Tribolium castaneum), in reference to the reproduction success rate, the number of viable offspring produced, and the mean offspring mass 13 days after mating. The most influential stage affecting reproduction differed between the diet and temperature experiments: adult temperature vs. parental growth diet. Generally, a yeast-rich diet or warmer temperature improved reproduction, supporting the silver-spoon hypothesis. However, interactions between life stages made the results more complex, also fitting the environment-matching hypothesis. Warm growth temperature positively affected reproduction success, but only when adults were kept under the same warm temperature. When the parental growth and adult diets matched, the mean offspring mass was greater than in a mismatch between the two. Additionally, a match between warm adult temperature and warm offspring growth temperature led to the largest offspring mass. These findings support the environment-matching hypothesis. Our results provide evidence for all these hypotheses and demonstrate that parental effects and plasticity may be induced by temperature and diet.The effects of different temperatures and diets experienced during distinct life stages are not necessarily similar. The silver-spoon hypothesis predicts that developing under favorable conditions will always lead to better performing adults under all adult conditions. The environment-matching hypothesis suggests that a match between developmental and adult conditions will lead to the best performing adults. Similar to the latter hypothesis, the beneficial-acclimation hypothesis suggests that either developing or acclimating as adults to the test temperature will improve later performance under such temperature. We disentangled here between the effect of growth, adult, and mating conditions (temperature and diet) on reproduction in the red flour beetle ( Climate has a major effect on all aspects of animal life, from development, through behavior, to reproduction –4. BecauThe beneficial-acclimation hypothesis  suggests that animals acclimated to a particular temperature perform better under such temperatures compared to animals acclimated to other temperatures , 16. ThiAcclimation always helps: According to BAH in its broad sense, growing or acclimating to inferior conditions will improve performance or enhance tolerance to similar or even harsher conditions later. (2) Only adult acclimation helps: Adult acclimation to inferior conditions will lead to improved performance, but development under superior conditions will always lead to individuals of higher quality and performance. (3) No acclimation: Larvae or adults exposed to the superior conditions will perform better under inferior conditions (no acclimation to the inferior conditions).Acclimation is not limited only to temperature. For example, some studies tested developmental acclimation to low- vs. high-water diets or to carbohydrate- vs. protein-rich diets , 23. TheRelatively few studies have disentangled the effect of growth and adult temperatures on adult performance, and the traits in such studies are usually those of physiological or behavioral responses , 19, 24.The same question applies regarding diet: What should be the relative contribution of rich vs. poor diet during the juvenile and adult stages? The "silver-spoon" hypothesis suggests that animals developing under a rich diet will perform better as adults under all diets. The "environment-matching" hypothesis suggests that the matching between developmental and adult diet is the most important factor dictating performance. In contrast to the previous hypothesis, developmental history is relevant in regard to adult conditions , 28. In Tribolium castaneum Herbst 1797) to separate between the effects of diets and temperatures experienced throughout life on reproduction, and to determine whether the silver-spoon or environment-matching hypothesis suits better. Specifically, the beetles were alternated between two food conditions and two temperatures during three life stages: growth, early adulthood, and mating. Our design (1) disentangled between the effect of growth and adult conditions, and (2) determined the possible contribution of a match or mismatch between these two stages and the larval growth conditions. We thus examined whether the silver-spoon or environment-matching hypothesis better fits our system in respect to both parental growth and adult conditions.We used the red flour beetle (Tribolium castaneum (Coleoptera: Tenebrionidae) is a common storage pest, living at high densities and demonstrating habitat preferences according to temperature and food type ),An effect, even a mild one, of adult diet in similar experiments separating between the juvenile and adult diets has been commonly found, and the effect is usually similar in direction to that of growth diet e.g., , 32). Ho. Ho32]).We could have expected anticipatory parental effects when offspring were grown under yeast-poor diets, after adults were either grown or moved at some stage, early adulthood or mating, to yeast-poor diets . Such adIn general, reproduction investment should be optimized by the mother in a way maximizing her own fitness and not the fitness of each individual offspring , 57. ParIn short, we separated here between the contribution of growth, adult and mating conditions for reproductive success, expressed in offspring number and growth rate. We applied the same experimental design for a thermal experiment and a diet experiment. While parental growth stage was the most important one considering diet, parental adult stage was the influential stage for the thermal experiment. We mostly bring evidence for the advantage of specific conditions (either warmer temperature or yeast-richer diet), but also some evidence for a higher reproductive success when growth and adult or adult and offspring conditions match. In any case, this study provides an example for parental effects and their interaction with the offspring growth conditions.S1 Data(XLSX)Click here for additional data file.


Despite the fact that the clinical features of migraine are well described, the relationship between migraine and cognitive performance is still poorly understood. Indeed, some authors have reported the presence of cognitive deficits in patients with migraine without aura (MwoA) and with aura (MwA) whereas others have not confirmed these findings. Although neuropsychological studies in migraine are not conclusive, the most likely pattern of neuropsychological impairment would relate to the cognitive domain of executive functions (EF) . Recent To further explore FPN functional connectivity in patients with MwA and patients with MwoA, in the interictal period.Using resting-state functional magnetic resonance imaging (RS-fMRI), we compared functional connectivity within the FPN in 20 patients with MwA, versus 20 sex- and age-matched healthy controls (HC). To examine the specificity of any observed differences in FPN functional connectivity between patients and HC, we further studied 20 age- and sex-matched patients with MwoA. Furthermore, we assessed the correlation between functional connectivity within FPN and EF in both migraine groups. Finally, we used voxel-based morphometry to assess whether between-group differences in functional connectivity were dependent on structural differences.Neuropsychological data revealed no significant executive dysfunction in both migraine groups compared to HC. RS-fMRI showed that both MwA and MwoA patients, compared to HC, had a significant functional connectivity decrease within the right FPN and specifically in the middle frontal gyrus and the dorsal anterior cingulate cortex. There were no structural differences between the three groups.Our data demonstrate that, even in the absence of clinically evident EF deficits, MwA and MwoA are associated with reduced FPN functional connectivity. We suggest that disrupted FPN functional connectivity might be only a part of a complex cascade that terminates in a migraine attack. In this context, FPN abnormalities may be the neuronal substrate on which biological, genetic and environmental factors could induce, and in turn correlate with, migraine attacks mostly characterized by high pain intensity in patients with MwoA and aura phenomenon in patients with MwA. In other terms, observed FPN connectivity changes may represent a migraine biomarker, probably related to well-known maladaptive stress response in migraine patients.Written informed consent to publish was obtained from the patient(s).


Hypercholesterolemia is a frequent metabolic disorder associated with increased risk for cardiovascular morbidity and mortality. In addition to its well-known proatherogenic effect, hypercholesterolemia may exert direct effects on the myocardium resulting in contractile dysfunction, aggravated ischemia/reperfusion injury, and diminished stress adaptation. Both preclinical and clinical studies suggested that elevated oxidative and/or nitrative stress plays a key role in cardiac complications induced by hypercholesterolemia. Therefore, modulation of hypercholesterolemia-induced myocardial oxidative/nitrative stress is a feasible approach to prevent or treat deleterious cardiac consequences. In this review, we discuss the effects of various pharmaceuticals, nutraceuticals, some novel potential pharmacological approaches, and physical exercise on hypercholesterolemia-induced oxidative/nitrative stress and subsequent cardiac dysfunction as well as impaired ischemic stress adaptation of the heart in hypercholesterolemia. Hypercholesterolemia, a frequent form of hyperlipidemia, is a metabolic disorder characterized by elevated levels of total cholesterol in the blood. Hypercholesterolemia may develop as a consequence of unbalanced diet, obesity, inherited (genetic) diseases , or other diseases . According to large clinical studies, hypercholesterolemia affects a significant population of adults in developed countries . For insOxidative/nitrative stress can be defined as an excess formation or insufficient removal of highly reactive molecules such as reactive oxygen and/or nitrogen species  including, for instance, superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrite . EnzymatAnother possible explanation for increased oxidative stress could be diminished endogenous antioxidant capacity. Indeed, hypercholesterolemia has been reported to be associated with decreased cardiac expression and activity of antioxidant enzymes –21. NeveIn addition to the effects of cholesterol and its derivatives on gene expression, membrane-related effects of cholesterol are also plausible ; howeverHypercholesterolemia has been shown to exert direct myocardial effects independent of the development of atherosclerosis in both clinical , 41 and Observations in human clinical trials and animal models suggest a direct effect of cholesterol on myocardial contractile function leading to impaired diastolic and in some cases also systolic function –8, 43–45The direct effects of cholesterol exposure on cardiomyocyte function were demonstrated in a cell culture model; that is, elevation of membrane cholesterol content in ventricular cardiomyocytes resulted in decreased cytosolic calcium levels and impaired cardiac myocyte contractility . This haIt has been shown previously that myocardial oxidative/nitrative stress induced by hypercholesterolemia significantly contributes to the development of cardiac dysfunction , 8, 50. Hypercholesterolemia facilitates the risk of atherosclerosis and subsequent myocardial infarction. The current treatment of myocardial infarction includes the attempt to reopen the occluded coronary artery (termed reperfusion) in a timely manner by coronary intervention procedures or thrombolytic therapies in order to reduce infarct size, which is one of the major determinants of long term complications and survival. Thus the majority of patients with acute myocardial infarction undergo ischemia/reperfusion injury. However, the myocardium is remarkably good at adapting to ischemic conditions by triggering endogenous adaptive mechanisms against ischemia/reperfusion injury. One of the most powerful endogenous adaptive cardioprotective mechanisms is ischemic preconditioning, that is, when brief exposure to repetitive ischemia/reperfusion cycles markedly enhances the ability of the heart to withstand a subsequent, potentially lethal ischemic attack . AnotherModulation of oxidative/nitrative stress in hypercholesterolemia can be approached by at least 3 different ways . First, To the best of our knowledge, there are no drugs on the market approved to specifically target cardiac oxidative/nitrative stress induced by hypercholesterolemia. Nevertheless, cholesterol-lowering drugs are excessively prescribed for patients presenting hypercholesterolemia and due to their cholesterol-lowering effect they should have a secondary attenuating effect on hypercholesterolemia-induced oxidative/nitrative stress. Interestingly, several of these drugs have also been implicated in directly modulating oxidative/nitrative stress. Moreover, large clinical trials have shown that antihyperlipidemic agents, for example, statins , fibrateStatins are a class of cholesterol-lowering drugs that inhibit the key enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, of