Higher Ester Levels Research Articles

Impact of sequential inoculation timing on the quality of wine fermented by indigenous Lachancea thermotolerans and Saccharomyces cerevisiae

Lack of acidity in wine is one of the main challenges in warm wine-producing regions. The non-Saccharomyces yeast, Lachancea thermotolerans (LT), is well known for its great potential in wine acidification through the production of lactic acid. In this study, an indigenous LT strain was sequentially inoculated with S. cerevisiae (SC). The influence of the timing of SC inoculation on wine quality was extensively studied. Inoculating SC 72 h after the inoculation of LT delayed the peak of fermentation rate, but had no significant influence on the duration of fermentation. The late inoculation (72 h) of SC effectively increased the production of lactic acid, which consequently increased the total wine acidity and decreased the pH. Higher levels of esters were also achieved by the late inoculation. Pilot-scale fermentations were carried out with both Chardonnay and Cabernet Sauvignon to verify the effect of late inoculation. Lactic acid produced in sequential fermentation was 2.73 g/L for Chardonnay wine and 5.35 g/L for Cabernet Sauvignon wine, and the production of ethyl esters was also enhanced. Overall, the results demonstrated that the late inoculation of SC in sequential fermentation with LT improved wine quality by increasing acidity and producing more balanced and aromatic wines.

Analysis for different flavor compounds in mature milk from human and livestock animals by GC×GC-TOFMS.

Breast milk plays a crucial role in the taste development of infants, which cannot be replicated by other mammalian milk or formulas. This study aimed to identify and characterize the flavor substances in 15 different types of milk and analyze the differences among them. The results showed that human milk contained high levels of esters, particularly fatty acid ethyl esters, which contribute to its unique flavor. The four substances that had the highest flavor contribution in all species were identified as 2,3-butanedione, trimethylamine, isophorone, and acetaldehyde. Furthermore, the analysis of differences revealed that thermal-oxidation of lipids could explain the variation between human milk and other species in terms of flavor compounds. The key differential flavor compounds identified in milk from all species were trimethylamine, propanal, 1-pentanol, pyridine 2-methyl, and 2-butanone. These findings can potentially aid in developing formulas that better meet the taste needs of infants.

Use of reconstituted kefir consortia to determine the impact of microbial composition on kefir metabolite profiles

Kefir is fermented traditionally with kefir grains, but commercial kefir production often relies on fermentation with planktonic cultures. Kefir has been associated with many health benefits, however, the utilization of kefir grains to facilitate large industrial production of kefir is challenging and makes to difficult to ensure consistent product quality and consistency. Notably, the microbial composition of kefir fermentations has been shown to impact kefir associated health benefits. This study aimed to compare volatile compounds, organic acids, and sugar composition of kefir produced through a traditional grain fermentation and through a reconstituted kefir consortium fermentation. Additionally, the impact of two key microbial communities on metabolite production in kefir was assessed using two modified versions of the consortium, with either yeasts or lactobacilli removed. We hypothesized that the complete kefir consortium would closely resemble traditional kefir, while the consortia without yeasts or lactobacilli would differ significantly from both traditional kefir and the complete consortium fermentation. Kefir fermentations were examined after 12 and 18 h using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) to identify volatile compounds and high performance liquid chromatography (HPLC) to identify organic acid and sugar composition. The traditional kefir differed significantly from the kefir consortium fermentation with the traditional kefir having 15–20 log2(fold change) higher levels of esters and the consortium fermented kefir having between 1 and 3 log2(fold change) higher organic acids including lactate and acetate. The use of a version of kefir consortium that lacked lactobacilli resulted in between 2 and 20 log2(fold change) lower levels of organic acids, ethanol, and butanoic acid ethyl ester, while the absence of yeast from the consortium resulted in minimal change. In summary, the kefir consortium fermentation is significantly different from traditional grain fermented kefir with respect to the profile of metabolites present, and seems to be driven by lactobacilli, as evidenced by the significant decrease in multiple metabolites when the lactobacilli were removed from the fermentation and minimal differences observed upon the removal of yeast.

