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Metabolic constituents of grapevine and grape-derived products
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The numerous uses of the grapevine fruit, especially for wine and beverages, have made it one of the most important plants worldwide. The phytochemistry of grapevine is rich in a wide range of compounds. Many of them are renowned for their numerous medicinal uses. The production of grapevine metabolites is highly conditioned by many factors like environment or pathogen attack. Some grapevine phytoalexins have gained a great deal of attention due to their antimicrobial activities, being also involved in the induction of resistance in grapevine against those pathogens. Meanwhile grapevine biotechnology is still evolving, thanks to the technological advance of modern science, and biotechnologists are making huge efforts to produce grapevine cultivars of desired characteristics. In this paper, important metabolites from grapevine and grape derived products like wine will be reviewed with their health promoting effects and their role against certain stress factors in grapevine physiology.
KeywordsGrapevine Medicinal importance Phytochemistry Resistance
Grapevine (Vitis spp.) is globally one of the most important fruit species due to the numerous uses of its fruit in the production of wine, grape juice and other foods. It has a connection with an ancient historical development of human culture. In the earliest writings and archives associated to all sorts of agricultural and religious activities, grapevine and its products were given a significant place (Thomas et al. 1993). The oldest records of the use of grapevine products by humans dated back to 3500–2900 B.C. (Bowers et al. 1999). Among the Vitis species, Vitis vinifera is currently the most cultivated fruit crop around the world because of its use in wine production (Lodhi and Reisch 1995).
The grapevine has been easily cultivated and across the world gives rise up to eight million hectares of vineyards (Vivier and Pretorius 2000). Up to now V. vinifera has reached all continents but is successfully cultivated only in temperate climate regions with sufficient rain, warm and dry summers and relatively mild winters. The Vitaceae family consists of almost one thousand species, grouped in seventeen genera. Grapevines are classified in the Vitis genus and it is generally accepted that the most cultivated V. vinifera comprises up to 5,000 true cultivars, used in wine, table (fresh fruit) and dried grape manufacturing around the world. Improvements to these cultivars initially relied largely on random selections of natural mutations which resulted in enhanced cultivation or improvement in some aspects of fruits and/or wine quality. This was later followed by more directed clonal selection schemes (Bowers et al. 1999).
The quality of grapevine, as with most plants, mainly depends on its metabolites. The production of these metabolites is especially sensitive to external conditions. In particular, the chemical diversity of grapevine is mostly affected by secondary metabolites. These secondary metabolites consist of a wide array of species-specific chemicals and belong to different phytochemical groups such as alkaloids, terpenes, antibiotics, volatile oils, resins, cardiac glycosides, tannins, sterols, saponins and phenolics, many of which have proved to be of great value to the pharmaceutical, agrochemical, food and fragrance industries (Zhang et al. 2005). In general, secondary metabolites are known to play key physiological functions in plants including their adaptation to the environment (Lewinsohn et al. 2001), acquired resistance to pests and diseases, pollinator attractant capacity and the building of symbiotic relations with microorganisms (Harborne 2001). They are also very often crucial in the determination of the quality of food attributes (color, taste, and aroma), and colors and pigments of ornamental plants (Dixon 2001; Iriti and Faoro 2006).
This review attempts to present detailed features of grapevine chemistry including primary and secondary metabolites and the biological activities associated to these chemical constituents. A detailed account of grapevine diseases and their resistance to them is also presented, followed by a brief discussion on grape biotechnology and its prospects. Grape seed oil, an important grape-derived product, is not included in this review. The nutritional highlights of the grape seed oil have been recently reviewed by Bertrand (2008).
Overview on phytochemistry of grapes and wine
Plant metabolism is mainly divided into two types, i.e. primary and secondary metabolism. Plant primary and secondary metabolites are known to perform completely different tasks in plants. A "primary metabolite" is directly involved in normal growth, development, and reproduction of plant species while a "secondary metabolite" is not directly involved in those processes, but usually has important ecological functions like defenses against predators, parasites and diseases, for interspecies competition, and to facilitate the reproductive processes (coloring agents, attractive smells, etc.). Many of these primary and secondary metabolites from plants are famous for their beneficial effects on human health.
The chemical composition of grapes and wine has been intensely studied in the recent decades and the number of compounds identified increased exponentially since the development of different analytical techniques such as gas chromatography (GC), high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) coupled with nuclear magnetic resonance (NMR) and mass spectrometry (MS) (Brun et al. 1986).
The study of the chemical composition of wine has so far allowed the identification of more than 500 compounds of which 160 compounds were esters. The concentration of these compounds ranges from 10−1 to 10−6 mg/l. Even at these concentrations many of the individual compounds are likely to play a very important role in the human taste perception. The carbonyls, phenols, lactones, terpenes, acetals, hydrocarbons, sulfur, and nitrogen compounds, although present in very low concentrations, contribute to the fragrance of wine. However, the taste and mouth-feel sensations are primarily due to compounds like water, ethanol, organic acids, amino acids, sugars, and glycerol.
Primary metabolites in grapes and wine
Sugars and amino acids
Glucose and fructose are the principal grape sugars while sucrose and other sugars are rarely found in V. vinifera grapes. Cultivars other than V. vinifera may contain up to a 10% of sucrose. Amount of sugars in V. vinifera can be affected by cultivar type, maturity and health of the berries (Crippen and Morrison 1986). High sugar concentrations can also result in the increase of the volatility of aromatic compounds. Generally, sweetness is detected at levels higher than 1% (w/v) of overall sugars, and this is influenced by other constituents such as ethanol, acids, and tannins.
The amino acids contribute to wine aroma, taste, and appearance (Hernandez-Orte et al. 2003). Carrying out studies of partial least-squares regression models, Hernandez-Orte et al. (2002) found that amino acid composition accounts for a high proportion of the variance in the volatile composition. Moreover, the composition of the amino acids in the wine has an influence on aromas during the maturing process (Escudero et al. 2000). In the wineries, obtaining young wines with fruity aromas is now gaining much interest. In order to improve the kinetics of wine fermentation, activators are usually added, mainly ammonium salts, also auxiliaries to avoid stuck and sluggish fermentations (Bisson 1999; Blateyron and Sablayrolles 2001). Daily-based research is carried out into new additives and therefore it is interesting to know how the addition of nitrogen sources affects amino acid uptake by the yeast and how the addition affects the synthesis of aroma compounds. The activators are metabolized by the yeasts and modify the consumption of amino acids and ammonium in the juice thus causing a variation in the wine aroma. As occurs with esters, the pool of intracellular nitrogen regulates the formation of higher alcohols (Large 1986).
Biogenic amines
Biogenic amines (BA) in wines are produced from the free amino acids by microbial decarboxylation. They can be originated from the different wine making processes or from the grape berries and have unpleasant effects at high concentrations and caused a health risk to sensitive individuals (Gardini et al. 2005). Studies on BA showed that yeasts not appear to be responsible for the production of BAs in commercial red wines (Marcobal et al. 2006). The wine contamination with BAs mostly occurred during natural or spontaneous malolactic fermentation by lactic acid bacteria present in low population in healthy grapes (Herbert et al. 2005; Landete et al. 2005). Although there are no legal limits for BAs but some countries, like Austria and Switzerland, have their own suggestions. The export of wines to those countries could be affected by the higher levels of BAs, making BAs a potential economic threat.
In comparison with other macromolecular fractions, wine polysaccharides have been less studied. Some of their properties were determined (Riou et al. 2002) along with the characterization of their structures (Vidal et al. 2000) during the past decades. Polysaccharides are generally low in final wine products, being partially water soluble and extracted into the juice during crushing and pressing. The effects of polysaccharides in wines are probably direct, i.e. 'mellowness' or indirect by modulating astringency. The main polysaccharides in wine include type II arabinogalactan-proteins and rhamnogalacturonans (from grapes) and mannoproteins (from yeast) (Pellerin et al. 1995, 1996).
According to Fraile et al. (2000), the formation of different alcohols takes place at the end of the fermentation, when most of the amino acids have been consumed, whereas, according to Rapp and Versini (1991), this synthesis occurs at the same time as ethanol production. Ethanol is the most abundant and important alcohol in wine. Ethanol concentration in wine generally ranges from 10 to 13 ml/100 ml, depending mainly on sugar content and lesser on temperature and yeast strain. Ethanol is crucial to the stability, aging, and sensory properties of wine. It also influences the type and amount of aromatic compounds produced by affecting the metabolic activity of yeast. The dissolving action of ethanol is also probably involved in the reduction of the evaporation of aromatic compounds during fermentation (Williams and Rosser 1981). Moreover, it is also involved in the production of acetals by reacting with the aldehydes.
Methanol is also present in wines in fairly large amounts (0.1–0.2 g/l) (Jackson 2000). In wines methanol is formed by the degradation of pectic substances, present in crushed grapes, by enzyme action (Ribereau-Gayon et al. 2000). The methanol content in wines is dependent on number of factors like the variety and condition, maceration treatment, fermentation temperature, and pectolytic enzyme treatment. The in vivo oxidation of methanol produces formaldehyde and formic acid which are toxic to the central nervous system. Due to this methanol content is one of the critical control point parameters which should be controlled during the alcoholic fermentation stage to produce wine safe for human consumption (Kourtis and Arvanitoyannis 2001).