the endogenous cholesterol biosynthesis. Statins have therefore become established in the treatment of hypercholesterolemia and attained a central place in cardiovascular medicine . HoweverRegarding the antioxidative effect of statins, a recent study showed that, beside the cholesterol-lowering effect, simvastatin is able to ameliorate endothelial dysfunction through increasing NO bioavailability and through suppression of oxidative stress in a rat model of hypercholesterolemia . SimilarEzetimibe, another lipid-lowering drug, is used in monotherapy or in combination with statins and is responsible for lowering intestinal cholesterol absorption by inhibiting the Niemann-Pick C1-Like 1 (NPC1L1) sterol transporter. Clinical studies showed that ezetimibe attenuates the markers of oxidative stress in hyperlipidemic subjects –80. The 3) is a potent lipid-lowering agent when used in pharmacologic doses, its clinical use is limited by side effects, so it can be applied particularly in combination to treat marked dyslipidemia with strict monitoring , leading to transcriptional changes [ in vivo administration of fenofibrate and clofibrate before ex vivo myocardial ischemia/reperfusion reduced infarct size and oxidative stress [α, the ligand of fibrates, leads to oxidative stress associated with cardiac dysfunction [Fibrates (amphipathic carboxylic acid derivatives) lower plasma triglyceride and small LDL level and increase high density lipoprotein (HDL) concentration; thus fibrates are particularly used to treat combined dyslipidemia related to diabetes or metabolic syndrome . This cl changes . Studies changes . In acco changes . Fibratee stress . Others e stress , 99. Fure stress , 101. Itfunction . In contfunction , and higfunction . Taken tThere are some available drugs, which are basically used to treat other diseases than hypercholesterolemia and which have beneficial effects on high-cholesterol-induced cardiac consequences.Rosiglitazone is an antidiabetic drug; however, it also improves blood cholesterol level in hypercholesterolemic models , 106. ItIncreasing the level of glucagon-like peptide-1 by administration of analogues or by inhibiting dipeptidyl peptidases is used for the treatment of diabetes and has recently emerged as a potential cardioprotective approach . SpecifiThe vasodilator fasudil (a potent rho kinase inhibitor) was shown to lower serum cholesterol level and decrease cardiac oxidative stress by enhancing antioxidants in hypercholesterolemic rat hearts . The groIn spite of the extensive research in cardiology, particularly in ischemic heart diseases, only very few new cardioprotective drugs have found their way into clinical practice . TherefoNatural food substances have the potential to alter biological functions of the cells by mechanisms enhancing the endogenous antioxidant systems or through altering the redox signaling status of the cell. This is often related to the unique composition of different antioxidant compounds in the various nutraceuticals. These products could be beneficial in pathological conditions where oxidative stress plays an important role.9) vitamin C, and vitamin E administered alone or in combination with other drugs or in multivitamin preparations could improve serum lipid profile and myocardial oxidative/nitrative stress in hypercholesterolemia [A number of preclinical studies have demonstrated that classical antioxidant vitamins including beta carotene (Vit A precursor), folic acid , also called ubiquinone or ubiquinol, is an isoprene derivative and an alternative product of the mevalonate/cholesterol pathway. CoQ10 is an endogenous nonenzymatic lipophilic antioxidant and free radical scavenging molecule playing a role in the mitochondrial electron transport in the inner mitochondrial membrane . MoreoveDue to the common biosynthetic pathway of cholesterol and CoQ10, the HMG-CoA reductase inhibitor statins may potentially reduce the levels of CoQ10 in different tissues . Indeed,Flavonoids are a large family of natural polyphenolic compounds in the human diet and their beneficial effect on cardiovascular diseases is widely studied. These compounds favorably affect a wide range of biological processes. Beside their antioxidant capacity, flavonoids improve lipid profile and have anti-inflammatory, antiplatelet, and antithrombotic effects as well.Green Tea Catechins (GTC). Catechins are abundant polyphenols in green tea. GTC were shown to reduce blood cholesterol level in both animals [ animals –143 and  animals –147. In  animals . In a do animals . Mehra e animals . GTC wer animals . Age-rel animals . The eff animals , 153 in  animals  and in a animals .Troxerutin. Troxerutin can be isolated from the Japanese pagoda tree (Sophora japonica). To the best of our knowledge, the antioxidant effect of troxerutin on the heart was first described in an ischemia/reperfusion model [on model . Geetha on model . Moreoveon model .Quercetin. The cardioprotective effect of the dietary flavonoid quercetin  against ischemia/reperfusion injury is well studied [ studied –163; how studied . In that studied  and lowe studied  and high studied  diet. Ul studied . In a ca studied .Rutin. Rutin (the glycoside between the quercetin and the disaccharide rutinose) improves cholesterol level in different hypercholesterolemic [erolemic , 171 anderolemic , 173 roderolemic . It was erolemic .Silymarin. Silymarin is a mixture of three flavonolignans  extracted from milk thistle seeds (Silybum marianum). Krečman et al. demonstrated that silymarin prevented the development of hypercholesterolemia in high-cholesterol-fed rats [fed rats . They hafed rats –178. Thefed rats  and silifed rats . Moreovefed rats . In thisNaringin and Hesperidin. Naringin and hesperidin are natural flavonglycosides in citrus fruits. Many studies reported hypocholesterolemic effect of naringin and hesperidin in animal models [l models –188 and l models . Howeverl models . Alam etl models . Recentll models . The stul models . Althougl models  or ischel models , 194, itPleiotropic beneficial effects of the polyphenol resveratrol  have been extensively studied and many publications indicate its protective function on cholesterol-induced cardiac oxidative stress. A direct antioxidant effect of resveratrol was demonstrated on H9c2 cardiac cells . Louis e In vivo pretreatment with grape seed proanthocyanidins improved postischemic functional recovery and reduced ROS production in normocholesterolemic rats [Grape seed and skin are rich in natural antioxidants, so these are possible supplements to alleviate hypercholesterolemia-induced oxidative stress. In a high-fat-induced obesity model, grape seed extract was shown to improve lipid profile and prevent postischemic heart dysfunction and cardiac lipid accumulation along with attenuated oxidative stress , 203. Lemic rats  and partmic rats .Prunus cerasus) may contain different bioactive constituents [Based on the observation that cherries contain bioactive phytochemicals, for example, phenolics and anthocyanins, which are reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic, and antiobesity properties, Tosaki and his group hypothesized that the seed kernel of sour cherry , ginger , and cayenne pepper (Capsicum frutescens) as well as their mixture were shown to attenuate cardiac lipid peroxidation induced by high-cholesterol, high-fat diet in a rat model of hypercholesterolemia [Aqueous extracts of certain spices including garlic (erolemia . Moreoveerolemia .Elaeis guineensis). RPO depending on the producer consists of about 51% saturated fatty acids (SFAs), 38% monounsaturated fatty acids (MUFAs), 11% polyunsaturated fatty acids (PUFAs), and a spectrum of antioxidative carotenoids with tocopherols and tocotrienols as the major constituents [Red palm oil (RPO) is a product from the fruits of the oil palm tree  protected the heart against ischemia/reperfusion injury in rats. They used isolated heart models, perfused both in Langendorff technique –218 and The proposed mechanism by which RPO exerts its cardioprotective effect in animals fed high-cholesterol diet is not fully understood. Supplementation with RPO in the presence of potentially harmful cholesterol showed no significant difference in serum cholesterol level; therefore, its protection cannot be explained by the cholesterol-lowering effect of RPO. It is proposed that the protective effect of RPO in high-cholesterol diet may be associated with either the RPO antioxidant characteristics and/or changes in the fatty acid composition of the myocardium during ischemia/reperfusion.Only 3% of the human genome codes for proteins and the remaining part consist of noncoding RNAs including short microRNAs  , 222. miIn recent years, a growing body of evidence has demonstrated that miRNAs play a role in the development of numerous cardiovascular diseases. Several excellent reviews focus on miRNAs as diagnostic markers and potential therapeutic targets in cardiac pathologies including acute coronary syndrome , 225 and in vivo inhibition of miR-25 by a specific antagomir resulted in the spontaneous development of cardiac dysfunction and sensitized the myocardium to develop heart failure in a Hand2-dependent manner [2+-ATPase (SERCA)2a activity and Ca2+ handling in chronic heart failure induced by transverse aortic constriction surgery in mice as well as in failing human heart samples; however, data on the presence of hypercholesterolemia in humans were lacking in this study [Hypercholesterolemia is a well-known risk factor of cardiovascular diseases and it leads to increased oxidative/nitrative stress in the myocardium. Experimental data are very limited on the regulatory role of miRNAs in hypercholesterolemia-induced oxidative/nitrative stress in cardiac pathologies. We have previously shown that the myocardial downregulation of miR-25 results in the upregulation of NADPH oxidase 4 (NOX4) mediating oxidative/nitrative stress and subsequent myocardial dysfunction in male hypercholesterolemic rats  Table 1Table 1. t manner . In contis study .∗ have been shown to be resistant to high-fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues [∗ [It is well known that hyperlipidemia, obesity, metabolic syndrome, and heart failure are associated with abnormal cardiac metabolism. In obese mice, a heart specific miRNA, miR-208a, has been reported to negatively regulate mediator complex subunit 13 (MED13), which controls transcription by thyroid hormone and other nuclear hormone receptors  Table 1Table 1.  tissues  . We have recently demonstrated that a cholesterol-enriched diet inhibited cardioprotection induced by KATP activators and that cholesterol diet may impair cardiac KATP channels [The exact explanation why physical activity, which similarly to hyperlipidemia leads to an increased oxidative stress, is able to protect the heart in hypercholesterolemia is rather difficult. One possible answer is that during exercise an intensive but only a temporary increase of oxidative stress occurs resulting in a possibility for cardiac adaptation by an improved enzymatic and nonenzymatic antioxidant capacity, cytoprotection, aerobic capacity, training-induced muscular adaptation, mitochondrial biogenesis, and so forth . In hypechannels . It is wIncreased physical activity can be also mimicked in experimental animal models by applying ventricular overdrive pacing of the heart, a method that was reported to induce both preconditioning and postconditioning by increasing the oxygen demand (relative hypoxia) instead of limiting oxygen supply (absolute hypoxia) in isolated heart models –285. PerIn addition to emphasizing the importance of regular exercise, novel future directions have been implicated to take advantage of exercise-induced benefits. Thus development of new “exercise mimetics” has a promising role in the future for treatment of patients .Oxidative and nitrative stress has been implicated as a pathophysiological mechanism of cardiovascular diseases; however, there is still no breakthrough regarding the use of general antioxidant therapies in clinical practice. The possible reasons for these disappointing results and some promising aspects of potential antioxidant therapy have been discussed in detail recently . Althoug