Effects of Solid-State Fermentation with Eurotium cristatum YL-1 on the Nutritional Value, Total Phenolics, Isoflavones, Antioxidant Activity, and Volatile Organic Compounds of Black Soybeans

In this study, black soybean was firstly processed by solid-state fermentation (SSF) with a probiotic fungus Eurotium cristatum YL-1. The effect of SSF on the nutritional components (including proximate, amino acids, minerals, and fatty acids), total phenolics, isoflavones, antioxidant activity, and volatile organic compounds of black soybeans were revealed. Results of this work demonstrated that black soybean processed by SSF with E. cristatum greatly increased the contents of protein, essential amino acids, and some minerals (e.g., calcium, phosphorus, and magnesium). GC results revealed that more than 80% of the total lipids from both fermented and non-fermented black soybeans were unsaturated fatty acids, and SSF influenced the fatty acids composition. Higher contents of total phenolics and aglycone isoflavones (i.e., genistein, daidzein, and glycitein) of fermented black soybeans were achieved by SSF. Furthermore, SSF with E. cristatum considerably augmented the ferric reducing antioxidant power, scavenging effects against ABTS·+ and DPPH radical, reducing power, and chelating ability of black soybeans, which evaluated with various polarity solvent extracts. HS-GC-IMS analysis detected a total of 66 volatile compounds in FBS and BS, and 56 volatile organic compounds were successfully identified. The intensities of main volatile compounds (i.e., 10 esters, 11 alcohols, and 19 aldehydes) differed remarkably by fermentation with E. cristatum. The intensities of seven alcohols and nine aldehydes considerably decreased, whereas higher levels of esters were achieved by SFF. Thus, our results confirmed that black soybeans processed by SSF with E. cristatum is a promising approach to substantially improve its nutritional value, flavor characteristics, and biological effect, and might have great potential in the development of new functional foods or be used as a new nutritional ingredient applied in food design.

Изследване на летливата фракция на дестилат с добавени екстрактиот магарешки бодил (Onopordum acanthinum L.)

ethanol extracts of donkey thistle (Onopordum acanthium L.) was conducted. The extracts were added to the pure distillate from the grapes of Melnik-55 grapevine variety in amounts of 20, 50, 80, 100, 200, 400 and 600 ml. The remaining amount to liter was a pure distillate. A distillate of Melnik-55 grapes (without extracts) was used as a control. The addition of 50% ethanol extract of Onopordum acanthium L. increased the total volatile composition of variants 1000:20 and 1000:50, in comparison to the control sample. Higher levels of esters and higher alcohols were found in the same variants. Incorporation of the 50% and 70% ethanol extracts of donkey thistle resulted in increased final total terpene content of the distillates. By increasing the amount of the added 50% ethanol extract, a reduction in the concentrations of acetaldehyde and ethyl acetate was found in the distillates. The basic ester in all the distillates analyzed was ethyl acetate, and the major higher alcohols were 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol and 2-butanol. By applying of 50% and 70% ethanol extracts of the plant, a reduction in methanol content in the final distillates was observed. A prospect was the application of 50% ethanol extracts of Onopordum acanthium L, especially variants 1000:20 and 1000:50. This two variants showed higher final levels of aromatic compounds (beverage quality) and slightly higher final levels of terpenes (biological value) compared to the control. The other variants of this group had proven lower levels of methanol (improved toxicological quality) compared to the control.