Other potentially significant higher alcohols in wine are n-propanol, 2-methyl-1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol (Rapp and Mandery 1986). The higher alcohols are produced as a by-product of yeast fermentation and their concentration is markedly influenced by vinification practices such as temperature, presence of oxygen, suspended solids and yeast strain (Sponholz 1988). Higher alcohols may produced by deamination of the amino acids and grape-derived aldehydes, and by the denitrification of amino acids (Chen 1978).
Organic acids
In grape juice and wine, organic acids composition is very important as it highly influences the organoleptic properties, microbiologic control and a parameter of critical control in wine stabilization. The processes like alcoholic fermentation, malolactic fermentation, and oxidation of ethanol are involved in the production of these compounds or they may directly come from grapes. Tartaric and malic acids are the major organic acids in grape juice while it also contains succinic and citric acid in low amounts. Wine contains both volatile and fixed acids. Volatile acids, principally acetic acid, can be readily removed by reverse osmosis. The content of fixed acids such as tartaric, malic, and citric acid (from grapes) along with lactic, succinic, oxalic, fumaric, and citric acid (from fermentation process) influences the pH of wine (Singleton 1982). Also small amounts of different acids like galacturonic, citramalic, pyruvic acid, and ketoglutaric acid, etc. are there (Castellari et al. 2000).
Secondary metabolites in grapes and wine
Simple phenolics and phenylpropanoids
Simple phenolics of grape origin are initially synthesized from phenylalanine (Hrazdina et al. 1984), whereas those of yeast origin are derived from acetic acid (Packter 1980). Simple phenolics are derivatives of hydroxycinnamic acids (HCA) and hydroxybenzoic acids (HBA). Primarily, they are stored in cell vacuoles of grape cells and can easily be released by crushing. Common hydroxycinnamic acids are p-coumaric acid, caffeic acid, sinapic acid, and ferulic acid. In wine esterified form of HCA with tartaric acid is more common than free HCA. The caftaric acid (caffeic acid conjugated with tartaric acid) is predominant (up to 50%) of the total HCA. Since the concentration of HCA depend on factors like grape variety, growing conditions, climate, etc., different studies shows different concentrations. Compounds like p-coutaric acid (p-coumaric acid conjugated with tartaric acid) and fertaric acid (ferulic acid conjugated with tartaric acid) are found in the concentrations of 55 and 16 mg/l, respectively (Ricardo-Da-Silva et al. 1993). Derivatives of HCA like ethyl esters of caffeic and coumaric acid along with ethyl esters and diethyl esters of caftaric acid were also reported (Baderschneider and Winterhalter 2001). Evidence of glucosides like 4-O-glucosides of ferulic and coumaric acids (both cis- and trans-forms), glucosides of caffeic, coumaric, ferulic, and sinapic acids have been shown by some studies (Cooper and Marshall 2001; Monagas et al. 2005; Baderschneider and Winterhalter 2001). Previous reports of NMR characterization showed cis-isomers of coumaric and coutaric acids in Chardonnay grape pomace and Riesling wine (Baderschneider and Winterhalter 2001). The hydroxycinnamic esters are more concentrated (2- to 100-fold) in grape skin than in pulp. Differences in the total amount and proportion (Cheynier et al. 1986) have been reported based on the varietal differences. In addition, HCAs are also important constituents of acylated anthocyanins. The relation of acetylated and coumaroylated anthocyanins can be used in the authenticity control of red wine.
When compared with HCA, wines have lower levels of hydroxybenzoic acids (HBAs) and their derivatives. The most common HBAs are gallic acid, gentisic acid, protocatechuic acid, and p-hydroxybenzoic acid, which are mainly found in their free form (Pozo-Bayon et al. 2003; Vanhoenacker et al. 2001). Hydroxybenzoic acids are mostly represented by gallic acid, which is found free as well as acyl substituent of flavan-3-ols. Two forms of gallic acid, i.e. 3-O-β-glucopyranoside and 4-O-β-glucopyranoside, have been reported in grape (Lu and Foo 1999). In wine the level of gallic acid has been shown by many studies (Pozo-Bayon et al. 2003; Pena-Neira et al. 2000; Sladkovsky et al. 2004) that gallic acid is ranging from 0.3 to 4.8 mg/l. Other benzoic acids such as protocatechuic, vanillic, and syringic acids are reported in Riesling wine from Germany (Baderschneider and Winterhalter 2001). Several derivatives of HBA were also identified including the ethyl esters of vanillic and p-hydroxybenzoic acid, methyl esters of vanillic and protocatechuic acid, ethyl esters of protocatechuic acid, the glucose ester of vanillic acid, from German Riesling wine (Baderschneider and Winterhalter 2001).
Flavonoids are found primarily in the skins and seeds of the grapevine fruit. Flavonoids can be further divided into several sub-groups like flavones, flavonols, dihydroflavanols, flavanols, and anthocyanidins, with the same skeleton but different oxidation state of their central pyran ring. The most common flavonoids in wine are flavonols, catechins (flavan-3-ols), and anthocyanins.
Flavonoids are synthesized from the combination of the shikimic and polyketide pathway. Polymerization of polyhydroxy flavan 3-ol units, (+)-catechin and (−)-epicatechin, and their gallate esters produces oligomers and polymers called proanthocyanidins (often referred to as procyanidins). Even though procyanidins occur primarily as dimers in grapes (Kennedy 2008), they tend to be polymerized and predominate in wine as condensed tannins (mid-size procyanidin polymers containing three to five subunits). In some cultivars, structural differences exist among skin, stem, and seed procyanidins. Also, there are considerable differences in types and concentrations between cultivars (Boselli et al. 2004). In grape seeds and skin, about twenty procyanidin dimmers and trimers have been identified (Fulcrand et al. 1999). Seeds contained procyanidin of catechins, epicatechin, and epicatechin gallate units while grape skin and wine showed procyanidins mostly based on epicatechin and epigallotcatechin units (De Pascual-Teresea et al. 2000). The total amount of procyanidins is reported to vary from 1.7 to 4.4 g/kg of berries in skin, 1.1 to 6.4 g/kg in seeds, and 0.2 to 1 g/kg in pulp (Mane et al. 2007).
Anthocyanins, which are responsible for the red or pink color in grape berries, belong to the flavonoid family. They are glycosylated derivatives and their parallel aglycones are known as anthocyanidins. In V. vinifera, 3-monoglucosides are the major anthocyanins where glucose can be acylated by acetic, p-coumaric, and caffeic acid (Alcalde-Eon et al. 2006; Vidal et al. 2004). Some additional anthocyanins, like 3-caffeoylglucosides of peonidin, cyanidin and delphinidin, have also been reported in grapes using HPLC–MS (Vidal et al. 2004). The anthocyanin profiling can be use for the chemotaxonomic classification as the total amounts (from 500 mg/kg to 3 g/kg of berries) and proportions of the various anthocyanins are the characteristic of each variety (Mattivi et al. 2006). Grape anthocyanin composition depends not only on the maturity but also different vine growing parameters like soil and climate together with practices such as pruning, fertilization, or watering (González-Neves et al. 2002). The accumulation of anthocyanins starts at the Veraison stage with occasional decrease towards the end of Ripe stage, especially in hot climates (Fournand et al. 2006).
In wine, apart from the anthocyanins, other class of compounds has been evolved known as pyranoanthocyanins. They are basically derived from anthocyanins having an additional pyran ring attached to position 4 and 5 of anthocyanin. These pyranoanthocyanins, e.g. vitisin A and B, varied in concentration at the different stages of wine (Asenstorfer et al. 2003). Pyranoanthocyanins are found more resistant against color loss due to sulfites and pH variation (Hakansson et al. 2003). The contribution of the pyranoanthocyanins in the color of red wine has been estimated by number of studies but resulted with different conclusions (Alcalde-Eon et al. 2006; Schwarz et al. 2003; Boido et al. 2006) which might be due to the wine type and the way to calculate the pigments. A particular type of pyranoanthocyanins derivatives are vinylpyranoanthocyanin-flavanol pigment, also known as Portisins (Mateus et al. 2004) as they isolated from the Port wine. These pigments have increased stability towards sulfites and pH-induced color loss (Oliveira et al. 2006). The formation of this new class of pigment involves pyranoanthocyanins and vinyl-flavanols, represents an evolution in the wine pigments having pyranoanthocyanins as a main precursor.
Flavonols are also reported in grape berries in the form of 3-O-glycoside of quercetin, myricetin, kaempferol, isorhamnetin, laricitrin, and syringetin, both in red and white cultivars (Mattivi et al. 2006; Talcott and Lee 2002). Mostly 3-O-glucoside and 3-O-glucuronide of flavanols are present on grapes but other mono and diglycosides are also reported present in lower amounts (Downey et al. 2003). Both grape and wine have also been reported for the presence of dihydroflavonols like astilbin and engeletin (Guebaila et al. 2006).
Stilbenoids (1,2-diarylethenes) are another class of phenolic compounds (non flavonoids) belonging to the phenolic group and attracted great interest because of their potential health valuable effects (Guebaila et al. 2006). Stilbenes are essentially located in grape skin (Creasy and Coffee 1988; Roggero and Garcia-Parrilla 1995) but also reported in grape seeds (Sun et al. 2006) and grape stem (Bavaresco et al. 1997). Grapes and red wine are two of the major dietary sources of stilbenes but their concentration is relatively low as compared to other phenolic compounds (Pace-Asiak et al. 1995). Grape seeds contain low amounts of stilbenes (Pezet and Cuenat 1996), but a substantial amount of these compounds may be found in grape stems (Sun et al. 2003). However, grape stems contribute little to these compounds in wine due to their poor diffusion property (Sun et al. 2006).