Both SES and supermarket choice have been associated with diet quality. This study aimed to assess the contributions of supermarket choice and shopping behaviors to the healthfulness of purchases and social patterning in purchases.Observational panel data on purchases of fruit and vegetables and less-healthy foods/beverages from 2010 were obtained for 24,879 households, stratified by occupational social class . Households’ supermarket choice was determined by whether they ever visited market-defined high- or low-price supermarkets. Analyses also explored extent of use within supermarket choice groups. Shopping behaviors included trip frequency, trip size, and number of store chains visited.Households using low-price (and not high-price) supermarkets purchased significantly lower percentages of energy from fruit and vegetables and higher percentages of energy from less-healthy foods/beverages than households using high-price (and not low-price) supermarkets. When controlling for SES and shopping behaviors, the effect of supermarket choice was reduced but remained significant for both fruit and vegetables and less-healthy foods/beverages. The extent of use of low- or high-price supermarkets had limited effects on outcomes. More-frequent trips and fewer small trips were associated with healthier purchasing for both outcomes; visiting more store chains was associated with higher percentages of energy from fruit and vegetables.Although both supermarket choice and shopping behaviors are associated with healthfulness of purchases, neither appears to contribute to socioeconomic differences. Moreover, differences between supermarket environments may not be primary drivers of the relationship between supermarket choice and healthfulness of purchases. There are substantial socioeconomic inequalities in the prevalence of non-communicable diseases, key determinants of which are behavioral risk factors, including unhealthy diets.6Neighborhood food environments may drive some of the socioeconomic differences in diet quality. Those who are more deprived may have less physical access to healthier food outlets and greater exposure to unhealthy outlets.Differences in food costs between supermarkets are another potential driver of supermarket choice, and may contribute to socioeconomic differences in diet quality.Beyond the type of supermarket patronized, food shopping patterns may affect diet quality. Previous work suggests that shopping behaviors may be associated with differences in purchasing, such as unplanned purchases being more likely during larger shopping trips compared to fill-in trips,Together, these studies suggest that both supermarket choice and shopping behaviors may shape food purchases, and subsequently the healthfulness of diets. This study aims to explore supermarket choice (including extent of use of different supermarkets) alongside shopping behavior, to provide a more nuanced account of how these factors may impact healthfulness of and social patterning in purchasing.Data were obtained from the Kantar WorldPanel (KWP) UK household survey from 2010, which comprised purchase records of 26,922 households over 52 weeks . Households were recruited by KWP via postal mail and e-mail, using quotas determined by the UK Office of National Statistics census and the UK Broadcasters’ Audience Research Board Establishment survey to ensure national representativeness in terms of UK region of residence, age group, and household size. Households received vouchers for high street retailers or leisure activities  as an incentive toward participation.Participating households were asked to record all food and beverage purchases brought home. Purchases were recorded using barcode scanners (using showcards with barcodes for non-barcoded products like fruit). Panelists also uploaded digital images of cash register receipts, which were used to verify the accuracy of households’ data. Data included volume purchased, nutritional content, and the retail chain from which products were purchased.To be included in the data set, households had to meet quality control criteria. These included meeting minimum volume and spending for purchases based on household size, every 4 weeks. In addition, households were included only if they reported at least 12 weeks’ data and at least 13 trips over 52 weeks . This gave a final sample of 24,879 households.n=5,332); white collar and skilled manual ; and semi-skilled and unskilled manual .The KWP data set categorizes head-of-household occupation using the UK Registrar General’s social class classification,We categorized all supermarkets in the KWP data set as high, medium, or low cost based on market definitions, whereby high-cost stores prioritize product quality over price, low-cost stores operate as “discounters,” and those falling between are medium-cost stores.Households were then classified according to the types of supermarkets they reported visiting. As nearly 100% of households shopped at medium-cost supermarkets over the year , supermarket choice groups were determined by whether or not households additionally ever patronized high- or low-cost supermarkets. This gave four groups: used low-cost supermarkets or low- and medium-cost supermarkets (termed “low-cost” for succinctness); used medium-cost supermarkets only (“medium-cost”); used high-cost supermarkets or medium- and high-cost supermarkets (“high-cost”); and used high-, medium-, and low-cost supermarkets .The extent of use of different types of supermarkets was characterized in terms of the percentage of trips that took place at each supermarket price tier.1.the number of trips recorded by each household each month;2.the percentage of small trips ;3.the mean number of different store chains used per month; and4.whether or not households used the same primary store chain  every month.Four characteristics of shopping behavior were examined:1.percentage of energy from less-healthy foods and non-alcoholic beverages, as classified by UK Food Standards Agency Nutrient Profile2.percentage of energy from fruit and vegetables—this included fresh, canned, frozen, and dried fruit, vegetables, and legumes but excluded juice, potatoes, and fruit and vegetables present in processed products.We used two outcome variables assessing healthfulness of food and beverage purchases, comprising less-healthy and healthier indices:Multiple regression analyses were conducted in 2014 to estimate the percentage of energy purchased from less-healthy foods/beverages and fruit and vegetables by supermarket choice and the four shopping behavior variables. To address the hypotheses relating to social patterning, analyses were conducted with and without controlling for SES .Analyses  used robust SEs, given evidence of heteroscedasticity, and the percentage of energy from fruit and vegetables was logged to address a positively skewed distribution. Reported significance levels were adjusted for multiple testing using Bonferroni correction.Analyses were also conducted within supermarket choice groups to examine whether extent of use of different supermarket tiers affects healthfulness of purchases (over and above being identified as an “ever shopper” at these store chains).Regressions controlled for age of main shopper, gender of main shopper, ethnic group of main shopper (white/non-white); number of adults in household, number of children in household, and dummies for region of residence . All households meeting study eligibility criteria had complete demographic data, so no households were excluded because of missing values.Sensitivity analyses were conducted (1) using a different cut off threshold for small trips (20 items) and (2) including trips to convenience stores. Both these sets of analyses found very similar results to those in the main analyses, so these results are not included here.The descriptive data presented in n=4] than high-cost [n=3] store chains).However, the supermarket choice groups may also show somewhat different patterns of shopping behavior: the low-cost supermarket group had on average a slightly higher number of trips per month and visited more different store chains than the high-cost supermarket group  compared with one at the 25th percentile (3.9 trips/month), whereas a 10 percentage point increase in the percentage of small trips per month was associated with a 0.4 percentage point increase in the percentage of calories obtained from less-healthy foods/beverages. Including shopping behaviors had little effect on the coefficients for SES.b coefficients). The high-cost supermarket group purchased 9% higher percentages of energy from fruit and vegetables on average compared with the low-cost supermarket group, when controlling for shopping behaviors and SES. The coefficients for supermarket choice, shopping behavior, and SES were largely similar when controlling for one another.In terms of shopping behavior, results suggest a 3% increase in the percentage of calories from fruit and vegetables for households at the 75th compared with the 25th percentile for trips per month, whereas for a 10 percentage point increase in the percentage of small trips per month, the percentage of calories from fruit and vegetables decreased by 6%. For each additional store chain visited per month, results suggest the percentage of calories households obtain from fruit and vegetables increased by 6%.The above analyses examined the effect of ever shopping at supermarkets in different price tiers (meaning there was considerable variability in the extent of use of each supermarket price tier within groups). For the high-cost supermarket group, there was no association between percentage of trips at high-cost supermarkets and percentage of calories for less-healthy foods/beverages. The effects of extent of use of high-cost supermarkets were again similar for the all-types supermarket group. A greater percentage of trips at high-cost supermarkets was associated with a greater percentage of calories being obtained from fruit and vegetables, but suggested a small effect: For the high-cost supermarket group, for a 10 percentage point increase in the percentage of trips at high-cost supermarkets, the percentage of calories from fruit and vegetables increased by 2%. The social patterning in purchasing was reduced within the high-cost supermarket group.Our analyses of a large UK data set indicate that supermarket choice might have implications for diet quality and health. Compared with households that ever used lower-cost supermarkets, households that ever used high-cost supermarkets purchased 9% higher percentages of energy from fruits and vegetables and 0.8 percentage points less energy from less-healthy foods/beverages. At the population level and over time, such differences in fruit and vegetable purchasing may be clinically significant. These results are consistent with previous studies, which found that consumers who patronized low-cost supermarkets consumed less nutritious diets and fewer vegetables and fruit.18Comparing the “ever use” analyses to the “extent of use” analyses—showing smaller (sometimes counterintuitive) or non-significant effects of supermarket use—suggested the latter had limited effects on purchasing. The lack of a dose–response relationship here suggests that differences in supermarket environments  may not be contributing greatly to differences in purchasing by supermarket price tier. Although we did not directly examine individual or cultural factors beyond SES, this may suggest that such factors  may be more-influential drivers of disparities in the healthfulness of purchases by supermarket price tier. Differences largely were not affected by socioeconomic adjustment, however, suggesting that supermarket choice and SES have independent effects on purchasing behavior, consistent with previous U.S. results.Unique to this study was the assessment of shopping behavior in the context of supermarket choice and SES. Though the measures of shopping behavior available in this data set were relatively limited, constraining the potential for comparisons with previous research, more shopping trips per month and a smaller percentage of small trips in particular were associated with more healthful food patterns. The inclusion of shopping behaviors in these analyses provided initial evidence that these variables were not confounding the association between supermarket choice and healthfulness of purchasing. In addition, including shopping behavior variables did not alter SES coefficients, suggesting these do not contribute to the socioeconomic differences in purchasing. This ties in with previous workThere are several limitations that need to be noted. First, these results reflect purchasing and as such may not translate directly to diet. In addition, we used market definitions to determine supermarket price tiers, which may reflect perceptions of store differences rather than, or in addition to, differences in price or other store characteristics. Regarding the representativeness of the data set, the overall low recorded volumes of food and beverages suggest under-reporting ,35Nonetheless, this study offers a more robust account of the association between supermarket choice and food/beverage purchases than previous work, by employing detailed scanner data. In addition, the novel inclusion of shopping behavior variables provides initial evidence as to their potential to influence healthfulness of purchases. Investigating indices of both healthier and less-healthy purchasing, the drivers of which may be different,Further research investigating the way in which households conduct their shopping would be beneficial, to establish whether encouraging certain patterns of shopping behavior may help to promote healthier purchases. However, the present results  suggest that addressing supermarket choice or shopping behavior is unlikely to substantially impact socioeconomic differences in healthfulness of purchases, and other public health measures may need to be considered to target these inequalities.Supermarket choice is associated with small differences in the healthfulness of purchases. However, differences between supermarket environments may not be primary drivers of this association—that is, high-cost supermarkets may not be differentially encouraging healthier purchasing nor low-cost supermarkets differentially encouraging less-healthy purchasing.