Abstract P6-05-05: The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen

Abstract Background: The premise that estrogen promotes the growth of all estrogen receptor positive (ER) + breast cancers has led to anti-estrogen therapies as the standard of care for all ER+ tumors. Paradoxically, estrogen (E2) has demonstrated a therapeutic effect in the adjuvant, palliative and prevention of breast cancers in some women suggesting that there is a subset of ER+ breast cancers that may be growth-suppressed by E2. Our companion window-of-opportunity trial (abstract submitted) found E2 is growth suppressive for some post-menopausal women with high ER expressing tumors. We utilized an in vitro cell model to investigate whether differential growth responses to E2 could be due to transcriptional differences mediated by the level of the ER. Method: We generated a stable MCF-7 transfectant (MCF7-ER) using a fluorescently labelled lentiviral plasmid with a doxycycline inducible ER and compared the response to E2 against an MCF-7 mock transfectant (MCF7-EM). We assessed changes in proliferation (S-phase fraction), gene regulation (RNA-Seq) and differential ER-DNA binding (chromatin immunoprecipitation (ChIP-Seq)). To determine if high levels of ER were causing changes in the underlying chromatin configuration, we compared our ChIP-Seq profiles against previously mapped DNA loop anchor regions obtained by chromatin interaction analysis by paired end-tag sequencing (ChIA-PET) on the MCF-7 parental cell line treated with E2 (Fullwood M et al. Nature 2009). Changes in DNA loop formation were assessed by fluorescence in situ hybridization (FISH) and chromatin conformation capture (3C). Results: We confirmed previously published findings that increased ER expression in the presence of E2 leads to significant decreases in S-phase fraction (P = 0.003) with impaired transition at the G1/S and G2/M checkpoints. RNA-Seq studies defined 342 basally-upregulated genes in the MCF7-ER cells that became down-regulated after E2 treatment indicative of inverse regulation compared to the effect of E2 in the MCF7-EM mock transfectants. In E2-deprived conditions, there was an increased number of ER-DNA binding peaks in the high ER expressing cells (2,776 in MCF7-ER vs. 1,906 in MCF7-EM), suggesting unliganded ER may mediate basal gene transcription. Only 17 (5%) of the basally upregulated genes had ER binding at a proximal or distant anchor associated with previously mapped ER-DNA loops. This suggests that high ER expressing cells may develop a novel chromatin configuration that is distinct from those of parental MCF-7 cells. FISH analysis confirmed a significant increase in loop formation at the promoter region of the TFF1 gene in the MCF7-ER cells compared to MCF7-EM mock transfectants (P = 0) in the absence of E2. This DNA loop was maintained, though slightly decreased, after E2 treatment. Conclusions: These results support a theoretical model where increased ER expression may enable a novel chromatin configuration which mediates a basal level of gene expression that switches to a growth-suppressive transcriptional response upon E2 treatment. Further elucidation of an anti-proliferative DNA configuration signature using MCF7-ER cells may generate a novel set of interactions that could serve as a strategy to predict a patient’s response to estrogens and endocrine therapies. Citation Format: Lacey Haddon, Xiuying Hu, Hosna Jabbari, Judith Hugh. The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-05.

Sourdough derived strains of Saccharomyces cerevisiae and their potential for farmhouse ale brewing

The Finnish farmhouse ale sahti is unique in that it is fermented with baking, rather than brewing strains of Saccharomyces cerevisae. The custom of maintaining farmhouse yeast cultures is however no longer practiced in Finland, and much yeast derived diversity in sahti beers has presumably been lost as a consequence. Here, the brewing potential of a number of sourdough derived strains was tested with respect to a number of different fermentation traits. Seven strains originally isolated from Finnish or Italian sourdough cultures were used to ferment high gravity sahti wort (20°P), and fermentation performance together with production of volatile compounds were assessed and compared with a reference baking yeast. Strains differed in terms of fermentation rate, yield, yeast viability and beer flavour profile. All were maltotriose positive, but utilisation varied so that alcohol yield could be greater or lower than that of the reference strain, with values ranging from 6.6 to 7.9% (v/v). Production of aroma compounds was also variable so that it was possible to identify strains producing high levels of esters and those with lower production, which could be used to emphasise flavours originating from raw materials. All strains generated 4-vinyl guaiacol and so would be suitable for other beers where this is a part of the normal flavour profile. Results suggest that sourdough isolates of S. cerevisiae are suitable for sahti production, but could also be applied to other beer styles as a way to differentiate products. © 2020 The Authors. Journal of the Institute of Brewing published byJohn Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling

Selection of an autochthonous yeast starter culture for industrial production of Primitivo “Gioia del Colle” PDO/DOC in Apulia (Southern Italy)

The aim of the present study was to isolate and characterize yeast strains as good candidates for driving the industrial fermentation process, from natural must fermentations of “Primitivo” grape cultivar, grown in the PDO/DOC “Gioia del Colle” (Apulia, Southern Italy). The selection protocol was based on parameters such as low production of acetic acid and hydrogen sulphide, complete sugar consumption during fermentation, significant production of some classes of volatile molecules responsible for wine aroma. Three Saccharomyces cerevisiae strains, named ITEM14088, ITEM14090 and ITEM14093, successfully dominated the fermentation process and contributed to increase organoleptic quality of the produced wines. The best performing strain, namely ITEM14093, was used as fermentation starter for three different industrial vinifications. The wines obtained were characterized by high levels of esters, associated to fruity nuances, as well as of alcohols responsible for vinous, sweet and floral notes. Furthermore, from a sensory point of view, all wines were positively judged, being characterized by frankness, gustatory persistence and intensity, good balance and body wine.