Resveratrol (a monomeric stilbene) is the major stilbene of grapes. It is synthesized from phenylalanine and can exist in two isomeric forms. In wines, both isomeric forms were detected (Sun et al. 2003) due to photochemical isomerization of trans-form into cis-form during wine making (Jeandet et al. 1995; Roggero and Garcia-Parrilla 1995).
Stilbenes can also occur in oligomeric and polymeric forms, known as viniferins. They are induced by oxidative polymerization of monomer resveratrol through the activity of a peroxidase (Jean-Denis et al. 2006). Many viniferins have been reported so far in grapes including ε-viniferin and δ-viniferin, dimeric resveratrol (Pezet et al. 2003; Vitrac et al. 2005) and α-viniferin, trimeric resveratrol (Pryce and Langcake 1977). Stilbenoids such as ampelopsin-A (dimeric resveratrol) and hopeaphenol (dimeric ampelopsin A) have also been identified (Jeandet et al. 2000; Guebaila et al. 2006).
The stilbene contents in wines varied considerably due to several factors like climate, grape variety, fungal infection (Perrone et al. 2007), UV light, metal ions (Pussa et al. 2006) and enological methods (Gambuti et al. 2004). It has been shown that red wine is usually more concentrated in stilbenes than rose and white wine as this depends on the skin contact of the must and the high phenolic content of grape cultivars during fermentation (Perrone et al. 2007). Many reports have been published targeted towards the quantification of stilbenes in different wines (Vitrac et al. 2005; Guebaila et al. 2006; Naugler et al. 2007).
Importance of grape phenolics to human health
The medicinal and nutritional value of grapes has been proclaimed for thousands of years. Several ancient Greek philosophers praised the healing power of grapes usually in the form of wine (Sun and Spranger 2005). Grape leaves have been used to stop bleeding, inflammation, pain, and diarrhea. Unripe grapes used to treat sore throats and dried grapes (raisins) to cure constipation and thirst. Round, ripe, and sweet grapes have been used to treat a range of health problems including cancer, cholera, smallpox, nausea, eye infections, skin, kidney, and liver diseases (Shi et al. 2003). Grape berries are prescribed as laxatives and as a diuretic in case of gastrointestinal and circulatory disturbances, or for the treatment of liver or kidney diseases (Stervbo et al. 2007). Grape phenolics have been proved to possess several health promoting properties playing an important role in the inhibition of carcinogenesis, mutagenesis, and cardiovascular diseases. These activities have been associated to their in vivo and in vitro antioxidative activities. Flavonoids in grape seeds have also been reported to exhibit activities against peptic ulcer (Saito et al. 1998) and several dermal disorders (Bomser et al. 1999).
Antioxidant activity
Phenolics including flavonoids and related polyphenols from wine and grape seeds have generated remarkable interest based on several reports (both in vivo and in vitro) of their antioxidant and free radical scavenging properties. A clinical report has shown that procyanidin oligomers from grape seeds are 20 and 50 times more potent antioxidant than vitamin C and E, respectively (Uchida 1980). Silva et al. (1991) found that monomeric and polymeric grape seed proanthocyanidins have superoxide and hydroxyl radicals scavenging activity. They also reported that certain polyphenols, for example, epicatechin-3-O-gallate, procyanidins B2 and B2-3-O-gallate, had higher superoxide radical trapping activity at pH 9 than at pH 7.5.
Kanner et al. (1994) found that red wine phenolics inhibit myoglobin, cytochrome C and iron ascorbate catalyzed lipid peroxidation. They also compared the antioxidative capacity of wine phenolics and α-tocopherol and concluded that wine phenolics were twice more potent antioxidants than that of α-tocopherol. It was shown that gallic acid, resveratrol, and tannic acid exerted same inhibition on lipid peroxidation which was greater than d–l-α-tocopherol. Cuendet et al. (2000) proved that trans-piceide, a glucoside derivative of resveratrol, could effectively scavenge free radicals.
An in vivo study regarding the antioxidant effect showed that grape seed proanthocyanidins exhibited a superior protective effect than vitamin E, vitamin C, vitamin E plus C and β-carotene against lipid peroxidation and DNA fragmentation in mice (Bagchi et al. 1998). Resveratrol was also reported to inhibit platelet aggregation and LDL oxidation, protecting liver from lipid peroxidation in rats, apart from its anticarcinogenic activities (Teissedre et al. 1996). Another in vitro study showed that grape seed procyanidins might reduce the oxidation of polyunsaturated fatty acids in mouse liver microsomes (Bouhamidi et al. 1998). Tedesco et al. (2000) found antioxidant activity of nonalcoholic constituents of red wine.
Anti-cardiovascular diseases activity
Coronary heart disease (CHD), a serious health condition affecting a significant amount of the population of the developed countries, is usually associated to high cholesterol levels in blood. In particular, high levels of low density lipoproteins (LDL) in plasma may play a role in the initiation of atherosclerotic plaque. Grape, wine, and grape seed extracts inhibit the oxidation of LDL and platelet aggregation and preventing CHD have been shown by several in vitro and clinical studies (Frankel et al. 1993, 1995; Gryglewsk et al. 1987; Mangiapane et al. 1992). Polyphenols (mainly catechins and procyanidins) of grape seeds play a major role in the observed inhibition of LDL oxidation due to their antioxidant activity and platelet aggregation inhibition (Manthey et al. 2002).
The consumption of purple grape juice offered a good protection against the oxidation of LDL cholesterol, as shown by an in vivo study with dogs, monkeys, and humans, from which it could be inferred that the flavonoids of purple grape juice and red wine may inhibit the initiation of atherosclerosis (Folts 2002). Another study shows that moderate red wine consumption for 4 weeks is associated with desirable changes in high density lipoprotein-cholesterol (HDL-C) and fibrinogen compared with water with or without red grape extract (Hansen et al. 2005). The impact of wine on the measured cardiovascular risk factors thus seems primarily explained by an alcohol effect. A study done by Sato et al. (1999) showed that animals fed with grape seed phenolics (GSP) had a lower myocardial infarction rate due to scavenging of free radicals produced during ischemia and reperfusion. Sato et al. (2001) also showed that in rats, fed with 50–100 mg/kg of GSP, the number of apoptotic cells could be reduced along with the lessening in free radicals production.
An in vitro study of Kaur et al. (2007) on peripheral blood mononuclear cells showed that resveratrol derivatives are 20- to 10-fold more efficient in inhibiting the tissue factor induction which is responsible for blood coagulation and thrombosis. The same group previously reported the same effect of these derivatives on the vascular (Pendurthi et al. 1999) and endothelial cells (Pendurthi and Rao 2002).
Some studies were also carried out to determine whether the alcohol in wine is responsible for the reduced levels of LDL in blood of moderate wine drinkers. Frankel et al. (1993) diluted red wine till 10 μmol/l total phenolics (1,000 times dilution) in order to evaluate in vitro effect of wine phenolics on human LDL oxidation inhibition. Diluted wine and quercetin at 10 μmol/l evenly repressed the human LDL oxidation, proving that this inhibition was from constituents other than alcohols. Interestingly, the inhibition was considerably higher than that induced by α-tocopherol. Another study indicated that several red grape polyphenols inhibit cardiac voltage-gated sodium channel and that this effect may contribute to the documented cardioprotective efficacy of red grape products (Wallace et al. 2006).
Anti-inflammation and anti-ulcer activity
Apart from the other described activities, grape seed polyphenols (procyanidin) are also capable of inhibiting some enzymes which catalyze the release of many substances promoting inflammation, such as, histamine, serine protease, prostaglandin, and leukotrienes (Amella et al. 1985; Middleton and Drzewieki 1983; Pearce et al. 1984). Procyanidins was also proved to be able to inhibit the activation of hyaluronidase, a proteoglycan splitting enzyme that attacks various tissues during inflammation. This activity was associated to the antihistaminic effect and to their ability to strengthen cell membranes of basophils and mast cells, which contain the allergens, thus preventing the hypersensitivity to pollens and food allergens (Hansen 1995).
In rats, grape seed extracts containing high or low flavonol contents showed anti-ulcer activity (Saito et al. 1998). This protective effect was higher in high flavonol containing extracts than in those with a low content. The authors also found that catechin, procyanidin B3 and dimeric and trimeric procyanidins did not show any defensive activity against ulcer while tetramers, pentamers, and hexamers of these were active. The authors postulated that the activity shown by longer oligomeric procyanidins might be due to their ability to bind to the proteins present on the stomach surface.
Anticancer and antimutagenic activities
A variety of procyanidins from grapes and their seeds have been shown to prevent the growth of cancer cells. The anti-mutagenic activity of procyanidins from V. vinifera on Saccharomyces cerevisiae was examined by Liviero and Puglisi (1994). They found that procyanidins from grapes at 0.5 mg/ml induced a 65% decrease of the mitochondrial spontaneous mutability in Saccharomyces strain S288C. At the same concentration of procyanidins from grapes, 92% reduction rate was observed in the mutation rate for the nuclear genetic determinant canavanine sensitivity. Another report (Tyagi et al. 2003) also shows that grape seed extract has a possible role in antiproliferation and apoptosis of human prostate carcinoma.