Hemangioma is a common benign tumor in the childhood; however our knowledge about the molecular mechanisms of hemangioma development and progression are still limited. Currently, microRNAs (miRNAs) have been shown as gene expression regulators with an important role in disease pathogenesis. Our goals were to identify miRNA-mRNA expression networks associated with infantile hemangioma.We performed a meta-analysis of previously published gene expression datasets including 98 hemangioma samples. Deregulated genes were further used to identify microRNAs as potential regulators of gene expression in infantile hemangioma. Data were integrated using bioinformatics methods, and genes were mapped in proteins, which were then used to construct protein-protein interaction networks.Deregulated genes play roles in cell growth and differentiation, cell signaling, angiogenesis and vasculogenesis. Regulatory networks identified included microRNAs miR-9, miR-939 and let-7 family; these microRNAs showed the most number of interactions with deregulated genes in infantile hemangioma, suggesting that they may have an important role in the molecular mechanisms of disease. Additionally, results were used to identify drug-gene interactions and druggable gene categories using Drug-Gene Interaction Database. We show that microRNAs and microRNA-target genes may be useful biomarkers for the development of novel therapeutic strategies for patients with infantile hemangioma.microRNA-regulated pathways may play a role in infantile hemangioma development and progression and may be potentially useful for future development of novel therapeutic strategies for patients with infantile hemangioma. Infantile Hemangioma, a common benign tumor in childhood, occurs in 10 % of children, more frequently in prematures and females , 3. In aThe body area more frequently affected by hemangioma is the head and neck, mainly the face, with an association with the embryological development of the face . AvailabTherefore, the identification of genetic and epigenetic alterations in proliferating and involuting infantile hemangioma lesions will likely contribute to better understand the underlying molecular mechanisms of development and progression of this disease, which is a leading cause of morbidity in affected children. Indeed, differences in the expression of genomic biomarkers have been reported in infantile hemangioma; e.g., insulin-like growth factor 2 (IGF-2) was found as highly expressed in proliferative lesions compared to involuting lesions .http://www.mirbase.org/cgi-bin/browse.pl?org=hsa) [In infantile hemangioma, neural crest markers (NG2 and nestin), pericytes markers  and stem cell markers  are frequently over-expressed both at mRNA and protein levels. In addition, pericytes (perivascular cells surrounding microvessels and that are related to the development and regulation of angiogenesis) and the derm of the face are derived from neural crest, suggesting that the neural crest may be involved in disease pathogenesis . Importaorg=hsa) –26.Different mechanisms can lead to deregulated miRNA expression, including genomic alterations, such as DNA gains or amplifications and mutations, epigenomic changes including DNA methylation and defects in miRNA biogenesis, including transcription and processing of miRNAs , 28. miRThe understanding of genetic and epigenetic mechanisms, such as deregulated miRNA and target gene expression, as well as molecular pathways regulated by miRNAs, may contribute for the development of new strategies for diagnosis and treatment of complex human diseases , 14, 16.To the best of our knowledge, there are no studies on global miRNA expression in infantile hemangioma. A recent PubMed search  showed only one published study on the involvement of miRNAs in senile hemangioma , which rHerein, we performed a comprehensive meta-analysis of gene expression data in infantile hemangioma and identified miRNAs as potential regulators of target genes in these tumors. Gene expression datasets were integrated with miRNAs for the identification of molecular pathways potentially involved in infantile hemangioma development and progression. These data may be clinically valuable to predict which infantile hemangioma lesions may respond and which lesions will be resistant to currently available treatment modalities. Furthermore, these data are useful for the identification of robust biomarkers applicable in the development of novel and better molecularly-targeted treatment strategies in infantile hemangioma.http://www.ncbi.nlm.nih.gov/pubmed). Key words used were: “infantile hemangioma AND global gene expression”, “infantile hemangioma AND gene signature”, “infantile hemangioma AND microRNAs”, “microRNA in infantile hemangioma”, “infantile hemangioma AND microarray”, infantile hemangioma AND mRNA expression”. Meta-analysis searches comprised studies published between the years of 2000–2015. Considering that our searches did not retrieve any records on miRNA studies in infantile hemangioma, we included only gene expression studies in this meta-analysis. Deregulated genes reported in selected studies were further used for bioinformatics prediction of miRNAs as potential regulators of gene expression, as described below.Meta-analysis study design followed the stages of the PRISMA Statement  Fig. 1)1). HereiInclusion criteria were: gene expression data in primary patient samples of infantile hemangioma or pure cell populations of infantile hemangioma, any subtype of disease, inclusion of normal tissues for comparison, data subjected to independent validation. Exclusion criteria were: non- infantile hemangioma, patients treated before molecular genetic analysis and in vivo model studies.Deregulated genes identified in the meta-analysis were used for bioinformatics prediction of miRNAs as regulators of gene expression. We used microRNA Data Integration Portal, mirDIP , a compuAccording to the meta-analysis study design and the inclusion and exclusion criteria Fig. , we seleEnrichment pathways analysis showed information on the biological role of differentially expressed genes in infantile hemangioma. Gene Ontology (GO) categories were divided into 3 hierarchically structured groups, in order to identify proteins encoded by deregulated genes in infantile hemangioma, and associated with biological processes, molecular functions and cellular components. The top 10 statistically significant enriched GO terms are shown in Fig. Molecular pathogenesis of infantile hemangioma is not well understood. Advances in methods of global genetic and epigenetic analyses represent an extremely valuable approach for the identification of disease development mechanisms and have the potential to identify biomarkers and/or pathways that may be useful for the development of better treatment approaches, including molecularly-targeted therapies.Our meta-analysis approach allowed us to integrate mRNA expression data in infantile hemangioma and to predict which miRNAs are potential regulators of gene expression. Among the different mechanisms that can lead to gene expression alterations; miRNA alteration is an important mechanism of over- or under-expression of target genes . Herein,Gene enrichment analysis showed that deregulated genes previously reported in infantile hemangioma –37 are mIt is known that the formation of vascular tumors including infantile hemangioma is partly related to increased expression of angiogenic growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which lead to the development of a disorganized blood vessel mass . Indeed,miRNAs control and modulate cell response of vascular endothelium to angiogenic stimuli; for example, miR-126 is a positive regulator of angiogenic signaling and vascular endothelial integrity. Vascular development defects were demonstrated in an in vitro model of miR-126-depleted cells, which did not respond to bFGF and VEGF angiogenic factors . AngiogeVEGFA plays an important role in vascular development and in pathological angiogenesis and its protein is highly expressed in vessels of proliferating infantile hemangioma . InteresTo our knowledge, the only available previously published study on miRNAs in hemangioma identified miR-424 under-expression in senile hemangioma . miR-424Several genes with roles in vascular and matrix remodeling, hematopoiesis, cell growth and differentiation and transcriptional regulation were shown in the PPI and miRNA-gene interation network. Among these, ANGPT2, MXRA5, JAG1, VEGFA and TBX2 showed a large number of interactions. Interestingly, some of these genes also play roles in cell signaling pathways that have been linked to the pathogenesis of infantile hemangioma ; namely,Herein, miRNA-gene interaction networks generated by integrative meta-analysis showed miRNAs with a large number of interactions ; these miRNAs are likely acting as main regulators in the network. Notably, miR-9 has been demonstrated as pro-metastatic and suppressor of E-cadherin in breast cancer cells, promoting cell motility and increasing invasive potential of carcinoma cells, besides activating β-catenin signaling, which in turn contributes to high VEGFA expression and consequently to induction of angiogenesis . IncreasA recent meta-analysis suggested that the let-7 family of miRNAs are potential biomarkers for tumor grade prediction in breast cancer  as well Among other miRNAs with a significant number of interactions, miR-637 links to main networks through direct interactions with VEGFA and CEACAM; the latter interacting with miR-9 and miR-939. Functional data has shown that miR-637 is one of the effective regulators of HER2 signaling; in HER2-positive trastuzumab non-responsive cell lines, miR-637 was efficient to inhibit breast cancer cell growth .Herein, we identified several interconnected genes and miRNAs as potential regulators of gene expression. Such miRNAs and genes may play important roles in the development and progression of infantile hemangioma. Additionally, these molecules show potential to be targets for drugs that may be clinically useful in the development of new therapies for infants and children affected by this tumor. Data generated herein may be used for validation of expression of miRNAs and genes regulated by miRNAs in infantile hemangioma. Validation analysis in a large representative cohort of primary untreated patient samples is necessary in order to establish robust biomarkers for prediction of treatment response and for the development of better treatment modalities.


