficiency, and renal failure. Perioperative myocardial infarction (MI) was defined as either new Q waves or ischemic ST segment changes with concomitant elevations of creatine kinase isoenzyme (CK-MB) > 5 times the upper limit of the reference range or a CK-MB to total creatine kinase (CK) ratio > 10% occurring within 48 hours after surgery or troponin I (TnI) > 1 ng/mL. Renal dysfunction was defined as rise of serum creatinine above 2.5 mg/dL and/or a need for hemodialysis. The surgical team examined all patients about 4-6 weeks after discharge and annually thereafter for two years.2 after separation from CPB. At sternal closure, an infusion of propofol was started (25-75 μg/kg/min), and the isoflurane was discontinued. Propofol sedation was continued in the ICU until weaning of ventilatory support was initiated.Anesthesia was maintained with isoflurane. Hypertension was treated by increasing the concentration of isoflurane or by the administration of nitroglycerin if increasing the depth of anesthesia was ineffective. Hypotension was corrected using volume replacement or phenylephrine, as clinically indicated. An additional dose of 5 mg of midazolam was provided during rewarming from CPB. Inotropic agents (dobutamine 5 μg/kg/min) were initiated for a cardiac indices (CI) <2.0 L/min/mAll patients had coronary artery bypass grafting (CABG) with the use of CPB, which was conducted with a roller pump and a membrane oxygenator primed with a solution consisting of 1 L of Ringer’s lactate, 5000 IU of heparin, 750 mL of Pentaspan , and 44 mEq of bicarbonate. During CPB, pump flow was set at 2.4 times the body surface area, and mean arterial pressure maintained between 50 and 60 mm Hg. The temperature was allowed to drift with active rewarming at the end of CPB. Cardioplegia solution , which was used at the discretion of the cardiac surgeon, was free of glucose and consisted of high-dose (100 mEq/L) and low-dose (40 mEq/L) potassium. Cardioplegia was given in an anterograde fashion with blood in a ratio of 1:4. Blood cardioplegia was also administered with each successive distal vein graft anastomosis. The proximal vein graft anastomosis were performed after the distal ones under tangential aortic clamp. The left internal thoracic artery was used in all cases to bypass the LAD, while other coronary arteries received saphenous vein grafts. In patients with severe aortic disease where intermittent cross-clamping may be harmful, we used the MMPOC technique immediately after completion of the proximal anastomosis and weaning off CPB, in the MMPOC group we applied bulldog clamps to the grafts anatomized to the coronary arteries as in a similar manner and duration explained by Zhao et al before 2.A repeated measures design has MMPOC and control groups of 39 and 40 subjects respectively for a total of 79 patients. Each subject is measured 6 times. The between-subject standard deviation is 0.41 and the within-subject standard deviation is 1.00. This design achieves 96% power when an F test is used to test the group-factor at a 5% significance level and the actual standard deviation among the appropriate means is 0.17 (an effect size of 0.42), achieves 91% power when an F test is used to test the time-factor at a 5% significance level and the actual standard deviation among the appropriate means is 0.19 (an effect size of 0.19), and achieves 83% power when an F test is used to test the (group X time) interaction at a 5% significance level and the actual standard deviation among the appropriate means is 0.17 (an effect size of 0.17)[t test and Mann–Whitney U test, respectively. Two-way analysis of variance (ANOVA) for repeated measures was used to assess the main effect of time as within-factor and post conditioning versus control as grouping factor and interactions between them (group X time). If a significant effect was detected, pair wise comparisons were evaluated using the Bonferroni test. A p value less than 0.05 was considered significant. All analyses were performed using SPSS® Statistics 18.0 .Nominal variables were assessed by Chi square test or Fisher’s Exact test, where applicable. Continuous and ordinal variables were evaluated between two groups by Student’s th and 12th post-conditioning hours till the end of the study respectively in the MMPOC group (p<0.001). Although TnI levels remained lower in the study group, the values reached statistical difference after the 24th hours (p<0.001)  Table A,B,C. T= 0.062) nor was the aortic cross-clamp time (p = 0.082). The mean number of grafts was similar between the groups (p = 0.974). The operative time was shorter in the MMPOC group . Under the similar preload filling status, patients in MMPOC group had higher post-conditioning CI in all time points (p<0.001). The actual values of PCWP, MPAP and CVP remained similar between the two groups in all time points (p>0.05) . The MMPOC group patients had lower length of stay in the ICU and in the hospital compared to the control group (p<0.001). Importantly, use of MMPOC shortened postoperative hospitalization by more than one day. Postoperative complications were similar in both groups. There were three and five perioperative myocardial infarctions in MMPOC and control groups respectively (p = 0.712). While two hospital deaths were seen in control group, none of the patients died in MMPOC group (p = 0.499). Wound infection rates, amount of bleeding and postoperative atrial fibrillation incidences were similar between the groups as shown in TableThe median length of intubation was similar between the groups  in CABG operations. Significantly shorter hospital stays will certainly result in lower total hospital costs and nosocomial infections. It can be an adjunct to the other regimes of cardioprotection. Furthermore, our study includes moderately high risk patients with impaired left ventricular ejection faction, medium EuroSCORE and high New York Heart Association (NYHA) status. We assume that improvement in clinical outcome after CABG for patients treated with MMPOC could be demonstrated in a larger study involving patents with worse left ventricular function.Briefly, our MMPOC technique adjusted after CPB promotes early metabolic recovery of the heart during elective CABG and leads to better myocardial protection and functional recovery.POC: Post-conditioning; MMPOC: Modified type of mechanical post-conditioning; LAD: Left anterior descending; CPB: Cardiopulmonary bypass; ICU: Intensive care unit; MI: Myocardial infarction; CK-MB: Creatine kinase isoenzyme; CK: Creatine kinase; TnI: Troponin I; CI: Cardiac indices; CABG: Coronary artery bypass grafting; CVP: Central venous pressure; MPAP: Mean pulmonary artery pressure; PCWP: Pulmonary capillary wedge pressure; NYHA: New York Heart Association.There is no undisclosed ethical problem or competing interests related to the submitted manuscript.SD, MS, DC participated in the design of the study and drafted the manuscript. EO, MBI, ARA, LY, SE, AU participated in performing trial patients and data collection. AHE, DC participated in the statistical analysis. SD, MS conceived of the study, participated in its design and coordination, helped to draft the manuscript and give final approval of the version to be published. All authors read and approved the final manuscript.


Lepidoptera encompasses more than 160,000 described species that have been classified into 45–48 superfamilies. The previously determined Lepidoptera mitochondrial genomes (mitogenomes) are limited to six superfamilies of the lineage Ditrysia. Compared with the ancestral insect gene order, these mitogenomes all contain a tRNA rearrangement. To gain new insights into Lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to the non-ditrysian lineage Hepialoidea and conducted a comparative mitogenomic analysis across Lepidoptera.Thitarodes renzhiensis and T. yunnanensis are 16,173 bp and 15,816 bp long with an A + T content of 81.28 % and 82.34 %, respectively. Both mitogenomes include 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and the A + T-rich region. Different tandem repeats in the A + T-rich region mainly account for the size difference between the two mitogenomes. All the protein-coding genes start with typical mitochondrial initiation codons, except for cox1 (CGA) and nad1 (TTG) in both mitogenomes. The anticodon of trnS(AGN) in T. renzhiensis and T. yunnanensis is UCU instead of the mostly used GCU in other sequenced Lepidoptera mitogenomes. The 1,584-bp sequence from rrnS to nad2 was also determined for an unspecified ghost moth (Thitarodes sp.), which has no repetitive sequence in the A + T-rich region. All three Thitarodes species possess the ancestral gene order with trnI-trnQ-trnM located between the A + T-rich region and nad2, which is different from the gene order trnM-trnI-trnQ in all previously sequenced Lepidoptera species. The formerly identified conserved elements of Lepidoptera mitogenomes (i.e. the motif ‘ATAGA’ and poly-T stretch in the A + T-rich region and the long intergenic spacer upstream of nad2) are absent in the Thitarodes mitogenomes.The mitogenomes of T. renzhiensis and T. yunnanensis exhibit unusual features compared with the previously determined Lepidoptera mitogenomes. Their ancestral gene order indicates that the tRNA rearrangement event(s) likely occurred after Hepialoidea diverged from other lepidopteran lineages. Characterization of the two ghost moth mitogenomes has enriched our knowledge of Lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements.The