Nakagawa et al. (2001) reported the effect of resveratrol on the inhibition of the growth of breast cancer cells. Suppression in the growth of these cells by resveratrol was reported to occur through apoptosis. Another anti-carcinogenic study showed that resveratrol was capable to inhibit tumor initiation, promotion and progression and also explaining how it intervened in each stage (Jang et al. 1997). At first stage, resveratrol acted as an antioxidant and drug metabolizing enzymes of phase II were induced. At the second step, it mediated anti-inflammatory effects and the enzymes like cyclooxygenase and hydroperoxidase were inhibited. Finally human leukemia cell differentiation was induced by resveratrol (antiprogression activity) along with the inhibition of the development of preneoplastic lesions in carcinogen treated mouse mammary glands.
Bomser et al. (2000) noted that ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis could have an essential role in diverse biological processes including cell proliferation and differentiation. High levels of ODC are associated with increased risk for cancer. Grape seed polyphenols extract containing mainly oligomeric and polymeric proanthocyanidins was shown to inhibit epidermal ODC activity in mice. Previously, Bomser et al. (1999) had found that grape seed extract containing mainly oligomeric and polymeric proanthocyanidins showed anti-tumor activity in mouse skin epidermis. A methylated derivative of resveratrol is also reported for the human colon cancer cell lines inhibition, about 100-fold more active than resveratrol (Schneider et al. 2003).
The p53 gene suppresses tumor formation and induces apoptosis. Modulation of p53 expression by red wine and its polyphenolic contents was studied on three human breast cancer cell lines and one colon cancer line by Soleas et al. (2001), which showed that these polyphenols, including quercetin, catechin, trans-resveratrol and caffeic acid did not affect p53 gene expression in two of the breast cancer lines. Resveratrol, on the other hand, did decrease the expression of this gene in breast cancer cells of a wild type (MCF-7), while catechin and caffeic acid increased its expression but in a concentration independent manner. Caffeic acid and resveratrol reduced the expression of p53 in colon cancer cell lines, but this reduction again was not dose-responsive. Soleas et al. (2001) concluded that anticarcinogenic properties of wine should not be attributed to the modulation of p53 gene expression by these wine polyphenolic constituents. An in vitro study showed that methylated derivative of resveratrol induces apoptosis in human lymphoblastoid cell independently of their p53 status (Schneider et al. 2004).
Basly et al. (2000) studied the estrogenic/antiestrogenic effects of resveratrol isomers on the in vitro human breast cancer cells, finding that the cis form was less effective than trans in both cell lines. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and FeCl3 reduction assays showed that these isomers could be both antioxidant and prooxidant, depending on their concentrations.
Other biological activities
The effect of plant flavonoids on intestinal microflora has also been investigated. Tebib et al. (1996) reported that monomeric proanthocyanidins of grape seeds did not affect the activities of fecal bacterial enzymes of rats. However, polymeric proanthocyanidins showed 'a beneficial colonic protective effect' by reducing colonic enzymatic activity of β-glucosidase, β-glucuronidase, mucinase and nitroreductase due to a dilution effect.
Polymeric tannin supplementation was shown to stimulate fermentative activities without increasing the activity of harmful enzymes on animal models. Chung et al. (1998) reported that tannic acid inhibited the growth of intestinal bacteria such as Clostridium perfringens, Enterobacter cloacae, Escherichia coli and Salmonella typhimurium while showing no inhibitory effect on B. infantis or lactic acid bacteria Lactobacillus acidophilus.
A fluorescence assay was developed to determine the bacterial degradation of flavonoids by Schoefer et al. (2001). This assay was proved to be able to distinguish the ability of colonic bacteria to degrade various flavonoids with the exception of catechin which could not be determined due to the lack of the quenching effect of the fluorescing compound. Future studies may reveal details on the mechanisms of flavonoid degradation by colonic microflora.
Khanna et al. (2001) showed that grape seed proanthocyanidin extract containing 5 mg/g trans-resveratrol induced the expression of vascular endothelial growth factor (VEGF) in keratinocytes. Therefore, GSP extract containing resveratrol can be used to treat dermal wounds and other dermal disorders.
Polyphenols in grape seed extract have been shown to reduce food intake in animal models. A study on humans showed that grape seed reduced energy intake without further effects on satiety, mood or tolerance (Vogels et al. 2004). Flechtner-Mors et al. (2004) also showed that moderate consumption of white wine is effective in weight loss in overweight and obese subjects.
Major grape diseases
As a crop, grapes are susceptible to many diseases. Downy mildew, powdery mildew, grey mold, anthracnose, black rot, and crown gall are the major grape diseases around the world. All of these-with the exception of crown gall, are caused by fungi that attack the berries, reducing yield and quality. Crown gall is caused by a bacterium and can kill the plant. The degree of susceptibility varies depending on the variety, but damage can be harsh if no pest management is carried out.
The most threatening diseases to the grapevines are powdery and downy mildew caused by two different fungi, Uncinula necator and Plasmopara viticola, respectively. Downy mildew, caused by P. viticola (oomycete fungi), is the disease which affects both leaves and fruit (Madden et al. 2000). Powdery mildew is another widespread and destructive disease of grapevines worldwide. The damage caused by the pathogen, U. necator, has been noticed from the studies of the disease on cultivars of the European grape species (David et al. 2001).
Botrytis cinerea, the causal agent of grey mould or botrytis bunch rot in grapes, is responsible for considerable economic damage in vineyards worldwide. During the last 50 years, management of B. cinerea has relied heavily upon the use of synthetic chemicals (Rosslenbroich and Stuebler 2000). Grapevines are also attacked by viruses such as the nepovirus, grapevine fan leaf virus (GFLV). GFLV is responsible for an important and widespread degeneration of grapevine, causing a substantial loss in grape yield (up to 80%), low fruit quality, and a progressive decline of infected vines that can eventually lead to plant mortality (Martelli 1993; Martelli and Savino 1990).
Pest and disease control
In nearly all parts of the world, pest and disease control in grapevines is generally achieved by widespread application of fungicides and pesticides, which results in high costs and a negative environmental impact. Scientists have been working hard to obtain grapevine varieties with an increased resistance against pathogens (Peixe et al. 2004). A great effort has been made to obtain improved grapevines through controlled cross-breeding between American and Asian cultivars and the resulting plants have been selected and their resistance evaluated (This et al. 2006). While this method presents unquestionable advantages, it is a slow process which also contributes in the reduction of genetic variability and loss of important quality of wine. Therefore, the use of biotechnological methods for this purpose is gaining much attention now.
Resistance in grapes
Plants like other organisms have developed different mechanisms to respond to different stresses such as unfavorable environmental conditions, nutrition deficiency and pathogen attack (Downie et al. 2004; Matsumoto et al. 2004). Plant defense system (resistance) against pathogens comprises a number of mechanisms, the most significant of which is the biosynthesis of secondary metabolites. A certain group of compounds have been found to be involved or associated with plant defense mechanisms. These compounds, known as phytoalexins, are low molecular weight, antimicrobial secondary metabolites (Harborne 1999; Kuc 1995; Purkayashta 1995).
Examples of inducing agents with demonstrated cellular and biological activity on grape tissues (partially adapted from Elmer and Reglinski 2006)
Inducing agent
Reported activity
Methyl jasmonate
Accretion of phytoalexins and defense-related proteins along with the induction of the oxidative burst
Repka et al. (2004), Belhadj et al. (2006), Vezzulli et al. (2007)
Aluminium chloride
Accumulation of phytoalexins in grapevine leaves
Laminarin (β-1,3-glucan from Laminaria digitata)
Induction of defense-related genes and oxidative burst. Enhancement is resistance against B. cinerea was also observed
Aziz et al. (2003)
Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester
High levels of resveratrol and anthocyanins with the induced resistance against B. cinerea with the accumulation of chitinase class III transcript
H2O2 production, induction of defense-related genes in cell suspensions, and elevation of resistance to B. cinerea in detached leaves
Phenylalanine ammonia-lyase activity induction and elevation of resistance to B. cinerea
5-Chlorosalicylic acid
Enhanced production of phenolics, and field efficacy against B. cinerea
Reduced B. cinerea infection on plantlet leaves
Ait-Barka et al. (2004)
Phytoalexins from grapes
Stilbenes are generally biologically active compounds that also have antifungal activities against various pathogens such as Cladosporium cuccumerinum, Pyricularia oryzae (Langcake and Pryce 1976), Plasmopara viticola (Dercks and Creasy 1989; Langcake 1981), and Sphaeropsis sapinea (Celimene et al. 2001).
Resveratrol is the major phytoalexin produced in grapevines along with its derivatives such as piceide, pterostilbene and ε-, α-, β-, and δ-viniferin (Jeandet et al. 2002; Pezet et al. 2004). Resveratrol was found to accumulate in the abaxial surface of leaves and in skin of berries in the range of 40–400 μg/g fresh weight (Adrian et al. 2000; Jeandet et al. 1991). A study on grape plantlets proved the existence of a positive correlation between resveratrol synthesis in leaves (induced by UV) and field resistance (Sbaghi et al. 1995). Regarding fungal infection resistance, an in vitro study showed that at the concentration of 90 μg/ml, resveratrol was able to inhibit conidial germination by 50% while reduction in mycelial growth was observed in the range of 60–140 μg/ml (Adrian et al. 1997).
Pterostilbene is the dimethylated derivative of resveratrol. A study showed that it is fivefold more toxic than resveratrol, completely inhibiting conidial germination at concentrations ranging from 52 to 60 μg/ml (ED50) and 18 to 20 μg/ml (Adrian et al. 1997; Langcake 1981; Pezet and Pont 1990). The methylation of the hydroxyphenyl groups in phenolics could potentially resulted in an enhanced biocidal activity as these two different stilbenes indicated (Pezet and Pont 1990). The oxidative dimers of resveratrol have formed a new class of compounds known as viniferins. A study by Pezet et al. (2004) showed that δ-viniferin exhibited toxicity similar to that of pterostilbene against P. viticola.