Biological functions are carried out by groups of interacting molecules, cells or tissues, known as communities. Membership in these communities may overlap when biological components are involved in multiple functions. However, traditional clustering methods detect non-overlapping communities. These detected communities may also be unstable and difficult to replicate, because traditional methods are sensitive to noise and parameter settings. These aspects of traditional clustering methods limit our ability to detect biological communities, and therefore our ability to understand biological functions. To address these limitations and detect robust overlapping biological communities, we propose an unorthodox clustering method called SpeakEasy which identifies communities using top-down and bottom-up approaches simultaneously. Specifically, nodes join communities based on their local connections, as well as global information about the network structure. This method can quantify the stability of each community, automatically identify the number of communities, and quickly cluster networks with hundreds of thousands of nodes. SpeakEasy shows top performance on synthetic clustering benchmarks and accurately identifies meaningful biological communities in a range of datasets, including: gene microarrays, protein interactions, sorted cell populations, electrophysiology and fMRI brain imaging. Molecules, cells and tissues carry out biological processes through physical interaction networks124568310111213101516182021a priori community titles. In this context, labels are unique bits of information that are assigned randomly and used to track cluster membership. SpeakEasy differs from previous label propagation algorithms, because nodes update their labels on the basis of their neighbors’ labels, while subtracting the expected frequency of these labels, based on their popularity in the complete network. This process combines a bottom-up approach to clustering (using neighboring information) with a top-down approach (using information from the whole network). This dual approach facilitates accurate community detection in many types of biological networks  clusters in biological networks. SpeakEasy is related to earlier label propagation algorithms2324networks  because not require manual tuning of clustering parameters for good results. Second, it can cluster networks with any type of links  or any type of network structure . SpeakEasy is highly scalable and can quickly cluster networks with hundreds of thousands of nodes. Third, because it is very efficient, the stochastic clustering process can be repeated many times to detect robust clusters that are not generated by data artifacts or noise. The repeated clustering process also allows SpeakEasy to identify multi-community nodes, whose membership tends to oscillate between different clusters. Finally, users can select overlapping or non-overlapping output, as is appropriate for their applications.In addition to accurate cluster detection (see Results section), community detection via SpeakEasy has several practical advantages for biological applications. For instance, since the number of communities in a dataset is rarely known in advance, SpeakEasy automatically predicts the number of communities and does For an intuitive example of how SpeakEasy identifies communities, we illustrate the clustering process on a demonstration network . This neWe use three approaches to determine the accuracy of SpeakEasy community detection. First, we test its performance on a large set of synthetic networks with carefully controlled characteristics, wherein the true clusters are known. Then we apply it to real-world networks, wherein the true clusters are unknown . In thisds)  benchmarks, which are widely used to test overlapping and non-overlapping clustering methodsds) . SpeakEa 25303132. Additio 25303132. Thus, Sm). When multi-community nodes are tied to many communities , community detection becomes more difficult The LFR benchmarks accurately represent certain aspects of social and biological networks, but are limited in other aspects. For example, networks in the LFR benchmarks have low transitivity and null assortativity (propensity for hubs to connect to hubs)ds metric that was shown to be more consistent with other metrics than original Q metric38ds values from SpeakEasy  indicate that it tends to detect more small and highly dense clusters than GANXiSds scores for the two biological networks in this test set (‘dolphins’ and ‘c.elegans’). These modularity values are approach those of methods that directly attempt to maximize modularityWe compare modularity values from SpeakEasy to those from another label propagation algorithm, GANXiS, because that method showed the best overlapping clustering performance in a recent comparison of clustering methodset al.et al.42433233Because a single protein may be part of more than one protein complex (set of bound proteins that work as a unit), Discovery of protein complexes directly benefits from development of methods which detect overlapping communities. We test SpeakEasy community detection of overlapping protein complexes, using two well-studied high-throughput protein interaction networks 46We find that applying SpeakEasy once again, to each of these broad categories of cell types, identifies sub-communities with higher correspondence to the tissue of origin and cell type, considered together . Thus, sSeveral cellular or molecular processes can generate correlated gene expression , including cell-type variation, transcription factors, epigenetic or chromosome configuration21Therefore, we use SpeakEasy to detect overlapping and non-overlapping coexpressed gene sets in two datasets that are commonly used to address many biological questions: The Human Brain Atlas (HBA)We also generate overlapping clusters from both the HBA and CCLE datasets. Overlapping coexpressed gene sets may be useful in biological studies because gene coexpression is driven by overlapping mechanismsExtracellular neuronal recording with single electrodes, tetrodes, or high density multichannel electrode arrays can detect the activity of multiple nearby neurons. However, these combined responses must be separated into responses of specific neurons. This blind source separation process is known as “spike sorting”, because each spike is assigned to a particular theorized neuron. Single neurons often generate relatively unique signatures (i.e. spike waveform shapes and amplitude distributions on multiple adjacent electrodes), and emerge as clusters in the matrix of waveform correlations.To realistically test spike sorting, it is important to match noise levels in real brain recordings. Therefore, we use real depth-electrode recordings generate a simulated time-series of spikes in which the true spike times and unique neuronal sources are known see . CompariFunctional magnetic resonance imaging (fMRI), obtained while a subject is at rest (rs-fMRI), is a valuable tool in understanding of systems-level changes in a variety of domains, including neurodegenerative diseaseCommunities identified in control and PD groups contain biologically similar sets of brain regions  from repeated runs with different initial conditions. The ability to generate many partitions is useful because it can quantify the stability of each cluster . It is a1858How to cite this article: Gaiteri, C. et al. Identifying robust communities and multi-community nodes by combining top-down and bottom-up approaches to clustering. Sci. Rep.5, 16361; doi: 10.1038/srep16361 (2015).


Our biodynamics laboratory group has conducted large cohortbiomechanical-epidemiological studies targeted at identifying the complexinteractions among biomechanical, biological, hormonal, and psychosocial factors thatlead to increased risk of anterior cruciate ligament (ACL) injuries. The findingsfrom our studies have revealed highly sensitive and specific predictors for ACLinjury. Despite the high incidence of ACL injuries among young athletes, largercohorts are needed to reveal the underlying mechanistic causes of increased risk forACL injury. In the current study, we have outlined key factors that contribute to theoverall success of multicenter, biomechanical-epidemiological investigations designedto test a larger number of athletes who otherwise could not be recruited, screened,or tested at a single institution. Twenty-five female volleyball players wererecruited from a single high school team and tested at three biodynamicslaboratories. All athletes underwent three-dimensional motion capture analysis of adrop vertical jump task. Kinematic and kinetic variables were compared within andamong laboratories. Reliability of peak kinematic variables was consistently ratedgood-to-excellent. Reliability of peak kinetic variables was consistently ratedgoodto-excellent within sites, but greater variability was observed between sites.Variables measured in the sagittal plane were typically more reliable than variablesmeasured in the coronal and transverse planes. This study documents the reliabilityof biomechanical variables that are key to identification of ACL injury mechanismsand of athletes at high risk. These findings indicate the feasibility of executingmulticenter, biomechanical investigations that can yield more robust, reliable, andgeneralizable findings across larger cohorts of athletes. Our multicenter, multidisciplinary research group has conducted several collaborative,multi-institutional studies that included reliability comparisons of biomechanical andneuromuscular data from three different sites - the Biodynamics Laboratories atCincinnati Children's Hospital (CCH or Site A), The Ohio State University (OSU or SiteB) and the University of Kentucky (UK or Site C) - using identical data collection,reduction techniques, data processing methods, and data analyses. Reliability studies ofthis kind, as with any such impactful study, are important for the establishment ofwidespread generalizability, reliability, reproducibility, and acceptability ofmulticenter collected data. The authors have previously tested and measured thelongitudinal reliability and validity of all of the data collected during testing fromone site at Cincinnati Children's Hospital (CCH)1. In order to conduct a propermeasurement of the validity of these prospective cohort study findings, we trackinjuries prospectively so that we can effectively use these data for widespread injuryrisk assessment.The primary objectives of our Multicenter Biomechanical-Epidemiologic studies are todetermine how individuals become more susceptible to injury, prospectivelyidentifying those athletes who are more susceptible to injury and to determine theunderlying mechanistic cause(s) of increased risk at the biomechanical level and tooptimize the effectiveness of treatments designed to prevent these injuries. Towardsthese goals, we test hypotheses related to multiple biomechanical variables: lowerextremity bone length and body mass maturational stage; neuromuscular performance;whole limb and whole body posture; trunk, hip, and knee joint loading; and injuryrisk in subsets of athletes.InSim approaches that combine multiple in vivo, invitro, cadaveric, computer modeling, and animal model approaches toanswer the most pertinent questions in our field; 2) High-Risk Individuals Studiesusing evidence-based medicine (EBM) datasets to determine which athletes are atincreased risk for anterior cruciate ligament (ACL) injuries; and 3) PreventiveStudies using Randomized Controlled Trial (RCT) designs to determine whichinterventions decrease risk for ACL injuries in large cohort populations. We alsoemploy dual identifying and preventive studies using EBM techniques and datasets andRCT designs to determine which interventions are most efficacious in specificathletes, both individuals and groups, which are at increased risk for ACLinjuries.Our research group has many ongoing studies and our research interests and activitiescan be broken down into three areas of study: 1) Mechanistic Studies using Over the past two decades, we have collaborated