mitogenomes of  Drosophila yakuba mitogenome, the first insect mitogenome to have its sequence determined, is shared by the majority of insect species, and this is therefore considered to be the ancestral order for the entire class Insecta  and T. yunnanensis [GenBank: HM744695]). Because of expansion of the A + T-rich region, the mitogenome sequence of T. renzhiensis is longer than any other complete Lepidoptera mitogenome sequenced to date. The 1,584-bp mtDNA sequence comprising partial rrnS, the A + T-rich region, trnI, trnQ, trnM, and partial nad2, was also determined for Thitarodes sp. [GenBank: HQ883371].The complete mitogenomes of T. renzhiensis and T. yunnanensis mitogenomes both include the entire set of 37 genes 3CTTTTTCAACTA whereas there is no such intergenic spacer in T. renzhiensis. There is a 15-bp intergenic spacer between trnS(UCN) and nad1 in both mitogenomes. In this region, a conserved motif ATACTAA is present in all previously sequenced Lepidoptera mitogenomes , AUU (Ile), and UUU (Phe). As a consequence, Leu (UUR), Ile, and Phe are the three most frequently used codon families  and one truncated repeat unit (101 bp). However, the A + T-rich region of Thitarodes sp. consists entirely of non-repetitive sequences  [Papilio maraho (252 bp) [Spilonota lechriaspis (124 bp) [The A + T-rich region of (126 bp) , Papilio(252 bp) , and Spi(124 bp) ,59. ThesrrnS gene in the previously sequenced Lepidoptera mitogenomes, there is a widely conserved structure that includes the motif ‘ATAGA’ and a 16–22 bp poly-T stretch. It has been suggested that this structure might function as a signal for mtDNA replication initiation [T. yunnanensisT. renzhiensis, and Thitarodes sp., indicating that these structural motifs are not conserved in the non-ditrysian Lepidoptera species. The Thitarodes mitogenomes may adopt a different strategy for replication initiation. The absence of the conserved elements in the A + T-rich region, together with above mentioned structures, such as the presence of the ancestral gene arrangement, and the absence of the intergenic spacer upstream of nad2, demonstrates that the Thitarodes mitogenomes have unusual features compared with the previously determined Lepidoptera mitogenomes. Therefore, in future studies, more attention should be paid to non-ditrysian lineages when comparing Lepidoptera mitogenomes.Downstream of the itiation ,60,61. HThitarodes renzhiensis and T. yunnanensis mitogenomes are the first representatives of non-ditrysian lineages of Lepidoptera. The arrangement of the tRNA genes between the A + T-region and nad2 is trnI-trnQ-trnM, different from those of previously sequenced Lepidoptera mitogenomes but identical to the ancestral gene order. Therefore, the previously identified tRNA rearrangement is not a synapomorphy for Lepidoptera mitogenomes. This result indicates that the tRNA rearrangement event(s) likely occurred after Hepialoidea diverged from other lepidopteran lineages. In addition, compared with other Lepidoptera mitogenomes, T. renzhiensis and T. yunnanensis mitogenomes have other unique structural characters such as the lack of the intergenic spacer upstream of nad2. Therefore, non-ditrysian lineages should be taken into consideration in future comparative mitogenomic studies of Lepidoptera. Characterization of the two mitogenomes has contributed to our understanding of Lepidoptera mitogenomes and provided insights into mitogenome evolution, especially gene rearrangements.T. renzhiensis and T. yunnanensis were obtained from cultures in Diqing, Yunnan Province, China. Specimens of Thitarodes sp., whose complete binomial name has not been specified, were collected in the Qilian Mountain, Qinghai Province, China. All the specimens were preserved in anhydrous ethanol and stored at −20 °C until used for DNA extraction. Whole genomic DNA was extracted from an ethanol-preserved larva with the DNeasy Blood & Tissue kit . DNA quality was assessed by electrophoresis in a 1 % agarose gel stained with ethidium bromide.Samples of T. renzhiensis and T. yunnanensis were amplified in 10 overlapping fragments (F1 to F10) using long PCR. All primers were designed based on the conserved nucleotide sequences of the known Lepidoptera mitogenome sequences (Table The entire mitogenomes of both es Table . Primer Raw sequence files were proof read and assembled into contigs using ContigExpress included in the Vector NTI (9.1) package.PCGs were determined and translated into putative proteins using the ORF finder in the DNAStar package . The identity of these polypeptides was established using the BLAST program available on the NCBI web site. To determine the gene boundaries, the 5' ends of PCGs were assumed to be at the first legitimate in-frame start codon  