Microbiology of high-sugar must fermentation by novel yeasts from the chihuahuan desert.

This work reports important results of aromatic profiles produced by native yeasts isolated from desert-grown wine grapes from the south of Chihuahua, México. These grapes stand very high temperatures during the ripening season, developing high sugar concentration and high pH. Yeast species found in grapes were identified by polymerase chain reaction and sequence analysis of the 5.8S internal transcribed spacer ribosomal RNA region. Aureobasidium namibiae, Sporobolomyces johnsonii, Candida apicola, Hanseniaspora uvarum, Candida thaimueangensis, Hanseniaspora opuntiae were identified. All of them can grow at glucose concentration of 35% (w/v) and 100 ppm of SO2, and produce low volatile acidity (0.2-1.0 g acetic acid/L). Volatile organic compounds analysis showed that C. thaimueangensis and one strain of C. apicola produce high levels of esters, and Hanseniaspora species produces high levels of higher alcohols and carbonyl compounds. The results of this study contribute to the knowledge about yeast communities associated with desert-grown winegrape yeasts.

The use of indigenous Saccharomyces cerevisiae and Starmerella bacillaris strains as a tool to create chemical complexity in local wines

The performance of two vineyard strains, Saccharomyces cerevisiae SacPK7 and Starmerella bacillaris StbPK9, was evaluated in laboratory and pilot scale fermentations of Cretan grape must under the following inoculation schemes: single inoculation of SacPK7 (IS), simultaneous inoculation of StbPK9 and SacPK7 (SM), and sequential inoculation of StbPK9 followed by SacPK7 (SQ). Un-inoculated (spontaneous) fermentations (SP) and fermentations inoculated with control S. cerevisiae strains (CS) were also conducted as reference. Star. bacillaris not only did not restrict but also slightly promoted the growth of S. cerevisiae when the two strains were co-inoculated at equal quantities. On the contrary, the SQ inoculation scheme conferred a competitive advantage to Star. bacillaris over S. cerevisiae, which maximum population was reduced, while increased levels of Star. bacillaris were recorded. The fermentation kinetics were also affected, accordingly. The completion of fermentation was faster in SM, IS and CS ferments than in SQ and SP. Ethanol accumulation had a predominant role in the early death of Star. bacillaris, since its growth was similarly arrested irrespective of the dominating yeast species, the magnitude of yeast population or the availability of energy sources. Interestingly, the inoculation scheme applied significantly affected the chemical profiles of the resulting wines. SQ produced the most divergent chemical profile in sterile must, with glycerol, acetic acid, acetaldehyde, residual glucose, malic acid, ethyl acetate and higher alcohols being the key compounds affected by the prolonged activity of StbPK9. In pilot scale ferments, the indigenous S. cerevisiae produced twice as high levels of esters and higher alcohols compared to the commercial starter. Star. bacillaris further increased the levels of ethyl esters in the respective ferments. The use of a mixed S. cerevisiae/Star. bacillaris starter culture instead of S. cerevisiae alone enhanced the chemical complexity of Cretan local wine. The magnitude of differentiation was even higher when the addition of Star. bacillaris preceded that of S. cerevisiae. The highest divergence in analytical profiles was recorded between wines produced by native strain combinations and commercial S. cerevisiae. Present results show that the use of indigenous yeast formulations provides significant diversification to local wines, in line with the microbial terroir concept and recent observations that indigenous yeast strains may confer regional characters to wines.

Abstract P3-04-15: The PI3K-inhibitor, copanlisib, has selective activity in luminal breast cancer cell lines and shows robust combined activity with hormonal blockade and CDK-4/6 inhibition in ER+ breast cancer cell line xenografts