The above reports constitute clear evidence of the substantial antifungal activity of stilbenes, which in turn suggests their participation in the resistance against fungal grapevine diseases, thus supporting their proposal as indicators of resistance. Pool et al. (1981) studied the relationship between phytoalexins production potential and resistance to diseases in grapevine, concluding that there was a positive correlation in both the speed and the intensity of resveratrol synthesis and the resistance of grapevine varieties to fungal diseases caused by B. cinerea or P. viticola. Unfortunately, the production of resveratrol in field-growing grapevines has proved to be very sensitive to a wide range of environmental factors, thus limiting its use as a marker for disease resistance (Barlass et al. 1987).
Pathogenesis-related proteins from grapes
Pathogenesis-related proteins can also be induced in leaves and berries as part of a defensive response to the classical PR protein gene-inducers (wounding, chemical elicitors, pathogen attack, or abiotic stress) by the expression of specific PR genes (Jacobs et al. 1999; Robinson and Davies 2000). These processes modulate the levels and proportions of the PR proteins in grapes, apparently depending on the factors like cultivar, region, climate, and agricultural practices (Monteiro et al. 2003).
Biotechnology of grapevine
Grapevine biotechnology is one of the most promising developments but on the other hand increasingly faced with many contradictions. As with plant biotechnology in general, several technical factors dictate the speed of the current progress in this field. These factors include the easiness with which the relevant plant species can be operated in tissue culture, be transformed with foreign DNA and be regenerated into new plantlets.
Vitis vinifera, being a woody perennial, initially proved to be reluctant to these manipulations. Very few of the early attempts, to transform grapevine, were successful. Now, several groups have produced transgenic grapevines that are presently at varying stages of evaluation. Still there is no universal protocol to transform or genetically engineer all grapevine cultivars. Significant progress has been made, however, for the establishment of this technology over the world and the success rate should increase with the coming years (Kikkert et al. 2001).
Targets for the genetic improvement of grapevine cultivars
Desirable properties
Examples of current potential target genes
Focus area
Pathogen tolerance
Virus coat proteins, glucanase- and chitinase-encoding genes, anti-microbial peptides
Pathogen-derived resistance strategies, defence and defence signaling against fungal and bacterial pathogens
Yamamoto et al. (2000), Vidal et al. (2006), Mauro et al. (1995)
Resistance to abiotic stress
Tonoplast integral proteins, carotenoid biosynthetic genes, antifreez genes
Carotenoid biosynthesis and control, polyamines and their role in stress, aquaporins
Tsvetkov et al. (2000)
Improvement in quality factors
UDP-glucose:flavanoid 3-O-glucosyltransferase, Silencing of polyphenol oxidase, sucrose transporters, baranase gene
Phloem loading and unloading, isolation of seed-specific promoters, anthocyanin biosynthesis and control, oxidation reactions
Fillion et al. (1999), Kobayashi et al. (2001, 2002), Boss et al. (1996)
Conclusion and perspectives
Economically, grapevine is one of the most important crop in the world and has a deeply rooted significance in human culture. The functional ingredients of grapevine include several flavonoids which have been reported to exhibit different activities including antioxidant, anti-cancer, and prevention of cardiovascular disease and the treatment of several skin disorders. Resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defense reactions comprising both constitutive and inducible barriers. In grapevine, the best characterized defense mechanisms are the accumulation of phytoalexins and the synthesis of PR-proteins. Particular attention has been given to stilbene phytoalexins (e.g. resveratrol) produced by Vitaceae. Despite the enormous amount of research carried out on grapevine phytochemistry, there is still a lot to be done in this field. The current advances achieved in analytical platforms and methodologies can and in fact will, really facilitate the isolation of new phytochemicals from grapevine with higher medicinal potential or novel bioactivities. The identification and characterization of different disease-resistant grapevine cultivars, at the gene, protein or metabolite level, is also a focal point of the grapevine research today. By the integration of different 'omics' technologies, it will be possible to better understand the total network of plant responses to various external factors including pathogen attack. In this way, a systems biology approach will be developed to explore and exploit the possible synergism between genes, proteins and metabolites.
The authors would like to acknowledge The Higher Education Commission (HEC) of Pakistan for the support of Kashif Ali. This work was supported by ERA-PG under the project Genomic Research-Assisted breeding for Sustainable Production of Quality GRAPEs and WINE (http://urgi.versailles.inra.fr/projects/GRASP/).
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This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


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Lola Thank You Card
Personalised Lola wedding thank you cards.
Thank your guests for sharing your wedding day with you with this floral design thank you card from the Lola collection.
The thank you cards will be personalised with the bride and groom's names and date of wedding. Available with a black or white background and coral, lavender or jade coloured flowers. The inside is blank for you to write your own thank you message.
Once you have placed an order you will receive a digital proof within 3 working days. Once you have carefully checked and approved the design and text we will send the artwork to print. If we need to make any amends prior to printing we will do so and follow up with another proof for you to accept. Please note that we allow 5-7 working days for delivery from approval of the proof.
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Stop Touching My Hair!!!!!
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Monday, June 15, 2009 at 09:24AM
When i had really big kinky hair and fro's i used to have many situations like that .
For example im going into a club,and as im getting through the crowd i just feel hands touching my hair.
Sometimes when i met someone after the introduction and chit chat their hands end up touching my hair.
At some point i was flattered and i felt really good but after a while when i started to wear a bit shorter curly hairstyles and apply many products for defining and taming hair i would be totally pissed if someone touched my hair without aksing me .
Spending hours to make my hair look great and then someone has to mess it up and create frizz but touching it .
Thanx,but no thanx .


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SoBeWFF 2015: Broken Shaker Wins People's Choice at Art of Tiki
The art of the tiki: It's all about the flair.
Photo by Laine Doss
A blustery evening couldn't stop the Shore Club pool from resembling an exotic port of call at last evening's Art of Tiki.
This first-time event celebrated the art of the tropical cocktail, with master bartenders from some of the country's best tiki bars creating inspired drinks.
Guests were issued a sand dollar, to be thrown into the treasure chest of their favorite drink. Cocktails were also judged by a crack team of experts including Emeril Lagasse and Jeff "Beachbum" Berry.
See also: Spirited Events at SoBe Fest: Tiki Masters and a Bloody Good Time
Tiki cocktail from Mai-Kai.
Photo by Anita Roman
Francesco Lafranconi, executive director of mixology and spirits educator for Southern Wine and Spirits of Nevada, was tasked with curating the talent for this tiki showcase, said that while there are no "specific parameters" for making the perfect tiki drink, the cocktail should showcase the "holy trinity -- the rum, the exotic nature with fresh fruit like guava, pineapple, banana, and your spice: cinnamon, nutmeg, anise, or allspice."
Emeril Lagasse brought his Tchoup Chop bartender to SoBeWFF.
Photo by Laine Doss
After using that base as a start, the creativity kicks in. Emeril Lagasse's Tchoup Chop Bar presented a piña colada mojito, made with coconut and pineapple rums, coconut milk, and mint.
The Rum Line's Rob Ferrara.
Photo by Laine Doss
The Rum Line's Rob Ferrara's Port of Miami blended three rums, passion fruit, almond milk, lime, and cinnamon-chili syrup into a drink with a cool bite at the finish. Ferrara said that tiki cocktails are all about, "the art of blending various rums and ingredients to make a drink that's not too boozy, not too sweet. A well-balanced all-around refreshing cocktail."
Spike and partner bring back the drink umbrella.
Photo by Laine Doss
Spike Mendelsohn, who used drink umbrellas in his Haole punch, said, "it's all about the flair. You want to use fresh juices and make something not too strong because you're out in the sun having fun. It's got to be refreshing."
Tiki Mondays won best cocktail.
Photo by Laine Doss
When the votes were tallied, the judges picked Brian Miller's twist on a classic pain reliever, made with coffee infused Zacapa rum. Miller runs Tiki Mondays with Miller in New York City.
Broken Shaker is the people's choice.
Photo by Laine Doss
The people's choice went to Miami Beach favorites Broken Shaker for the bar's Paradise Found (which also came in third with the judges). The cocktail combined rum with fresh watermelon, mint, and a touch of bitters for depth to make a completely refreshing and ultimately drinkable cocktail. Shaker bartenders Randy Perez and Gui Jaroschy agreed that the perfect tiki potable is all about making, "something fresh, using different ingredients that make you rethink things." In the end, a tiki cocktail is more than a beverage in a glass. As Perez said, "It's the whole tiki culture."
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Recombination, together with mutation, is the ultimate source of genetic variation in populations. We leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing-over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P<10−245). We identify a 17 base pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of African-enriched alleles of PRDM9.
In humans and many other species, recombination is not evenly distributed across the genome, but instead occurs in "hotspots": two kilobase (kb) segments where the crossover rate is far higher than in the flanking DNA sequence1,2,3. The highest resolution genetic map in contemporary humans to date, the "deCODE Map", is based on about 500,000 crossovers identified in 15,000 Icelandic meioses4. However, a limitation of maps built in people of European descent4,5,6 is that they may not apply equally well in other populations, as suggested by comparisons of maps across ethnic groups4,7,8,9 and patterns of linkage disequilibrium (LD) breakdown which suggest that more of the genome may be recombinationally active in West Africans10. It is known that a major determinant of the positions of recombination hotspots is PRDM9, a meiosis-specific histone H3 methyltransferase whose zinc finger (ZF) domain binds DNA sequence motifs11,12,13. In Europeans, PRDM9 ZF arrays are predominantly of two similar types, "A" and "B", both of which bind the 13-bp motif CCNCCNTNNCCNC11. In contrast, 36% of West African alleles are not of the A or B type9,13. Sperm typing of males who carry neither the A nor the B allele has shown no evidence of crossover activity at recombination hotspots associated with the 13-bp motif9.