in an open reading frame (ORF) that was not located within an upstream gene encoded on the same strand; the 3' ends were inferred to be at the first in-frame stop codon encountered downstream of the start codon. When the stop codon was located within the sequence of a downstream gene encoded on the same strand, a truncated stop codon (T or TA) adjacent to the beginning of the downstream gene was designated as the termination codon ,11. ThisrrnL was annotated to extend to boundaries of the flanking trnL(CUN) and trnV. The 3' end of rrnS was annotated to be adjacent to the start of trnV, while the 5' end was determined via comparison with orthologous sequences of other Lepidoptera mitogenomes using MEGA ver4.0 [Identification of tRNA genes was performed using the tRNAscan-SE Search Server  with invA ver4.0 .http://amiga.cbmeg.unicamp.br/) [T. renzhiensis and T. yunnanensis mitogenomes were calculated with MEGA. To measure synonymous codon usage bias, RSCU values were also computed. Average codon usage and RSCU values were further analyzed for each superfamily used in this study. GC-skew = (G-C)/(G + C) and AT-skew = (A-T)/(A + T) were used to measure base compositional differences [Nucleotide sequences of the 13 mitochondrial PCGs of the previously determined 37 Lepidoptera species (Table amp.br/) . Nucleotferences . The tanferences .atp6 and atp8, ATP synthase subunits 6 and 8; cob, cytochrome b; cox1–3, cytochrome c oxidase subunits 1–3; nad1–6 and nad4L, NADH dehydrogenase subunits 1–6 and 4 L; rrnS and rrnL, small and large ribosomal RNA (rRNA) subunits; trnX, transfer RNA (tRNA) genes with X representing the one-letter abbreviation of the corresponding amino acid.The authors declare that they have no competing interests.DRY conceived and designed this study. JYC collected specimens and extracted DNA. YQC carried out molecular experiments. YQC and CM analyzed the data and drafted the manuscript. DRY thoroughly revised the manuscript. All authors contributed to the manuscript and approved the final version.Figure S1. Codon distribution in mitogenomes of currently used lepidopteran superfamilies. CDspT, codons per thousand codons.Click here for fileFigure S2. The relative synonymous codon usage (RSCU) in mitogenomes of currently used lepidopteran superfamilies.Click here for fileTable S1. PCR primer sequences.Click here for file


We evaluated cardiac autonomic modulation by heart rate (HRV), and arterial pressure variability (APV), and metabolic response in streptozotocin diabetic rats treated with green tea. Male Wistar rats were separated in groups: control, drinking tap water (C), green tea-treated (GT) group, diabetic, drinking tap water (D), and diabetic, treated with green tea (DGT). Kidney mass was greater in D and DGT than in C and GT, but reduced in DGT compared to D. Green tea prevented the increase in creatinine clearance and reduced hyperglycemia in DGT compared to D. Arterial pressure was increased in GT and decreased in D compared to C. HRV was reduced in D compared with all groups. APV was decreased in D compared to C and recovery in DGT. Sympathetic modulation of APV was decreased in D compared with all groups. Green tea reduced hyperglycemia, prevented renal injury and autonomic dysfunction, suggesting reduced cardiovascular risk and target organ damage in diabetes. Diabetes mellitus (DM) is a metabolic disorder characterized by an impairment of carbohydrate, fat, and protein metabolism caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin . This di A number of studies have been published concerning the association between chronic hyperglycemia and cardiovascular complications in DM, including systemic hypertension, atherosclerosis, nephropathy, coronary ischemia, vascular disease, and stroke –7. DysfuHeart rate and blood pressure variability (HRV and BPV), estimated in time or frequency  domain, have been widely used to evaluate the cardiac autonomic modulation in both clinical and experimental studies –16. HearCamellia sinensis, Theaceae) is a popular beverage in east Asia, and in recent years green tea has been widely studied to assess its beneficial effects in the treatment and prevention of human disease [Green tea , green tea-treated group , diabetic, drinking tap water , and diabetic, treated with green tea . The animals were housed in cages with free access to water and food, at a constant temperature of 23°C, on a 12-hour light/dark cycle. All experimental protocols were in accordance with the Guidelines for Ethical Care of Experimental Animals and were approved by the Institutional Animal Care and Use Committee (Protocol: 025/09/2008).Experiments were performed on adult male (eight-week-old) Wistar rats weighing 260–300 g. The animals were randomly separated in four experimental groups: control, drinking tap water  and blood glucose, and creatinine concentrations were determined in nonfasting animals using colorimetric enzyme kits . The kidneys were quickly removed and the ratios of kidney: body weight (kidney mass index) were determined.Diabetes was induced by a single injection of STZ  dissolved in 0.05 M citrate buffer, pH 4.5, administered 21 days before the treatment. Controls (C and GT) were injected with the vehicle  alone. Animals were fasted for 4 h before STZ or vehicle injection. During the 24-hr after STZ induction (D and DGT) or vehicle injection (C and GT), the animals were fed with glucose solution (12.5 g/L) to avoid ketoacidosis.