with large public geographiccounty-based schools. Our primary methods of recruitment were data-driven,state-of-the-art presentations and cutting-edge tools used in areas of particularinterest, as they fit nicely into school administrators' overall objectives. Theseschool administrators included superintendents, athletic directors, principals,teachers, and coaches.Our primary objectives with these studies have been to collaborate with schooladministrators, coaches, students, and parents to gain support for our coupledBiomechanical-Epidemiologic studies, to design and develop screening protocols toidentify high-risk athletes who demonstrate identifiable neuromuscular controldeficits that put them at risk, and to develop and implement neuromuscular traininginterventions to decrease injury risk. Our overall objectives for the currenttheoretical construct include understanding the United States' National Institute ofHealth (NIH) funding opportunities; technology options for recruiting and retention;tracking options for study/trial management; tips to help avoid time delays in studyimplementation and to detail technical research methods and tool options andmethods.The goals of our experimental approach are to determine the injury risk predictiverole of specific factors such as trunk, hip, and knee position; strength and musclerecruitment at the hip and knee; hip and knee load and neuromuscular control, i.e.adolescent growth and increases in tibia and femur length; and body mass in athletes.Our group utilizes what we term a 'Top-down & Bottom-Up' administrative approachto implement these studies. We begin our explorations of the school systems withschool administrators including superintendents, athletic directors, and principals.We then contact coaches, athletic trainers (ATs), and teachers to get their fullsupport with these research studies.For over a decade, we have conducted prospective longitudinal large cohort combinedbiomechanical-epidemiological studies. ACL injury risk has proven to be a complex,multi-faceted problem that involves biomechanical, biological, hormonal, andpsychosocial factors. We have tested the hypothesis that measures related to dynamiclower extremity valgus will prove predictive of ACL injury risk in high-risk femaleathletes. For example, Myer et al.We utilized a prospective longitudinal design in county-based school-sponsoredsoccer, volleyball, and basketball teams from multiple school systems that werecruited, tested, and tracked. Female and male subjects from high school and juniorhigh school were screened prior to the start of multiple consecutive soccer,volleyball, and basketball seasons. Graphs depicting the standardized means withstandard deviations from the demographic variables of the subject population areshown in  confidence interval bounds.For over two decades, our research group has recruited hundreds of high school teamsthat have yielded thousands of soccer, volleyball, and basketball players who werepre-screened, trained, and post-screenede scale. In addition, thecorrelations between variables needed to be accounted for, in particular maximum hipand knee angles and moments. We continue to examine biostatistical models and willcheck them against data to determine any emergent predictive profiles. Comparison ofmean variables, with and without adjustment for potential confounders, should also bethe focus of future analyses.Careful biostatistical analysis should be performed by experienced biostatisticiansand biomechanical profiles should be created for each of the screening movements.Standardized values should be used, as the variables are on different scales withvarying mean values. Due to underlying normality assumption, it was necessary totransform some relevant variables to the logA total of 25 volleyball players from a single High School team were screened in Augustof 2011. Testing was completed over two weeks in that month. All subjects providedinformed consent approved by the institutional review boards at the University ofCincinnati, Cincinnati, OH, USA, The Ohio State University, Columbus, OH, USA, andUniversity of Kentucky, Lexington, KY, USA . Each subject was instrumented with fortythree (43) 9-mmretroreflective markers by a different research assistant at each laboratory site.Markers were placed over the spinous process of C7; the midpoint between thesuprasternal notch and the second costal notch of the sternum; the L5-S1 spinaljunction; left posterior superior iliac spine; and bilaterally on the shoulders, upperarms, elbows, wrists, anterior superior iliac spines (ASIS), greater trochanters, midthighs, medial and lateral knee joint lines, tibial tubercles, distal and lateral shanks(i.e. lower part of leg), and medial and lateral ankles, as shown in Data collected at each of the three sites were obtained using similar equipment andsampling frequencies. Different personnel collected data and instructed subjects at eachsite. A detailed equipment list is specified in We had internally verified that 3-D marker position data collected simultaneously onVicon  and Motion Analysis  camera systems using the same 240 Hz sampling frequency andthe same data reduction techniques yielded no significant differences in computed jointanglesA two-step process was utilized to check the quality of the marker coordinates and todetermine proper tracking identification. First, a technician from each laboratoryperformed the quality control of each trial detailing any potential errors within aspreadsheet. Potential errors, such as marker misidentification and small gaps, wereimmediately fixed. Secondly, a senior researcher reviewed the trials and addressed anyadditional concerns with the data. Gaps in marker position data of less than 10% of themarker sampling frequency  wereinterpolated using a cubic spline fill. Due to the inherent high hip and trunk flexionin combination with clothing, small 9mm markers, and the rapid rate of data collection,larger gaps in ASIS marker position data were present in a number of subjects. Based onthe results of this study, we have now modified camera placement and include redundantpelvis markers. Gaps in ASIS marker position data greater than 10% but less than 25% ofthe marker sampling frequency were interpolated using a virtual marker fill based on thefixed relative distance from the contralateral ASIS marker and hip joint center and thesacrum. Our lab has internally validated this approach and verified that it creates anacceptable amount of root mean square error when joint angle curves are compared betweenreal and virtual filled ASIS marker data. It also creates no clinically relevantdifferences in peak computed joint angles.Motion data were subsequently processed through Visual 3D  using batch scripts via the motion capture software and Matlab. All marker and force data were subsequentlyfiltered using a low-pass fourth-order Butterworth filter, sampling at 12 Hz, andpost-processed using the same custom Visual 3D and Matlab coding to compute lowerextremity joint Euler angles. Force data and computed kinematic data were used tocompute joint moments. All joint kinematic and kinetics data from each trial wereexported from Visual 3D and plotted as an additional quality control step. We have foundthis identifies a small percentage of erroneous data from incorrect model based analysesthat would not be identified when batch processing large amounts of data.Peak hip, knee, and ankle angles and moments were calculated during stance from the DVJusing Visual 3D. Stance was defined as the time period between initial contact andtake-off  exceeded 10N andsubsequently fell below 10 N). The average of three trials was used in the currentanalysis. A two-way, random-effects model of intraclass correlation coefficients (ICC)were used to compute the reliability of peak angles and moments from trial-to-trial ateach site , between each combination of sites , and among all three sites. ICC values were classified as ICC>0.75 = excellent, 0.4≤ICC≤0.75 = good, orICC<0.4 = poorThe average (± standard deviation) age of subjects at the time of the first testingsession (Site A) was 15.3±1.0 years. The average height and mass at the same time point were 169.3±4.5 cm and62.3±6.8 kg, respectively. There were no significant changes in height (p=0.248) or bodymass (p=0.096) during the study period.The reliability of peak kinematic variables among all three sites was rated asexcellent  . ICCs weBetween sites, there was only one instance in which ICCs were rated as excellent. Theremaining comparisons were rated as good or poor. Kinetic waveforms for each variablehad CMC values that were either similar or slightly less than the respectivekinematic waveforms.Within each site, the reliability of peak kinetic variables from trial-to-trial wasconsistently rated as excellent with only five instances in which reliability wasrated as good . The kin--In general, the kinematic data were reliable and reproducible for use in largemulticenter trials. However, the reliability of the kinetic data did not appear to behigh, hence, this data may be best fit for use at individual sites to train individuals.This kinetic data may also be useful for biofeedback training. For instance, feedbackregarding position and technique during sports-related movements may increase anathlete's awareness and allow him or her to make adjustments during training. Usingreal-time kinematic biofeedback may provide an intriguing option for deliveringaugmented feedback and could maximize the effectiveness of traditional neuromuscularintervention programs. Recent studies with real-time gait retraining have reinforced theconcept of providing critical feedback with detailed real-time motion analysis dataWe have faced and met many challenges recruiting subjects (i.e. the athletes) inschool systems, but to a greater extent at the junior high school level than thehigh school level in the county school systems. For example, some of the countyjunior high schools do not have organized sports teams. We addressed thischallenge by recruiting teams from recreational leagues and parochial (religious)schools within the county and adjoining counties in order to capture thoseathletes who go on to play high school sports within the school system.Teams at all sites completed their data processing pipeline for the biomechanicalanalyses. With data collected and processed over multiple sites, it was found thata small percentage of movement trials had incorrectly tracked markers, even thoughall trials had been inspected by a human operator. Due to the high volume of data,it was not possible to do a more thorough inspection of the raw data; therefore,two data verification steps were added to the processing pipeline. The first stepinvolved a complete tracking quality control step at the home site. The secondstep processed the marker trajectories using a simplified skeleton model with afew degrees of freedom. When presented with incorrectly tracked markercoordinates, this analysis reported an error because the simplified skeleton wasunable to fit the data. Trials where this error was detected were sent back to thehuman operator for re-tracking, and the others were processed further into jointkinematics and kinetics. A third quality control step was added at the end of thebiomechanics pipeline. A human operator looked at groups of curves representingthe main biomechanical variables, each curve representing one trial. Outlyingcurves were identified and the marker tracking of those trials was re-examined forcorrectness. If correct, the data was used for further statistical analysis. Ifnot, marker tracking was corrected or, if the data was corrupted or incomplete,the trial was discarded. The authors are currently developing a fourth qualitycontrol step using a confidence interval-based approach for automatic detection ofoutliers.Our research group normally reports both peak and mean variable values across theentire stance phase in our studies,-How one decides to filter and analyze motion data is both an art and a sciencethat requires careful consideration of both the tasks being analyzed and theoutcome