To investigate differences in the crossover landscape across human populations, we built a genetic map in African Americans, who have an average of about 80% West African and 20% European ancestry, leading to genomes comprised of multi-megabase stretches of either West African or European ancestry14. Computational approaches, including HAPMIX15, have been developed to infer the probability of 0, 1 or 2 European or African alleles at each locus in individuals genotyped at hundreds of thousands of single nucleotide polymorphisms (SNPs)15,16,17. Positions where the inferred number of European or African alleles changes reflect crossover events that have occurred since admixture began (on average six generations ago15). The change in the probability of European ancestry between adjacent SNPs can be interpreted as the probability of such a crossover between them. We inferred crossover events in 29,589 apparently unrelated African Americans who had been genotyped on SNP arrays in genetic association studies (Methods; Figure 1A). To minimize false-positive crossovers, we restricted to crossovers that HAPMIX inferred with probability of >95%, and that were flanked by a minimum of 2 centimorgan (cM) stretches where the ancestry was inferred to be unchanging (Note S1). This produced 2,113,293 highconfidence crossovers, with a typical switch point resolved within 70kb with probability 50% (Note S1).
To build a high resolution African American genetic map (AA Map), we leveraged the fact that most crossovers occur in hotspots shared across individuals1 (Methods). Intuitively, while any crossover can only be roughly localized, inter-SNP intervals that are inferred to have an appreciable probability of crossover in multiple individuals are likely to contain recombination hotspots, allowing much better localization (Figure S1). To implement this idea, we modeled the recombination rate for each inter-SNP interval as shared across individuals, and used a Markov Chain Monte Carlo (MCMC) to sample rates consistent with the data (Methods). This provides well-calibrated estimates of the crossing-over rate between all pairs of markers as well as estimates of rate uncertainty (Note S1 and Figure S2). We find that the interval size at which the average recombination rate is equal to the standard error is 6 kb, which is the same accuracy that would be expected from a map based on 500,000 crossovers whose boundaries were precisely resolved (Note S1). Despite this high resolution, there are also some limitations. First, the AA Map does not separately infer male and female recombination rates (it is a sex-averaged map) and requires normalization by the total map length (like LD maps3,18). Second, the map has less resolution and may miss a higher fraction of true crossovers at loci where it is more difficult to detect and resolve crossovers due to low SNP density or low differentiation between West Africans and Europeans. Third, the map may be biased where ancestry deviates from the average, for example at 8q24, where the 10% of the people in this study who have prostate cancer have an elevated proportion of African ancestry19. Fourth, the map assumes that all individuals are unrelated, whereas in fact there is likely some shared ancestry, resulting in multiple counting of some crossovers and an overestimate of map precision.
To assess the accuracy of the AA Map, we generated an independent African American pedigree map by analyzing 222 nuclear families that included 1,056 meioses in which we could directly detect crossovers between parent and child (Methods; Figure 1A). Examination of the AA Map rate around directly detected crossovers confirms the high resolution: the rate around such crossovers shows at least as strong a peak as that observed in maps based on LD2,3,18 (Figure S3). We next computed correlation coefficients for both the AA Map and the deCODE Map4 to maps derived from the breakdown of LD in Europeans (CEU) and West Africans (YRI)18. At broad scales (>3 Mb) they are almost identical (ρ>0.97; Table 1) At fine scales, the AA Map is more accurate (Table 1 and Table S1), as reflected in a modest improvement in correlation to the CEU Map at a 3kb scale (ρAA,CEU(3kb) = 0.66 vs. ρdeCODE,CEU(3kb) = 0.58), and a major improvement for the YRI Map (ρAA,YRI(3kb) = 0.71 vs. ρdeCODE,YRI(3kb) = 0.53). The deCODE Map is more correlated to the CEU Map than to the YRI Map at scales <1 Mb, suggesting that this map, built in Icelanders, reflects more European recombination rates. The AA Map shows the opposite pattern, suggesting that it reflects more West African recombination patterns.
We compared the rate estimates for all four maps (AA, deCODE, CEU and YRI) over a 200 kb region within the MHC locus where recombination rates in European males have been characterized through sperm typing1 (Figure 1B). The AA Map detects five of six known hotspots, and localizes them to within 1 kb (the sixth hotspot is weak, with a peak male rate below the genome average1). Strikingly, the two maps based on samples with African ancestry (AA and YRI) found a hotspot not present in either map based on samples of European ancestry (deCODE and CEU) (Figure 1C; Figure S4 gives a second example). We confirmed that such "African-enriched" hotspots also occur genome-wide, by examining 2,375 loci with recombination rate peaks in the YRI Map (>5 cM/Mb) but not the CEU Map (<1 cM/Mb), and finding a rate rise in the independently generated AA Map, but not in the deCODE Map (Figure S5A). In the reciprocal experiment searching for European-specific hotspots, we find no such evidence for genuine ancestry specificity; at loci with recombination rate peaks in the CEU Map but not the YRI Map, there are weak peaks in both the deCODE and AA maps (Methods; Figure S5B). Thus, hotspots active in Europeans are consistently "shared" with YRI and African Americans, while populations with African ancestry harbor additional, non-shared hotspots we call "African-enriched".
To understand the features of recombination in West Africans that differ from Europeans, we estimated the degree to which each African American person's crossovers occur in "African-enriched" hotspots, compared with "Shared" hotpots, a phenotype we refer to as their "African-enrichment" (AE). We view each individual's crossovers as sampled from a mixture of two genetic maps—an "S Map" of shared hotspots based on the deCODE Map, and an "AE Map" of African-enriched hotspots that is learned from comparing the deCODE and AA Maps—so that the proportion of crossovers assigned to the AE Map is a person's AE phenotype (Note S4). We tested approximately 3 million SNPs (genotyped and imputed) for association with three phenotypes: AE, usage of LD-based hotspots known to be enriched for the 13-bp motif CCNCCNTNNCCNC20, and genome-wide crossover rate (in pedigrees) (Methods and Note S4). In crossovers detected in unrelated African Americans, the alleles a person carries are only sometimes descended from the ancestor in whom the crossover occurred, thus adding noise to the association signal (nevertheless there is useful signal given the large sample size; Note S4). In the Pedigree Map, association between alleles and AE can be tested directly because we have genotypes in the parents.
The SNP showing the strongest association with AE is rs6889665 (P=1.5×10−246; Figure 2A, Figure S6), which has a derived allele frequency of 29% in YRI and 2% in CEU, and is within 4 kb of the ZF array of PRDM94,9,11,12,13. This SNP is associated with AE in both the pedigree individuals and the unrelated individuals (Note S4), and is also the SNP most strongly associated with usage of LD-based hotspots (P=1.8×10−52) (Table S2). No locus outside PRDM9 is significant (P<0.01 after Bonferroni correction; Table S3). To better understand the association at rs6889665, we inferred the alleles in the PRDM9 ZF array carried by 139 individuals based on sequencing data from the 1000 Genomes Project21, using the reads to infer each individual's PRDM9 alleles among 29 alleles whose full sequences were previously determined9 (Note S5). Grouping PRDM9 alleles based on how closely their binding target predictions match the 13-bp motif, following Berg et al.9, we find that the ancestral "T" variant at rs6889665 is strongly correlated to "8/8 matches" to the 13-bp motif (including the "A" and "B" alleles ), while the derived "C" variant is almost perfectly correlated to a group of "5/8 match" alleles, all predicted to bind a common, different, 17-bp motif "CCgCNgtNNNCgtNNCC"9. This implies a common historical origin for alleles matching this 17-bp motif (Figure 2B; Figure S7; Note S5). We also experimentally measured the number of zinc fingers in PRDM9 in 354 individuals including 166 African Americans from the pedigree study (Methods). This showed, again, that rs6889665 differentiates PRDM9 alleles into two different classes, with 96% of haplotypes carrying the ancestral allele having <14 zinc fingers, and 93% of haplotypes carrying the derived allele having ≥14 zinc fingers (Figure S7). After conditioning on rs6889665, there is no evidence that ZF length is associated with the AE phenotype. Several SNPs near the PRDM9 ZF array show a conditional association signal that is much weaker than rs6889665, but still significant (Figure 2C; Figure S6; Note S4), with the strongest at rs10043097 (P= 8.3×10−14), upstream of the PRDM9 transcription start site. These SNPs may tag additional variation in PRDM9 ZF array, or potentially expression levels.