Abstract Background: Genetic and epigenetic alterations in the PI3K/mTOR and cyclin D:CDK-4/6:Rb signaling axes occur frequently in breast cancer and have been attributed to resistance to both ER- and HER2-directed therapeutics. Pharmacologically targeting CDK-4/6 in combination with hormonal blockade provides clinical benefit in patients with advanced ER+ breast cancer. In this study, we evaluated the activity of the pan-class I PI3K inhibitor, copanlisib (BAY-80-6946), with potent alpha and delta activity as a single agent or in combination with CDK-4/6 inhibition and hormonal blockade in a panel of breast cancer cell lines. Methods: The growth inhibitory activity of copanlisib was evaluated against a large panel of 48 breast cancer cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. The activity of copanlisib in combination with hormone blockade and CDK-4/6 inhibition, by palbociclib, was assessed in two cell line xenograft models of ER+ breast cancer; MCF7(PIK3CA-E545K) and ZR751(PIK3CA WT). For xenograft studies, tumor bearing mice were treated once weekly (BID) by intravenous injection with clinically achievable doses of copanlisib (10 mg/kg) as single agent or in combination with tamoxifen or fulvestrant with or without 75 mg/kg daily palbociclib for 21 days. Results: A broad range of IC50 values (0.491-895 nM), with a high degree of separation between sensitive and resistant histologically defined subgroups were determined for copanlisib, indicating the potential for a wide therapeutic window. Luminal subtype, the presence of activating mutations in PIK3CA, high levels of ER, HER2, HER3 and EGFR protein enriched for sensitivity to copanlisib. Activating mutations of KRAS and BRAF were associated with resistance to copanlisib. Single agent copanlisib induced significant tumor growth inhibition (TGI) relative to vehicle control in each of the xenograft models. Modest increases in anti-tumor activity were achieved when copanlisib was combined with hormonal blockade by either tamoxifen or fulvestrant. However, robust tumor regressions were observed with the triple combinations of copanlisib-palbociclib-tamoxifen and copanlisib-palbociclib-fulvestrant. Furthermore, these triple combinations achieved a statistically significant improvement in anti-tumor activity over the standard of care combination of palbociclib plus fulvestrant. Each of the single agent and treatment combinations tested were well tolerated in animals. Discussion: These preclinical data illustrate the potent and selective activity of the pan class I PI3K inhibitor copanlisib in luminal breast cancers and support the clinical investigation of copanlisib in combination with CDK-4/6 inhibition and hormonal blockade in ER+ breast cancer. Citation Format: O'Brien NA, Conklin D, Luo T, Ayala R, Issakhanian S, Kalous O, Von Euw E, Politz O, Wilhelm S, Childs BH, Hurvitz SA, Slamon DJ. The PI3K-inhibitor, copanlisib, has selective activity in luminal breast cancer cell lines and shows robust combined activity with hormonal blockade and CDK-4/6 inhibition in ER+ breast cancer cell line xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-15.

Evaluation of Zygosaccharomyces bailii BCV 08 as a co-starter in wine fermentation for the improvement of ethyl esters production

Zygosaccharomyces bailii BCV 08, a yeast isolated from red wine barrels in Brazil, was evaluated as co-starter in fermentations with Saccharomyces cerevisiae. Z. bailii BCV 08 was preliminarily shown to produce high levels of esters, and the production was optimized in bench and bioreactor scales using grape must. White wine vinifications were conducted with mixed cultures containing different proportions of Z. bailii BCV 08 and an enological strain of S. cerevisiae. In all trials that contained Z. bailii BCV 08, the production of ethyl esters was enhanced in comparison to the vinification control. Our results clearly show the potential of Z. bailii BCV 08 as a mixed starter with S. cerevisiae in order to increase the aromatic complexity of wine.

Progesterone-induced stimulation of mammary tumorigenesis is due to the progesterone metabolite, 5α-dihydroprogesterone (5αP) and can be suppressed by the 5α-reductase inhibitor, finasteride

Progesterone has long been linked to breast cancer but its actual role as a cancer promoter has remained in dispute. Previous in vitro studies have shown that progesterone is converted to 5α-dihydroprogesterone (5αP) in breast tissue and human breast cell lines by the action of 5α-reductase, and that 5αP acts as a cancer-promoter hormone. Also studies with human breast cell lines in which the conversion of progesterone to 5αP is blocked by a 5α-reductase inhibitor, have shown that the in vitro stimulation in cell proliferation with progesterone treatments are not due to progesterone itself but to the metabolite 5αP. No similar in vivo study has been previously reported. The objective of the current studies was to determine in an in vivo mouse model if the presumptive progesterone-induced mammary tumorigenesis is due to the progesterone metabolite, 5αP. BALB/c mice were challenged with C4HD murine mammary cells, which have been shown to form tumors when treated with progesterone or the progestin, medroxyprogesterone acetate. Cells and mice were treated with various doses and combinations of progesterone, 5αP and/or the 5α-reductase inhibitor, finasteride, and the effects on cell proliferation and induction and growth of tumors were monitored. Hormone levels in serum and tumors were measured by specific RIA and ELISA tests. Proliferation of C4HD cells and induction and growth of tumors was stimulated by treatment with either progesterone or 5αP. The progesterone-induced stimulation was blocked by finasteride and reinstated by concomitant treatment with 5αP. The 5αP-induced tumors expressed high levels of ER, PR and ErbB-2. Hormone measurements showed significantly higher levels of 5αP in serum from mice with tumors than from mice without tumors, regardless of treatments, and 5αP levels were significantly higher (about 4-fold) in tumors than in respective sera, while progesterone levels did not differ between the compartments. The results indicate that the stimulation of C4HD tumor growth in BALB/c mice treated with progesterone is due to the progesterone metabolite 5αP formed at elevated levels in mammary cells as a result of the 5α-reductase action on progesterone. The results provide the first in vivo demonstration that stimulation of breast cell tumorigenesis and tumor growth accompanying progesterone treatment is due to the progesterone metabolite 5αP, and that breast tumorigenesis can be blocked with the 5α-reductase inhibitor, finasteride.