variables of interest. For these reasons, there are several subtleties andsome possible flaws in other studies that warrant clarification. Our research teamunderstands the benefits of testing the reliability of kinematic and kinetic dataat matched cutoff frequencies and we have been filtering our motion data atmatched frequencies for several yearsThe effects of filtering may render measured biomechanical variables less reliableas injury prediction tool than previously thought. This is why it is importantthat authors report the reliability of their data. If investigators do not reportthe reliability of their measures in their study paper or elsewhere in theliterature, their conclusions should be interpreted with caution.It is likely incorrect for investigators to assume that differences in kinematicand kinetic (moment) calculations for one movement directly relate to all othermovements that involve high-impact accelerations. For example, it has been shownthat relative loads may vary greatly between a drop vertical jump (DVJ) and acutting movement. All movement tasks that are subject to large forces andaccelerations fall victim to a certain degree of specificity of angles, loads, andartifacts. For example, a run-cut task is subject to much larger frontal-planeforces and segment accelerations than a DVJ task; therefore, Knee Abduction Moment(KAM) measured during a run-cut task is likely more sensitive to cutoff frequencythan KAM measured during a DVJ. However, large artifacts are typically reservedfor the planes of motion in which these large forces and accelerations occur. Forexample, Kristianslund et al.In order to conduct a proper measurement of the validity of investigators'conclusions from a coupled biomechanical-epidemiologic cohort prospective trial,one would need to examine the fidelity with which injuries were prospectivelytracked before a study of any task can be effectively evaluated for injury riskassessment. There are also many potential bias problems introduced in poorlydesigned cohort and intervention trials. Potential biases abound such as selectionbias, reporting bias, and absence of blinding. For example, in a recent studypublished as a Level One trial in The American Journal of Sports Medicine,significant limitations in the design of the study may have affected the resultsand their interpretationIn order to conduct a proper measurement of the validity of our findings andconclusions, one would need to track injuries prospectively before a run-cut taskcould be effectively used for injury risk assessment. Many studies are not designedproperly to answer the questions upon which they speculate. A properly designed studyrequires an approach that includes apples-to-apples comparisons of groups and toother studies using identical data collection, reduction techniques, injury trackingmethods, and analyses.,-Replication of any study is important for its tenets to gain widespreadacceptability. ACL-injury risk factors have proven to be complex and multifacetedwith mechanical, biological, hormonal, and psychosocial components. KAM and KAA arecertainly prominent, predictive markers for ACL injury risk, and have been repeatedlyvalidatedThe findings of these coupled biomechanicalepidemiologic studies should provide afoundation for approaching both the mechanistic questions underlying injury riskdisparities between individuals and groups, such as sexes, as well as increase ourability to direct high-risk athletes to effective, neuromuscular interventionstargeted at specific, measured deficits related to pubertal growth anddevelopment.,We performed these parallel studies at the three sites all within two weeks of oneanother. The goals of these studies were to develop the reliability across sites in alarge cohort in order to conduct large multicenter randomized controlled trials. Wetested and cross-validated three different biodynamics laboratories  to collect data on the same medium-sized cohort of subjects. This resulted inadequate statistical power and allowed us to examine injury events as both secondaryand primary outcomes. Though KAA and KAM are prominent markers for ACL injury riskand have been demonstrated repeatedly to predict increased ACL injury riskThis extended study method developed with a multi-institutional, multidisciplinary teamwill likely yield more robust results with increased generalizability and applicabilityto diverse populations. The additional analyses will provide a foundation for addressingimportant mechanistic questions; however, they are extremely costly and time-consumingand require assistance. Nevertheless, the added approaches proposed in this supplementwill foster the development of a clinician-friendly assessment tool that will enhancethe translation of the study results into use in the medical community.We suggest future collaborative, multicenter, multi-institutional studies that includeapples-to-apples comparison of data grouped across sites using identical datacollection, reduction techniques, injury tracking methods, and analyses. Replication ofany study is important for establishing widespread acceptability. Injury risk factorshave proven to be a complex, multifaceted problem with biological, hormonal, mechanical,and psychosocial factors. For example, KAM is certainly a prominent marker for ACLinjury risk and has been demonstrated repeatedly, but it is only one of many importantfactors. We have new data that indicates that knee abduction angle may be as strong apredictor as KAM. This is important as we move forward with our secondary kinematic 2Danalyses and developing more comprehensive and generalizable clinic-based predictivemodels.Our research consortium continues to utilize a prospective longitudinal design forschool-sponsored soccer and basketball teams from multiple school systems, which arerecruited, tested, and tracked. Female and male subjects from high schools and juniorhigh schools are being screened prior to the start of each consecutive soccer,volleyball, and basketball seasons. We have tested the basketball players for severalconsecutive years. We have previously tested and measured the longitudinalreliability and validity of all of the data collected during testing from one site atCCH


BLI is a powerful biophotonic imaging technology that allows in vivo visualization of temporal and spatial progression of infections in living organisms. BLI relies on the detection of visible light arising from an enzymatic reaction of oxidation known as bioluminescence. Originally, bioluminescence referred to the light emission of living organisms , which results from the oxidation of organic substrates mediated by catalysts named luciferases  [1]. ThiLUC) as a reporter . However, the bacterial luciferase dependence on FMNH2 as a co-substrate makes it less suitable for eukaryotic cells. Finally, regardless of the luciferase system used, light emitted from reporter cells is externally monitored in real time, using sensitive photon detectors based on cooled or intensified charge coupled devices (CCD) In practice, an infectious agent is first genetically modified to stably express a luciferase gene  fLUC gene encoding a luciferase (Fluc), which converts the substrate luciferin to oxy-luciferin in an ATP-dependent manner, and (ii) the sea pansy (Renilla reniformis) rLUC and (iii) the copepod Gaussia (Gaussia princeps) gLUC, which both encode coelenterazine dependent luciferases   the firefly (ctively)  [2].Candida albicans, which adopted an unconventional genetic code For a long time, the use of luciferase reporters in pathogenic fungi has been restricted to gene regulation studies Gaussia luciferase-based BLI models. Basically, the Gaussia luciferase catalyzes an ATP-independent light emission and is naturally secreted from eukaryotic cells C. albicans cells is limited Gaussia luciferase allowed a cell surface exposure of the enzyme that potentiates access to the substrate coelenterazine and promotes a focalization of strong photonic emissions from infected sites. This advance now represents the spearhead of powerful Gaussia luciferase-based BLI currently available for fungal infections Recently, a breakthrough was also achieved via the rational optimization of some During the last decade, many efforts have been made to develop pioneering models of BLI for the most common opportunistic fungal infections such as candidiasis and aspergillosis.C. albicans strain that constitutively expressed a codon-modified version of the firefly luciferase as a bioreporter C. albicans in the vaginal lumen of infected mice when the substrate luciferin was applied to the genital tract C. albicans hyphae formed during infection has also been proposed. Both problems are now circumvented by using the cell surface–anchored Gaussia luciferase described above Candida bioluminescent strain does not appear to be a convenient reporter for imaging deep-seated C. albicans organ infections, because Gaussia luciferase emits light at 480 nm . Thus, complete adaptation of luciferases to the respective fungal host appears essential for maximum sensitivity of the reporter system.Finally, latest advances in BLI of deep-seated candidiasis look promising. With the above-mentioned knowledge, a completely synthetic firefly luciferase adapted to the codon usage of highly expressed The primary vocation of BLI for pathogenic fungal agents is to offer the possibility to follow with extreme accuracy, in real-time and in a non-invasive manner, the proliferation of microorganisms within intact living animals .C. albicansA. fumigatusCandida or Aspergillus reporters, BLI now offers the possibility to study a wide range of other host–pathogen interactions, such as biofilm formation From an ethical point of view, BLI thus allows a significant reduction in the number of animals required for such investigations , since multiple imaging of the same animal throughout an experiment can be easily carried out. As described above, the currently available fungal BLI infection models include cutaneous, subcutaneous, vaginal, oropharyngeal, and invasive candidiasis caused by Although recent advances clearly demonstrate the potential of BLI for monitoring cutaneous, subcutaneous, mucosal, and invasive mycosis, the available systems still suffer from major limitations, which have to be overcome to further expand their field of use.Gaussia luciferase offers several advantages  compared to the large and ATP-hungry firefly luciferase (Gaussia luciferase currently only allows visualization of superficial infections. For these reasons, whereas significant progress was made in recent years to develop pioneering bioluminescent systems to monitor fungal infections, solving the equation concerning the improvement of BLI for deep fungal infections seems difficult and, therefore, represents a strong issue to take up. In this way, red-shifted coelenterazine analogues for Gaussia luciferase, as well as red-shifted and thermostable firefly luciferases Aspergillus terreusCandida species, dimorphic fungi, Cryptococcus, Fusarium, and zygomycetes, will certainly become a main challenge in the field.All luciferases essentially require at least small amounts of oxygen , which mciferase , its cogciferase  strongly