μL of blood from tail vein) . Animals with blood glucose levels less than 250 mg/dL or greater than 400 mg/dL were excluded from the study to avoid great discrepancies in our data due to changes in glucose levels since it is well known the influence of glucose levels on metabolic and cardiovascular functions.Two days following STZ injection, diabetes induction was confirmed by blood glucose level  to 1000 mL of boiled water cooled to 90°C. The solution was filtered after 15 min, cooled to room temperature, and dispensed into clean drinking bottles. The total content of caffeine and polyphenols in the green tea solution was 0.07% and 422.80 n = 7; GT, n = 6; D, n = 6; DGT, n = 8), at the end of the second week (day 14), animals were housed separately for 48 h in metabolic cages  with free access to food and tap water or green tea solution. Hydric consumption, urinary excretion, and food intake during the last 24 h between 9:00 am and 10:00 am. Urine glucose and creatinine were measured using a colorimetric enzyme assay .In a subgroup of rats randomly selected , glucose was measured using an Accu-Check Advantage Blood Glucose Monitor . Animals were fasted for 4 h, given an intraperitoneal glucose load , and blood samples were taken at baseline and 15, 30, 60, and 90 min from a cut made on the tip of the tail.At the end of the study, in a subgroup of randomly selected rats . The catheter was exteriorized through the back of the neck.One day prior to arterial pressure (AP) recordings, an arterial catheter was placed in the right femoral artery of study animals under ketamine-xylazine anesthesia (50 : 10 mg/kg i.p.), for the direct measurement of AP in all groups  connected to the arterial catheter. Animals were conscious and moved freely during recording. The AP signal was fed into an amplifier  connected to a 16-channel analog-to-digital interface, and continuously sampled (2 kHz) on an IBM/PC . Beat-to-beat values of systolic, diastolic, and mean AP  were determined, and heart rate (HR) or pulse interval (PI) were calculated as the interval between successive systolic pressure values using the software application WinDaq .PI and SAP fluctuations were assessed in the time and frequency domains using autoregressive spectral analysis, as described elsewhere , 31, 32.P < 0.05.Data are reported as mean ± SEM. Statistical analyses were performed using two-way analysis of variance (ANOVA) followed by the Bonferroni test. Differences were considered to be significant at P < 0.05). There was no significant difference between the mean body weights of the D group compared with DGT showing that green tea consumption did not prevent weight loss in diabetic animals. In fact, while the groups C and GT showed a significant increase in body weight , a significant reduction in body weight was observed in D (7.5%) and DGT (9.6%) groups at the end of the study. The kidney mass index was greater in D and DGT than in C and GT animals (P < 0.05). However, green tea intake reduced the kidney mass index in DGT animals compared with D .The induction of diabetes mellitus in the experimental animals was confirmed by blood glucose value above 250 mg/dL 48 h after STZ induction. D and DGT animals developed hyperglycemia , their serum glucose concentrations being significantly higher than nondiabetic animals . Diabetic rats treated with green tea (DGT) showed lower hyperglycemia than diabetic rats not treated with green tea (D), suggesting that green tea consumption protects from severe hyperglycemia in diabetic rats. It is important to note that green tea did not change the serum glucose level in GT animals . DespiteP < 0.0001). Green tea consumption prevented the strong increase in creatinine clearance in the DGT group (0.75 ± 0.1 mL/min/Kg) compared with D group (1.1 ± 0.3 mL/min/Kg). However, in nondiabetic rats there was no statistically significant effect of green tea on creatinine clearance . Finally, diabetic rats treated with green tea showed a significant reduction in urine glucose compared with D. Although, there was a difference