To directly identify candidate African-enriched hotspot motifs, we selected 2,454 loci with a high crossover rate in the AE Map and YRI Map (>2cM/Mb over 2kb), and no more than half this rate in the S Map and CEU Map (this set is more powerfully enriched for higher recombination in people of African ancestry than the 2,375 above, as it includes information from the contemporary maps). We compared these to a "control set" of 7,328 candidate hotspots more active in the European than the African derived maps (Methods; Note S6). To identify sequence motifs associated with the African-enriched hotspots3,22, we identified short motifs that occurred at increased frequency in the African-enriched hotspot set (Note S6). Testing all motifs of length 5–9 bases revealed a 9-mer "CCCCAGTGA" (OR=1.79, P=2.24×10−8, Bonferroni corrected P=0.004) which exhibited a kilobase-scale rate peak near occurrences of this motif in African derived maps, but in neither of the European derived maps (Figure S8). Further analysis revealed a strong influence of downstream flanking bases (Figure S9), and degeneracy, yielding a 17-bp consensus sequence "CCCCaGTGAGCGTtgCc" (Figure 3A; more strongly signaled bases are uppercase) with the same consensus obtained when we considered flanking sequence for only odd or even chromosomes, and whether we based the analysis on AE-S or YRI-CEU map comparisons (Note S6). The 500 best matches to this motif have a ~3-fold increase in average rate in the AA and YRI relative to the deCODE and CEU maps (Figure 3B, Figure S8). Hotspots associated with the motif occur in both unique and repetitive DNA (e.g. L1PA10/13 LINE elements; Figure S10) (Note S6). We also compared the 17-bp consensus to the binding motif predicted for "5/8 match" alleles, and found that they match almost precisely (Figure 3A; 10 of 11 bases, P=8.1×10−6).
How much of the African-enriched recombination pattern can be explained by PRDM9? We estimated the fraction of variation in the AE phenotype explained by rs6889665 in our pedigree data after accounting for noise in the phenotype estimation (Note S4). Over 82% of map usage variability is explained by rs6889665 genotype alone. Given there are further influential PRDM9 variants (Figure 3C), this gene may thus explain almost all differences in local rate between the West African and European populations. We next examined rates around 82 narrowly defined (<10kb) crossover sites in 7 individuals homozygous for the derived allele at rs6889665. There is no evidence of hotspots at these loci in either the deCODE or CEU Maps (Figure 3C), in contrast to events in individuals carrying the ancestral allele at rs6889665 (Figure S11). Thus, crossover positions in individuals who are homozygous for the derived allele at rs6889665 are consistent with an entirely different recombination hotspot landscape, which would imply PRDM9 control of all hotspots9. Despite the strong correlation between maps at megabase scales, there is mounting evidence that PRDM9's influence on crossing-over may not be limited to fine scales4,11: we observe a weakly significant association of rs6889665 with the total number of crossovers genome-wide in pedigrees (P=0.04), corresponding to an average 1.3 crossovers more per meiosis per derived allele, exceeding the strongest previously known association23 at RNF212.
We have shown that PRDM9 alleles that bind a novel 17-bp motif and occur at greatly increased frequency in people of West African ancestry have led to a shift in the recombination landscape compared with people of non-African ancestry. The larger number of hotspots available to West Africans implies that at the population level, crossovers are more evenly distributed than in Europeans10, and thus the shorter extent of West African LD is not due to differences in demographic history alone (such as the lack of an out-of-Africa founder event)24. Our findings also have medical implications, as recombination errors leading to insertions or deletions are known to be associated with recombination hotspots9,22,25. Our results predict that the congenital abnormalities that have been associated with the recombination hotpots bound by PRDM9 "A" and "B" alleles will occur at a decreased rate in people of West African ancestry, whereas new diseases will arise due to recombination errors near African-enriched hotspots.
We assembled SNP array data from 29,589 unrelated people and 222 nuclear families genotyped at 490,000–910,000 SNPs from the Candidate Gene Association Resource (CARe), studies at the Children's Hospital of Philadelphia (CHOP), the African American Breast Cancer Consortium, the African American Prostate Cancer Consortium and the African American Lung Cancer Consortium. To build a recombination map, we used HAPMIX to localize candidate crossover positions15, and implemented a Markov Chain Monte Carlo (MCMC) that used the probability distributions for the positions of the filtered crossovers to infer recombination rates for each of 1.3 million inter-SNP intervals. We also implemented a second MCMC that models each individual's set of crossovers as a mixture of a Shared (S) Map similar to the European deCODE Map and an African-enriched (AE) Map, and then assigns each individual an "AE phenotype" corresponding to the proportion of their newly detected crossovers assigned to the AE Map. We imputed genotypes at up to three million HapMap2 SNPs8 using MaCH26, and then tested each of these SNPs for association with the AE phenotype and other recombination-related phenotypes. We identified 2,454 candidate African-enriched hotspots with increased recombination rates in the YRI vs. CEU maps, and in the AE vs. S maps, and searched for motifs enriched at these loci, thus identifying a degenerate 17-bp motif. To study the structure of PRDM9, we measured the length of the PRDM9 zinc finger array and genotyped rs6889665 in YRI, CEU and the CARe nuclear families; we also carried out imputation based on 1000 Genomes Project short read data10 to infer the alleles individuals carry, among 29 previously characterized in a sequencing study of PRDM99.
We are grateful to the participants who donated DNA samples, to David Altshuler, Jerome Buard, Kasia Bryc, Joseph Kovacs, Bernard de Massy, Gil McVean, Bogdan Pasaniuc and Sriram Sankararaman for conversations and critiques, and to Adam Auton for facilitating analysis of the 1000 Genomes Project data.
Analysis was supported by the Wellcome Trust and NIH grants HL084107 and GM091332.
CARe was supported by a contract from the National Heart, Lung and Blood Institute (HHSN268200960009C) to create a phenotype and genotype database for dissemination to the biomedical research community. Eight parent studies contributed phenotypic data and DNA samples through the Broad Institute (N01-HC-65226): the ARIC, CFS, CARDIA, JHS, and MESA studies, as well as the Cardiovascular Health Study (CHS), the Framingham Heart Study (FHS), and the Sleep Heart Health Study (SHHS). Support for CARe also came from the individual research institutions, investigators, field staff and study participants. Individual funding information is available at http://public.nhlbi.nih.gov/GeneticsGenomics/home/care.aspx.
All genome-wide genotyping of the CHOP dataset was supported by an Institutional Development Award to the Center for Applied Genomics from the Children's Hospital of Philadelphia, a research award from the Landenberger Foundation and the Cotswold Foundation. We thank all study participants and the staff at the Center for Applied Genomics for performing the genotyping.
AABCC was supported by a DoD Breast Cancer Research Program Era of Hope Scholar Award to CAH and the Norris Foundation, and by grants to the component studies: MEC (CA63464, CA54281); CARE (HD33175); WCHS (CA100598, DAMD 170100334, Breast Cancer Research Foundation); SFBC (CA77305, DAMD 17966071); CBCS (CA58223, ES10126), PLCO (NCI Intramural Research Program); NHBS (CA100374), WFBC (R01-CA73629) and CPS-II (the American Cancer Society).
AAPCC was supported by grants CA63464, CA54281, CA1326792, CA148085 and HG004726, and by grants to the component studies: PLCO (NCI Intramural Research Program), LAAPC (Cancer Research Fund 99-00524V-10258), both MEC and LAAPC (PC35139, DP000807); MDA (CA68578, CA140388, ES007784, DAMD W81XWH0710645); GECAP (ES011126); CaP Genes (CA88164); IPCG (W81XWH0710122); DCPC (GM08016, DAMD W81XWH0710203, DAMD W81XWH0610066); SCCS (CA092447, CA68485).
AALCC was supported by grants CA060691, CA87895, PC35145 and CA22453, CA68578, CA140388, ES007784, ES06717, CA55769, CA127219, CA1116460S1, CA1116460, CA121197, CA141716, CA121197S2, CPRIT RP100443, CA148127, DAMD W81XWH0710645, University Cancer Foundation, Duncan Family institute, Center for Community, Implementation, and Dissemination Research Core, and by grants to the component studies: PLCO and the Maryland Studies (NCI Intramural Research Program), LAAPC (Cancer Research Fund 99-00524V-10258), and both MEC and LAAPC (PC35139, DP000807).
AUTHOR CONTRIBUTIONSDR and SRM conceived the study. AGH, AT, NP, YS, NR CDP, GKC, KW, SGB, DR and SRM performed analyses. NR performed the experimental work (genotyping of polymorphisms at PRDM9). AGH, NP, JNH, BEH, HAT, ALP, HH, SJC, CAH, JGW, DR and SRM coordinated the study. AGH, DR and SRM wrote the paper. NR, CDP, GKC, KW, SGB, SR, JNH, BEH, HAT, HH, CJC, CAH, JGW, DR and all the alphabetically listed authors contributed to sample collection and generation of SNP array data. All authors contributed to revision and review of the manuscript.
Crossover rate estimates for the AA Map, Pedigree Map, AE Map and S Map can be found at http://www.well.ox.ac.uk/~anjali/AAmap/. We also provide estimates of uncertainty for each map based on samples from the Markov Chain Monte Carlo. Association testing results for each SNP are available from the authors on request.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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: Recalled/Assigned:
Schroeder or Ebbett (UPD: Canucks recall Schroeder from Chicago - Jan 22)
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This is how I see the 2nd line C battle being fought out.
Let's say Ebbett's performing at a 6 and Schroeder's performing at a 5 through camp. With Ebbett, you've got a 30 year old journeyman. A 6 is the best you can hope for him to accomplish. If the competition is close, I say you absolutely give it to Schroeder, because even if he's competing just a notch below, his potential to step up and surpass that is much higher than Ebbett. Schroeder could potentially reach a 7 or an 8, while Ebbett will always max out at 6.
I just don't see a reason to give Ebbett the job over Schroeder unless the latter completely bombs his camp.