Effect of ethylene and temperature conditioning on sensory attributes and chemical composition of ‘Bartlett’ pears

‘Bartlett’ pears are resistant to ripening after harvest. Ethylene and temperature conditioning have been successfully used to stimulate fruit ripening with improved eating quality over non-conditioned fruit. However, few studies have evaluated the effect of different conditioning treatments on the sensory attributes of the fruit. In this study, we compared a descriptive sensory evaluation with the chemical composition of ‘Bartlett’ pears after the fruit were exposed to the following conditioning treatments: 2d 100μlL−1 ethylene, 14 or 7d at 0°C, 7 or 3d at 10°C, or untreated control at 20°C. Fruit were softened to 27, 18 and 9N firmness before evaluation. At 9N, fruit conditioned at 0°C produced high levels of esters, and fruit conditioned at 0°C for 14d also were high in sweet taste and fruity flavor attributes. Fruit treated at 10°C had lower concentrations of esters, but fruit treated at 10°C for 3d was high in sweet taste perception. Ethylene treated fruit produced low levels of esters and high levels of aldehydes and were associated with apple aroma, similar to the untreated control fruit. Water soluble pectin levels were highly and positively correlated with juiciness and sweetness and negatively correlated with firmness, crunchiness, and grittiness. Future studies should determine whether consumer liking of ‘Bartlett’ pear fruit is also influenced by conditioning treatment.

Free and Esterified Carotenoids in Ornaments of an Avian Species: The Relationship to Color Expression and Sources of Variability

Many animal species show ornaments with yellow-orange-red colors produced by carotenoid pigments. Such traits have evolved as reliable signals of individual quality because of the costs inherent to their production or maintenance. In animal tissues, carotenoids are often found combined with free fatty acids, as carotenoid esters, which may confer more stability to coloration than free carotenoids. Surprisingly, the potential relevance of carotenoid esterification in the expression of animal sexual signals has been virtually ignored. Moreover, the sources of variability of esterified carotenoid levels are barely known, because most studies have not quantified their concentrations. Here, carotenoids in the ornaments (bill, eye rings, and legs) of red-legged partridges Alectoris rufa were quantified in their free and esterified forms. Carotenoid ester levels were the best predictors of leg color, whereas the redness of the other traits was better explained by free carotenoids. Nonetheless, total carotenoid levels (the sum of free and esterified forms) were always significantly correlated to redness. Young partridges had lower levels of free and esterified carotenids in the legs than did older individuals. Also, wild animals had higher ester levels and a higher proportion of carotenoids in esterified forms in all traits than did captive partridges. Probable physiological mechanisms explaining these patterns are discussed.

The impact of grape variety on the aromatic chemical composition of non-aromatic Peruvian pisco