Rapid eye movement (REM) sleep is generated and maintained by the interaction of a variety of neurotransmitter systems in the brainstem, forebrain, and hypothalamus. Within these circuits lies a core region that is active during REM sleep, known as the subcoeruleus nucleus (SubC) or sublaterodorsal nucleus. It is hypothesized that glutamatergic SubC neurons regulate REM sleep and its defining features such as muscle paralysis and cortical activation. REM sleep paralysis is initiated when glutamatergic SubC cells activate neurons in the ventral medial medulla, which causes release of GABA and glycine onto skeletal motoneurons. REM sleep timing is controlled by activity of GABAergic neurons in the ventrolateral periaqueductal gray and dorsal paragigantocellular reticular nucleus as well as melanin-concentrating hormone neurons in the hypothalamus and cholinergic cells in the laterodorsal and pedunculo-pontine tegmentum in the brainstem. Determining how these circuits interact with the SubC is important because breakdown in their communication is hypothesized to underlie narcolepsy/cataplexy and REM sleep behavior disorder (RBD). This review synthesizes our current understanding of mechanisms generating healthy REM sleep and how dysfunction of these circuits contributes to common REM sleep disorders such as cataplexy/narcolepsy and RBD. Rapid eye movement (REM) sleep is characterized by rapid eye movements, cortical activation, vivid dreaming, skeletal muscle paralysis (atonia), and muscle twitches –3. A disDisturbances in the normal control of REM sleep underlie cataplexy/narcolepsy and RBD, which are two common and serious sleep disorders. Narcoleptics not only experience pronounced sleep disturbances, but they also experience cataplexy – the sudden unwanted loss of muscle tone during otherwise normal wakefulness. Cataplexy is hypothesized to result from intrusion of REM sleep paralysis into wakefulness . By contThe core of the REM-generating circuit is localized at the mesopontine junction, medial to the trigeminal motor nucleus and ventral to the locus coeruleus (LC) Figures  and 2 66–8. The A/GABAB/glycine receptors on motoneurons prevents REM sleep atonia, indicating that both GABA and glycine-mediated inhibition of motoneurons underlies REM sleep atonia , a group of GABA-containing neurons, is also REM-active and may inhibit wake-promoting areas; hence, allowing the entrance into REM sleep . These mGABAergic neurons of the vlPAG region are divided into two subpopulations – REM-active and REM-inhibiting. REM-active neurons of this region are thought to silence wake-promoting neurons of the LC and DR. Luppi and colleagues have demonstrated that the vlPAG GABAergic REM-active neurons send projections to these wake-active regions , 37, 41.In addition to the REM-generating network of the brainstem, hypothalamic and forebrain structures project to and influence the core of the REM sleep circuit , 45. MelThree million people worldwide suffer from narcolepsy . A partiCataplexy is thought to result from inappropriate intrusion of REM sleep paralysis into wakefulness Figure  53, 61,,61, 62. Though cataplexy may occur spontaneously, most attacks are precipitated by strong positive emotions such as excited laughter, elation, or surprise . Even inBecause the amygdala is intimately involved in processing emotion, it may play a role in the mechanism triggering cataplexy . Single-The cessation of activity of LC noradrenergic neurons results in the disfacilitation of motor neurons, which in turn contributes to reduce muscle tone Figure  17). Dr. Dr17). In contrast to cataplexy, wherein muscle paralysis occurs inappropriately during wakefulness, REM sleep behavior disorder (RBD) is characterized by the absence of normal muscle paralysis during REM sleep . Loss ofDuring normal REM sleep, muscle paralysis is intermittently punctuated by muscle twitches. Since RBD may include an exaggeration of these natural motor events, identifying the functional and neurochemical mechanisms that control this phasic motor activity may help elucidate the pathological process that contributes to the RBD phenotype. Intracellular recording studies show that intermittent release of glutamate excites motoneurons and causes REM sleep muscle twitches , throughThe cholinergic system, which normally functions to promote REM sleep atonia, is altered in RBD patients. Neuroimaging studies reveal that individuals with RBD have significant degradation of cholinergic centers within the brain , 87. TakThe excess motor activity that occurs in patients with RBD is often highly coordinated and reflects stereotypical movements seen during wakefulness, an observation that implicates the motor cortex in potentially driving movements associated with RBD. Pyramidal tract neurons control voluntary limb movement and are highly active during both wakefulness and REM sleep . HoweverA major concern in RBD is that it precedes, in 80% of cases, development of synucleinopathies such as Parkinson’s disease (PD) by several decades , 82. ThiAlthough initial studies suggest that SubC neurons generate REM sleep , their nThe interaction between various neurotransmitter systems regulates REM sleep and its characteristics; however, one system has been understudied in relation to REM sleep and associated disorders – the dopaminergic system. While dopamine levels in the cerebrospinal fluid are highest during wake and lowest in sleep , several2-like receptors increases the frequency of cataplectic attacks in these mice, whereas receptor blockade reduces their occurrence , have also been shown to produce RBD symptoms in monkeys . ImmediaA receptor agonism and antagonism of the amygdala produce decreases and increases (respectively) in REM sleep amounts  and the REM sleep core. Indeed, anatomical tracing studies have established that the amygdala has both direct and indirect connections with the SubC region , 113. Im amounts , the app amounts , and cho amounts .Interaction between the core of the REM-generating circuit and other forebrain, hypothalamic and brainstem structures generate REM sleep and its characteristics . Both direct cholinergic activation , 32 and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Remarkably, we found that little to no attention has been devoted to examine the influence of personality traits on the neural correlates of cigarette cravings in fMRI studies. Impulsivity has been linked with craving and relapse in substance and tobacco use, which prompted our research team to examine the influence of impulsivity on cigarette cravings in an fMRI study. We found that the influence of impulsivity on cigarette cravings was mediated by fronto-cingulate mechanisms. Given the high prevalence of cigarette smoking in several psychiatric disorders that are characterized by significant levels of impulsivity, we conclude by identifying psychiatric patients as a target population whose tobacco-smoking habits deserve further behavioral and neuro-imaging investigation.Craving is a core feature of tobacco use disorder as well as a significant predictor of smoking relapse. Studies have shown that appetitive smoking-related stimuli  trigger significant cravings in smokers impede their self-control capacities and promote drug seeking behavior. In this review, we begin by an overview of functional magnetic resonance imaging (fMRI) studies investigating the neural correlates of smokers to appetitive smoking cues. The literature reveals a complex and vastly distributed neuronal network underlying smokers’ craving response that recruits regions involved in self-referential processing, planning/regulatory processes, emotional responding, attentional biases, and automatic conducts. We then selectively review important factors contributing to the heterogeneity of results that significantly limit the implications of these findings, namely  Frontiers in Psychiatry  and phasic urges for a drug, both of which have been associated with smoking relapse , 12, 13.cravings  in absti smokers . These eOver the last decade, considerable efforts have been spent on elucidating the neurobiological bases of cigarette cravings. Most studies used functional magnetic resonance imaging (fMRI) and presented pictures or videos (85% of studies) to depict smoking situations likely to provoke cravings. Relative to neutral material, appetitive smoking-related stimuli have been shown to consistently elicit brain activations throughout cortical and sub-cortical regions in both abstinent and non-abstinent smokers –25. A meIn addition to the vast neuro-imaging literature examining brain responses of smokers to appetitive smoking-related material, a few authors have investigated the specific neural correlates of appetitive drug-related processing by comparing it to the processing of appetitive stimuli other than drugs. As hypothesized by Volkow et al. , drug adOur team has further explored the specificity of the neural correlates of appetitive drug-related processing by contrasting the brain responses of smokers to appetitive and aversive smoking cues . The ratbetween studies, such as abstinence levels, smoking expectancy, and craving suppression. There is also high individual variability within studies in the brain activations associated with exposure to smoking cues. It is therefore crucial to further understand this variability in brain reactivity to appetitive cigarette stimuli. Smoking dependence severity, sex-differences, motivation to quit, and genetic factors have been studied as potential factors of inter-individual variability in cigarette craving-induced brain responses. Comparatively, little attention has been paid to anxiety, depression, and personality traits. Table When looking at individual studies assessing the neural correlates of cigarette craving, one finds that several factors contribute to the heterogeneity of findings moderate and uncontrollable cravings may trigger substantially different brain responses. Consistently with this idea, the meta-analysis from Engelmann et al.  prefrontal cortex, the (anterior and posterior) cingulate, the temporal cortex, the insula, and the dorsal striatum. The widespread brain reactivity to cigarette cues suggests that craving is a multidimensional construct.n = 15) and women (n = 19) who underwent an fMRI session, during which neutral and smoking-related images, known to elicit craving, were presented  , 57. Yetrapid action form, which refers to behaviors that are performed without consideration for their consequences and (2) a slower form involving the adoption of desired behaviors despite their negative consequences [Note: other researchers have proposed similar models , was significantly associated with cigarette smoking initiation, even after controlling for education level . Among second-order factors of the BIS , it was the non-planning subscale that was the most significantly correlated with craving ratings . A negative correlation  was also observed between the total BIS score and activations in the PCC. Among second-order factors, only the non-planning subscale was significantly correlated with PCC activations . Such results were consistent with previous findings showing that the PCC is involved in resisting cigarette cravings . Critically involved in cognitive control  or cognitive deficits  levels compared to control smokers  or top-down  neural mechanisms explain these findings. To explore this self-regulation hypothesis, impulsivity will need to be measured in these populations. Although most studies in the KEY CONCEPT 4. ComorbidityThe prevalence of cigarette smoking is elevated in several psychiatric disorders characterized by high or moderate levels of impulsivity, namely attention deficit hyperactivity disorder, bipolar disorder, cluster-B personality disorders, psychotic disorders, and substance use disorders.Given that craving is a core feature of tobacco use disorder and a significant predictor of smoking relapse, several fMRI studies have examined the neural correlates of (cue-induced) cigarette urges. The available literature has demonstrated that cigarette cravings elicit activations in regions involved in self-referential processing, planning/regulatory processes, emotional responding, attentional biases, and automatic conducts. Unfortunately, the implications of this literature are limited by the heterogeneity of results. Sources of heterogeneity include both methodological  and individual factors . Surprisingly, the potential influence of impulsivity has been largely ignored, although impulsivity is positively associated with cigarette cravings and smoking relapse. The influence of impulsivity on cigarette cravings is possibly mediated by fronto-cingulate mechanisms, as recently demonstrated by our team. Future behavioral and neuro-imaging studies will need to pay attention to the high prevalence of cigarette smoking in several psychiatric disorders, as these disorders are characterized by significant impulsivity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.