between C and GT in urine glucose, it was not statistically significant  and untreated (D) animals showed serum, and urine glucose levels elevated compared with C group. However, we observed a significant reduction in both serum and urine glucose levels in the DGT group as compared to the D group. Considering that hyperglycemia is the principal factor responsible for kidney and cardiovascular damage , the antWith regard to metabolic parameters, STZ rats showed polydipsia, polyphagia, and polyuria . HoweverGlomerular filtration rate assessed by creatinine clearance was measured in our study to provide information about renal function. Diabetic rats (D and DGT) increased creatinine clearance compared with nondiabetic rats C and GT. It is interesting that green tea consumption resulted in lower creatinine clearance in DGT compared with D, but did not affect nondiabetic rats (C versus GT), thus, our data suggests that green tea prevents glomerular hyperfiltration in diabetes. In support with our data, it has been reported that in rats with streptozotocin-induced diabetes, green tea consumption showed a significant reduction in renal injury associated with hyperglycemia .Diabetes caused an abnormal increase in kidney mass index in both diabetic groups D and DGT. Interestingly, green tea consumption by DGT group significantly reduced diabetes-induced hypertrophy of kidney by 20% as compared to the D group. These results suggest the importance of green tea in reducing kidney hypertrophy and corroborate another study with STZ rats . CreatinIn the present study, we observed in rats with STZ-induced diabetes a reduction in AP and HR associated with a decrease in HRV and APV as previously described , 41, 42.in vitro studies have demonstrated that green tea extracts and tannin mixtures have a direct scavenging activity against nitric oxide and superoxides [The favorable effects attributed to the green tea extract in the prevention of cardiovascular diseases are correlated with the antioxidant properties of the catequins, which are the major components of green tea . Experimeroxides . Thus, weroxides . TherefoIn summary, the major findings of this study are that green tea decreased hyperglycemia and prevented renal injury by improvement of glomerular filtration and kidney hypertrophy observed in diabetic rats. Moreover, green tea was able to prevent the autonomic dysfunction in diabetic rats by blocking the alterations in arterial pressure variability. In conclusion, our data suggest that green tea consumption has important effects in reducing the cardiovascular risk and in targeting organ damage observed in diabetes.


This work investigates the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2) and Bcl-2/Bax in rat skeletal muscles. A total of 18 Sprague-Dawley female rats were randomly divided into three groups: the control group (CON), the trained control group (TC), and the exhaustive trained group (ET). Malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XOD), ATPase, UCP2, and Bcl-2/Bax ratio in red gastrocnemius muscles were measured. Exhaustive training induced ROS increase in red gastrocnemius muscles, which led to a decrease in the cell antiapoptotic ability (Bcl-2/Bax ratio). An increase in UCP2 expression can reduce ROS production and affect mitochondrial energy production. Thus, oxidative stress plays a significant role in overtraining. The mechanism ofovertraining (OT) remains poorly understood.One common theory considered is glycogendepletion.Ourprevi2+ released from the endoplasmic reticulum-induced MPTP opening, leading to apoptosis . Bo. Bo11]. The TC and the ET groups were sacrificed after 36 h after the last training session to avoid acute exercise effects. The control group was sacrificed at the end of the nine weeks. All rats were anesthetized with pentobarbital (40 mg/kg body weight). Blood was rapidly collected from the abdominal aorta; plasma was separated by centrifugation and then stored at −78°C for further analysis . The samHb was analyzed by an automatic cytometer .2, 120 mM KCl, 5 g/L BSA, with pH of 7.4. The tissue fragments obtained were homogenized with the above medium . The homogenate was then centrifuged at 600 g for 10 min twice. The resulting supernate was centrifuged at 17000 g for 10 min. The pellet was homogenized with the above medium  and then centrifuged again at 7000 g for 10 min. The pellet was washed and resuspended in a suspension medium  and centrifuged at 3500 g for 10 min. The pellet was resuspended in the suspension medium described above to determine SOD, XOD, and ATPase activities and MDA [The red gastrocnemius muscles were freed of excess fat and connective tissue, finely minced, and washed in a medium containing 20 mM HEPES, 2 mM MgCl and MDA . The pelProtein concentrations were determined by Bio-Rad Bradford protein assay.Mitochondria SOD, XOD, ATPase activities, and MDA concentrations were measured using a commercial kit .