Pisco is a common beverage, originating in some South American countries (such as Peru and Chile), which is obtained by distillation of wine made from different varieties of grapes. In this paper, thirty one commercial Peruvian pisco samples made with Negra Criolla, Mollar and Uvina grapes have been analyzed by gas chromatography–flame ionization (GC–FID) and mass spectrometry detection (GC–MS). A representative sample of each variety was also evaluated by gas chromatography–olfactometry (GC–O). A sensory descriptive analysis also was carried out to evaluate the effect of these varieties on the sensory properties of pisco. All these analyses provide a novel and complete information about the aromatic composition of these three varieties of piscos. It is the first time that the aromatic profiles of Negra Criolla, Mollar and Uvina piscos have been evaluated.The Negra Criolla grape gave a neutral aroma to pisco with no particular aromatic notes. The Mollar variety was notable for the presence of β-phenylethyl acetate in high concentrations and low levels of volatile phenols and some esters. The Uvina samples showed significantly high levels of esters, cinnamates and terpenes and low levels of β-phenylethyl acetate. The Uvina piscos received the highest score in the sweet descriptor, which could be related to their high levels of esters, acetates and terpenes. In addition, the odor thresholds of 35 compounds have been estimated for the first time in a solution consisted of a 40% ethanol/water (v/v) mixture, simulating a real sample of pisco. Thirteen compounds were found in higher concentrations than their corresponding thresholds in some of the piscos analyzed. Four of these had aroma values higher than or equal to unity in more than 70% of all the non-aromatic varieties of piscos. These compounds are: ethyl isovalerate, 2-methyl-1-butanol, 3-methyl-1-butanol and β-phenylethanol. It is worth noting the low odor threshold of ethyl isovalerate, being only 5μgL−1.

Abstract 1403: Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer.

Abstract More than 70% of breast cancers express estrogen and progesterone receptors (ER and PR), and are thus susceptible to adjuvant endocrine therapy. Treatments for these patients are mainly focused on blocking ER function at multiple levels. In the last few years, evidence has linked progestin to breast cancer pathogenesis. According to that, PR would be a valid target for breast cancer therapy. In addition, up to 40% of hormone-receptor positive breast tumors have PI3K activating mutations, and then PI3K/AKT inhibition is becoming a new approach for breast cancer treatment. The aim of this work was to study early stages of tumor regression in an experimental model of breast cancer. We tested the effect of an antiprogestin and a PI3K inhibitor for 48 hs, alone or in combination. We used the medroxyprogesterone acetate (MPA)-induced breast cancer model. Tumors of this model express high levels of ER and PR. Initially, they are MPA-dependent, but after a few passages MPA-independent variants appear spontaneously. These variants may respond to an endocrine therapy or may progress to a hormonal resistant phenotype. We have previously demonstrated that the MPA-independent variant C4-HI regresses after 48 hs administration of the antiprogestin Mifepristone (MFP, 12 mg/kg/day) or the PI3K inhibitor Wortmannin (WN, 1 mg/kg/day), displaying an increase in ductal differentiation. We now explored the mechanisms involved in these inhibitory effects. Proliferation of tumor cells, determined as mitotic and Ki67 indexes, is reduced (p<0.01) similarly by MFP and WN respect to vehicle. Consistently, we detected a downregulation in the expression of cell cycle proteins c-MYC and Cyclin D1 determined by western blot and immunohistochemistry. However, after 48hs only WN increased necrosis, apoptosis (p<0.01) and the BAX/BCL-XL ratio. As expected, only WN decreased pAKT and pS6 levels. Furthermore, when given in combination, MFP and WN improved all the above mentioned parameters. In an antiprogestin resistant tumor variant, PI3K inhibition still suppressed tumor growth. We detected that after 7 days of treatment WN-treated C4-2-HI tumors were smaller than vehicle-treated tumors (p<0.001). However, no evident signs of regression were histologically observed. Ongoing experiments are being conducted to evaluate histologically tissue response at earlier time points (48hs) after WN therapy. Taken together, these results suggest that inhibiting PI3K pathway could represent a useful tool for the treatment of hormone receptor-positive tumors, even of those that have progressed to a hormonal resistant phenotype. Understanding the early mechanisms involved in tumor regression may improve the design and monitoring of cancer therapies. Citation Format: Maria Laura Polo, Marina Riggio, Claudia Lanari, Virginia Novaro. Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1403. doi:10.1158/1538-7445.AM2013-1403

Volatile Compounds and Sensory Properties in Various Melons, Which were Chosen from Different Species and Different Locations, Grown in Turkey

Gas chromatography coupled to mass spectrometry using a static head space technique was applied to analyze the volatile aromatic compounds of various melons collected from different locations in Turkey. Some physicochemical and sensory analyses were also made. A total of 33 volatile compounds were identified. Differences in the physicochemical and sensory properties of various Turkish melon samples were observed. The multivariate analyses by the volatile compounds separated the three varieties into groups successfully. The most abundant volatile compounds as mean value in all melon varieties were ethyl acetate (22.10%), acetaldehyde (13.65%), and ethanol (23.25%). The results showed that Cantaloupe melons were more preferred by panelists, which had relatively high levels of esters being responsible for strong fruit flavor, like melon, as well as high total soluble solid content and low titratable acidity compared with Inodorus and Simama.