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Faculty:Faculty of Arts, Law & Social Sciences
Department:Department of English and Media
Location: ARU London, Cambridge
Areas of Expertise: Creative writing , English literature , Sociology
Sally's areas of expertise are in the theory and practice of life writing (biography, memoir, autobiography); the theory, history and practise of narrative/literary non-fiction, the methodology of short stories, novellas, novels, American & English 20th Century literature, crime writing, feminist theory, women's studies and sociology.
Sally Cline (Fellow of the Royal Society of Arts, former Advisory Fellow of the Royal Literary Fund, and a Hawthornden Fellow) is an award-winning biographer and short fiction writer. Her two most recent books, the linked short stories One of Us is Lying (Golden Books) and her thirteenth book and latest biography Dashiell Hammett: Man of Mystery (Arcade) follow best-selling biographies on Radclyffe Hall (shortlisted for the LAMDA prize) and Zelda Fitzgerald. A short study on Lillian Hellman is forthcoming. She was 2013 Judge for the HW Fisher prize for First Published Biographies, and was presented in 2012 to the Queen at Buckingham Palace for her writing and work with the RLF. She is co-Series Editor for Bloomsbury's 9 volume Writers' and Artists' Companions about writing and has co-authored two volumes on life writing and on literary non-fiction.
She has taught at Cambridge University, was Writer in Residence at Anglia Ruskin University, Cambridge, holds degrees and Masters from Durham and Lancaster Universities and was awarded an Honorary Doctor of Letters at ARU for international writing.
Sally's research interests focus on biographical subjects (often women who were invisiblised or of special interest), notably American or UK 20th century (e.g. Radclyffe Hall, Scott and Zelda Fitzgerald, Hemingway, Lillian Hellman, Dashiell Hammett) but also short fiction, narrative non-fiction, women's studies and feminism.
Sally's Successful mentoring/supervising for Gold Dust Mentoring Scheme UK has resulted in five UK students/mentees getting their novels, short stories, memoirs published. Several more have been winners or shortlisted for the Tony Lothian Biographers Club Prize, London.
2013 Royal Literary Fund. Advisory Fellowship. (£6000)
2013. Judge of H.W. Fisher Best Biography Prize. The Biographers Club. UK
2012. RLF Advisory Fellowship. £6000
2012. Shortlisted Fish Prize for Memoir.
2012. Feb 14th. Buckingham Palace. Presented to the Queen for outstanding work for the Royal Literary fund and for contribution to literature.
2010-2011. RLF Advisory Fellowship. £6000
Cline Sally & Gillies Midge. (2016) Literary Non-Fiction. A Writers' & Artists' Companion. Writing About Everything from Travel to Food to Sex. Bloomsbury. London & New York
Cline Sally. (2016.) Dashiell Hammett: Man of Mystery. Arcade Publishing. New York.
Cline Sally (2014) Dashiell Hammett. Man of Mystery. Arcade Publishing. New York.
Cline Sally.(2013) One of Us is Lying. Golden Books. UK.
Cline Sally. & Angier Carole (2013) Life Writing. Writing Biography, Autobiography & Memoir. A Writers' & Artists' Companion. Bloomsbury. London. New York.
Cline Sally & Angier Carole. (2012) The Arvon Book of Life Writing. Writing Biography, Autobiography and Memoir. Methuen Drama. UK.
Cline Sally. (2012). Zelda Fitzgerald. The Tragic Meticulously Researched Biography of the Jazz Age's High Priestess. Arcade publishing. New York.
Angier Carole and Cline Sally (eds) (2016) Playwriting : A Writers' & Artists' Companion (Grace Fraser & Bayley Clare) Bloomsbury London & NY
Angier Carole & Cline Sally (eds) (2016) Writing for TV & Radio. A Writers' & Artists' Companion. (Teddern Sue & Warburton Nick) Bloomsbury London & New York
Angier Carole & Cline Sally (eds) (2016) Literary Non-Fiction. A Writers' & Artists Companion. Writing About Everything from Travel to Food to Sex. (Sally Cline & Midge Gillies) Bloomsbury London & New York
Angier Carole & Cline Sally (eds) (2015) Novel Writing . A Writers' & Artists' Companion. (Gunnesekere Romesh & Kennedy A.L.) Bloomsbury London & New York
Angier Carole & Cline Sally (eds) (2015) Writing Short Stories. A Writers' & Artists' Companion. (Newland Courttia & Hershman Tania.) Bloomsbury London & New York
Angier Carole & Cline Sally (Eds. 2014) Writing Historical Fiction A Writers' & Artists' Companion (Brayfield Celia & Sprott Duncan) Bloomsbury London & New York .
Angier Carole & Cline Sally (eds) 2013. Writing Children's Fiction. A Writers' & Artists' Companion. (Coppard Yvonne & Newbery Linda) Bloomsbury London & New York
Angier Carole & Cline Sally (eds) (2013) Crime & Thriller Writing. A Writers' & Artists' Companion. (Spring Michelle & King Laurie) Bloomsbury London & New York
Cline, Sally. (Dec 2012, June, July August 2013) Series of 4 articles about Memoir. Myslexia Magazine. London.
2015. Guardian Master Class on Life writing. Stratford upon Avon
2015. Biography and Memoir Talk & Workshop. Stratford Literary Festival.
2015. Life Writing Workshop. Anglia Ruskin University.
2014 U3A & RLF Presentation on my short stories. U3A offices Cambridge.
2014 Biography Talk and Workshop. Cambridge Literary festival.
2014. Talk on The Maltese Falcon and Dashiell Hammett. Arts Picture House. Cambridge.
2014 Launch talk for Dashiell Hammett: Man of Mystery. Heffers. Cambridge
2014 Launch talk for One of Us is Lying. Heffers. Cambridge.
2012. Royal Literary Fund Induction Event talk on History of Royal Literary Fund. London.
2011. Royal Literary Fund Induction Event. Talk on My Life as a Royal Literary Fund Fellow. London.
For many years Sally has recorded and written talks, stories and interviews for the BC Radio 4, especially but not exclusively for Woman's Hour.
Radio scripts, radio stories, radio plays, documentaries for BBC RADIO and CBC (Canadian Broadcasting Corporation):
2015-2016. Scripting and recording podcasts for the RLF web site.
1993 The Passenger in Pink. Radio Play. New London Playwrights Festival. LBC. Highly Commended.
1993. The Passenger. Radio story. Winner BBC Radio Story Contest.
1990-91. Canadian Broadcasting Corporation. Research and scripts for documentary based on my book Women, Celibacy and Passion.
1988 and 1989. Ideas Program. CBC Canada.
Just Desserts: Women and Food. three Radio Documentaries based on one of Sally's books. Scripted, edited, produced and recorded.


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Alice in Wonderland Framed Triple Film Cell (Series 1)
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18" x 14" | Film Cell Clips | Limited Edition of 2500 | Framed | From the mind of visionary filmmaker Tim Burton comes a unique new twist on the classic tale Alice in Wonderland. Alice Kingsleigh (Mia Wasikowska) is a beautiful 19 year-old, quite out of place in a world of rules and tradition. One day she is lured from a garden party by a White Rabbit and falls into a rabbit hole where she re-enters the outrageous dreamlike world of her childhood. A magical world where rabbits talk and cats disappear into thin air. Only this time, they've been expecting her. The fate of Underland lies within her hands. Alongside the Mad Hatter (Johnny Depp), Alice embarks on a fantastical journey to end the Red Queen's (Helena Bonham Carter) reign of terror and attempt to restore peace to Underland. | This item contains clips of film, an image portraying the movie(s), artist(s) or character(s) and a stunning descriptive plaque. These elements have been double mounted and placed within a beautiful black wood frame. Each item comes with an official ? Trend Setters Ltd. certificate stating its authenticity. The film strip portrayed in the image above is only an example of the film cells you may receive. Please note, the cells may vary from piece to piece because they are hand selected from reels of film. | ? Disney
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YUGOSLAV FILM ARCHIVE-package design-
The package design for Yugoslavfilm archive was part of my graduate project. I decided to design a fictionalpackage for this institution in a way how I would like it to be it and to make it more interesting forvisitors. Here you can see two different designs for popcorns (sweet and salty),for three different coffee flavors also, for soda and non-carbonated juice and packagefor peanut/sunflower seeds.
*For me, it was very interestingto experiment with colors, retro and modern shapes, textures as well as with Cyrillicand Latin letters that I used in this project as a symbol that alludes to theword Yugoslav in the project title.
*Enjoy the projection! :)
Thank You!


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The Long Road to the Bikini Bracket Final Four!
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The Long Road to the Bikini Bracket Final Four!
Our Hottest Bikini Body contest is down to the Final Four contestants, and today we're taking a look at what the ladies have been up to in the last year. Jennifer Aniston, Katy Perry, Brooklyn Decker, and Jessica Alba are currently battling it out, and tomorrow at midnight we'll be down to just two remaining stars. Make sure to read the official rules and then fill out your bracket, which enters you to win an iPhone 4. Click through to get background info on the Final Four!


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Jimmy Fallon Explains How He Injured His Hand... Again
Turns out he's not cursed.
The details from Jimmy Fallon's second hand injury of 2015 were fuzzy. Over the weekend, the Tonight Show show host was in Boston being honored with Harvard Lampoon's Elmer Award for excellence in humor. During the ceremony, he reportedly tripped over "a random girl" while clutching a bottle of Jägermeister. As he did when arriving to work with a giant bandage around his hand back in July, Fallon delivered an explanation for his tumble on last night's show, recreating the event on his stage. Errol Morris would be proud.
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