μg) were electrophoresed on sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinyl difluoride. Themembranes were blocked with 5% nonfat dry milk in Tris-buffered saline for 1 h at roomtemperature, and incubated overnight at 4°C with primary antibodies .The membranes were then incubated with appropriate horseradish peroxidase-linked secondary antibodies  for 2 h at room temperature and visualized using an enhanced chemiluminescence detection system  [After the red gastrocnemius muscles were homogenized in an ice-cold lysis buffer,whole-tissues homogenates were centrifuged at 14,000 g for 20 min,and the supernates were collected. After quantitation of proteinconcentration, equal amounts of proteins (30 μm) from each group were dehydrated in xylene and graded ethanol series and added in order with the primary antibody , biotinylated secondary antibody , SABC reagents, and diaminobenzidine solution . For negative control, the sections were treated with PBS instead of primary antibody. Ten sample sections from each group were selected for analysis. More than five visual fields were observed per section. The following equation was used: the positive percentage of each protein = protein − positive cells/all cells × 100%.Bcl-2 and Bax from the red gastrocnemius muscle of each group were determined by the streptavidin-biotin complex (SABC) method. The paraffin sections . The results are presented as means ± standard deviation (SD). We used one-way ANOVA followed by least significance difference post hoc test to compare the means of the three groups. If data failed the normality test, the Kruskal-Wallis one-way ANOVA on ranks and Tukey's post hoc test were used. P < 0.05), but no significant difference was indicated between the TC and the ET groups (Both trained groups exhibited a significant decrease in body weight relative to the CON group at the end of the ninth week of training (T groups .P < 0.001) (P < 0.05). SOD activity was significantly decreased in the ET group compared with the CON group (P < 0.05). The SOD/MDA ratio was significantly increased in the TC group compared with the CON group (P < 0.05), but the SOD/MDA ratio was significantly decreased in the ET group than that in the CON group (P < 0.001). XOD activity was significantly increased in the ET group compared with the CON group (P < 0.05). Meanwhile, ATPase activity was significantly decreased in the ET group compared with the CON group (P < 0.001).Hb was significantly decreased in the ET group compared with the CON and the TC groups (< 0.001) .MDA concP < 0.05). However, no difference in the UCP2 expression was indicated between the TC and the CON groups  decreased in the ET group compared with the CON group  of oxidative phosphorylation is identified as a major site for cellular ROS generation.As electrons pass through the complexes of the ETC, some of these electrons leak to molecular oxygen, thus forming superoxides . When en2•−  can be activated in the endometrial stromal side of uncoupling proteins (UCPs). This activation leads to proton transfer and mitochondrial membrane solution coupling, thus reducing ROS generation [Mitochondrial ROS production highly depends on the membrane potential generated by the proton gradient formed across the inner mitochondrial membrane . High meneration , 27. ROSneration , activatneration . In the neration . Continuneration , 27. 2+ release [UCP2 can affect mitochondrial energy production when ROS generation is inhibited. Studies found that the ATP content of UCP2-overexpressing islets was reduced by 50%  and that release .UCP2 does not only suppress mitochondrial ROS; it also regulates apoptosis in various cell systems . CultureApoptosis isa complexprocess involving several cellular proteins. A study suggested that Bcl-2/Baxexpressionbalance determinessurvival or death following apoptosis . We founIn summary, exhaustive training increases the ROS in red gastrocnemius muscles, decreases SOD activity, and causes oxidative stress.Extensive ROS production increases UCP2 expression, which modulates the membrane potential and decreases ROS production. However, the increase in UCP2 expression also leads to a decline in mitochondrial energy metabolism.These effects can significantly affect exhaustive training-induced OT. On the other hand, ROS accumulation can increase Bax expression and reduce Bcl-2 expression, promoting cell apoptosis.These behaviors can be related to a pathological change after OT.The currentproblem is thatunder normal circumstances,the ATP/ADP ratioregulates the cytochromec oxidase (CcO)activity . CcO is