Abstract 2386: Triple fluorescence analysis of estrogen receptor alpha (ER) and ErbB2 status of circulating tumor cells (CTC) in breast cancer: from cell models to metastatic patients

Abstract Purpose: Hormone therapy and anti-ErbB2 (HER-2) therapy are prescribed according to the hormone status (ER/PR protein expression) and ErbB2 expression of the initial tumor. Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. As it appears that the CTC, and consequently the metastatic cells, may have a very different receptor status than the related primary tumors, it is of the highest relevance to analyze ER and ErbB2 expression of the CTC. Experimental procedures: We developed a triple fluorescence technique to visualize simultaneously cytokeratin (8/18, 8/19), ER and ErbB2 on each individual cell. Using this protocol, we analyzed ER and ErbB2 expression in various breast cancer cell lines (MCF-7, T47D, Cama-1, ZR75, SK-Br-3, HCC 3153, MDA-MB-231), and compared ER expression in the different cell lines using also immunostaining. We were then able to test the CTC presence (CK positivity) and ER and ErbB2 expressions on blood samples (cytospins) collected from 15 metastatic breast cancer patients. Results: The 2-step triple fluorescence protocol was optimized to visualize CK in green (DyLight488 conjugate), ER in red (cy3 conjugate) and ErbB2 in blue (AMCA conjugate). Using in parallel this triple fluorescence protocol and the reference immunostaining technique, we could analyze that ER-positive cell lines express very different levels of ER and within one cell line, cells can express low, moderate and/or high levels of ER. The identification of cells that express a very low level of ER is compulsory as the CTC from one unique patient can potentially express differently ER. Consequently, ER-positivity and -negativity have to be clearly defined. CTC analysis from patient blood samples was then performed with an individual assessment, for each single isolated cell, of cytokeratin (epithelial marker that peripheral blood cells should not express) and of ER and ErbB2 expressions. We were able to identify, among the CTC positive patients, changes between primary tumor status and CTC status, e.g. ER-positive primary tumors with both ER-positive and ER-negative CTC. Those ER-negative CTC may direct towards tamoxifen resistance and have to be identified among other ER-positive cells, similarly as any ErbB2 negative CTC. Conclusion: We demonstrated that triple immunofluorescence is very relevant, first to identify CTC, and then to allow an individual assessment of ER and ErbB2 expression, at the protein level. Moreover, the approach we developed allows to characterize a population of CTC with a very low expression of ER. Large multicentric cohorts of breast cancer patients treated with adjuvant therapy should now be analyzed for both ER and ErbB2 status of the CTC, compared to the primary tumor profile. The expected results should lead to optimal individualized treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2386. doi:1538-7445.AM2012-2386

Prognostic utility of β-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: a translational Hellenic Cooperative Oncology Group (HeCOG) study

We evaluated the prognostic and predictive utility of β-tubulin isotype III (TUBB3) tumour gene transcription in early breast cancer patients enrolled in a randomised study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for assessment of TUBB3, ER, PgR, HER2 and MAPT messenger RNA and immunohistochemistry (IHC) for protein expression in 314 patients enrolled in trial HE10/97, evaluating epirubicin-alkylator adjuvant chemotherapy with or without paclitaxel. High TUBB3 mRNA status was associated with advanced T stage, high histological grade, low mRNA and protein levels of ER, PgR and MAPT, and high levels of HER2 (p < 0.001). At a median follow-up of 98 months, multivariate analysis showed high TUBB3 mRNA status to have prognostic significance for DFS (HR = 1.83, 95% CI 1.25-2.68, p = 0.002) and OS (HR = 1.71, 95% CI 1.03-2.83, p = 0.038), along with the number of involved axillary nodes, PgR mRNA status and tumour grade. TUBB3 mRNA levels did not predict benefit from inclusion of paclitaxel in adjuvant chemotherapy (test for interaction p = 0.96 for OS, p = 0.46 for DFS). Transcriptional activity of β-tubulin isotype III in early breast cancer is an adverse prognostic factor, though not a predictive one for taxane efficacy.