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The liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for determination of clenbuterol residues in liver tissue as a regulatory matrix for the control of clenbuterol abuse as an anabolic. To investigate the level of clenbuterol residues during the withdrawal period, male food-producing pigs were exposed to subchronic repeat oral administration of a clenbuterol growth-promoting dose for 28 days. The analytical procedure showed acceptable validation results for all liver spiked samples analyzed and proved to be useful as a quantification and confirmation method in supporting regulatory enforcement programs of clenbuterol misuse monitoring. The highest level of clenbuterol in the liver of treated animals was recorded on day 0 of treatment cessation (21.58±14.29 ng/g), followed by 6.59±3.11 ng/g on day 3, 0.83±0.27 ng/g on day 7, and 0.44±0.08 ng/g on day 14 of withdrawal. At the end of the study period (day 35), the concentration of clenbuterol was below the method limit of detection (

Main Conclusion: A multi-year study of perennial Z. dumosum shows a consistent seasonal pattern in the changes of petiole metabolism, involving mainly organic acids, polyols, phenylpropanoids, sulfate conjugates, and piperazines. GC-MS and UPLC-QTOF-MS-based metabolite profiling was performed on the petioles of the perennial desert shrub Zygophyllum dumosum Boiss (Zygophyllaceae). The petioles, which are physiologically functional throughout the year and, thus, exposed to seasonal rhythms, were collected every month for 3 years from their natural ecosystem on a southeast-facing slope. Results showed a clear multi-year pattern following seasonal successions, despite different climate conditions, i.e., rainy and drought years, throughout the research period. The metabolic pattern of change encompassed an increase in the central metabolites, including most polyols, e.g., stress-related D-pinitol, organic and sugar acids, and in the dominant specialized metabolites, which were tentatively identified as sulfate, flavonoid, and piperazine conjugates during the summer-autumn period, while significantly high levels of free amino acids were detected during the winter-spring period. In parallel, the levels of most sugars (including glucose and fructose) increased in the petioles at the flowering stage at the beginning of the spring, while most of the di- and tri-saccharides accumulated at the beginning of seed development (May-June). Analysis of the conserved seasonal metabolite pattern of change shows that metabolic events are mostly related to the stage of plant development and its interaction with the environment and less to environmental conditions per se., (© 2023. The Author(s).)

Elicitation treatments of grape cell cultures with methyl jasmonate (MeJA), ultraviolet-C (UV-C) irradiation, and sucrose induce mild production of stilbenes and flavonoids due to limited substrate availability. However, these treatments cause a synergistic boost of stilbenes production when applied to two phenylalanine (Phe)-enriched transgenic grape cell lines, AroG * + STS and AroG * + FLS . The combined treatment of UV-C elicitation on the Phe-fed AroG * + STS line resulted in the highest content of stilbenes (37.8-fold increase, 17.39 mg/g dry weight (DW)) mainly due to resveratrol (64-fold, 3.23 mg/g DW) and viniferin (1343-fold, 13.43 mg/g DW). The synergistic increase following either UV-C or MeJA elicitation was due to the induction of stilbene-related genes, while sucrose treatment had no effect on gene expression levels and served as an additional carbon source for phenylpropanoids. The combined strategy presented may enable future usage of grape cell cultures for the production of stilbenes and in particular viniferin.

Stilbenes and flavonoids are two major health-promoting phenylpropanoid groups in grapes. Attempts to promote the accumulation of one group usually resulted in a decrease in the other. This study presents a unique strategy for simultaneously increasing metabolites in both groups in V. vinifera cv. Gamay Red grape cell culture, by overexpression of flavonol synthase ( FLS ) and increasing Phe availability. Increased Phe availability was achieved by transforming the cell culture with a second gene, the feedback-insensitive E. coli DAHP synthase ( AroG *), and feeding them with Phe. A combined metabolomic and transcriptomic analysis reveals that the increase in both phenylpropanoid groups is accompanied by an induction of many of the flavonoid biosynthetic genes and no change in the expression levels of stilbene synthase. Furthermore, FLS overexpression with increased Phe availability resulted in higher anthocyanin levels, mainly those derived from delphinidin, due to the induction of F3'5'H . These insights may contribute to the development of grape berries with increased health benefits.

Dead organs enclosing embryos, such as seed coats and pericarps, are emerging as important maternally-derived components of the dispersal unit that affect seed performance and fate. In the face of climate change and increased incidents of heatwaves, we sought to investigate the effect of salinity (S), short episodes of high temperature (HS), and combination of S + HS (SHS), at the reproductive phase, on the properties of dead pericarps of Brassica juncea . Proteome and metabolome analyses revealed multiple proteins and metabolites stored in dead pericarps whose levels and composition were altered under single and combined stress conditions. The protein profile of SHS showed a higher correlation with salt than with HS indicating the dominant effect of salt over heat stress. On the other hand, the analysis of metabolites showed that the profile of SHS has better correlation with HS than with salt. The integration of metabolic and proteomic data showed that changes in TCA cycle intermediates and certain amino acids (e.g., proline) under salt treatments (S and SHS) are highly correlated with changes in proteins involved in their biosynthetic pathways. Thus, accumulation of proteins and metabolites in dead pericarps is differently affected by single and combination of salt and heat stresses. Salinity appears to dominate plant response to combined stresses at the protein level, while heat appears to be the major factor affecting metabolite accumulation in dead pericarps.

Stilbenes are phytoalexins with health-promoting benefits for humans. Here, we boost stilbenes' production, and in particular the resveratrol dehydrodimer viniferin, with significant pharmacological properties, by overexpressing stilbene synthase ( STS ) under unlimited phenylalanine (Phe) supply. Vitis vinifera cell cultures were co-transformed with a feedback-insensitive E. coli DAHP synthase ( AroG *) and STS genes, under constitutive promoters. All transgenic lines had increased levels of Phe and stilbenes (74-fold higher viniferin reaching 0.74 mg/g DW). External Phe feeding of AroG * + STS lines caused a synergistic effect on resveratrol and viniferin accumulation, achieving a 26-fold (1.33 mg/g DW) increase in resveratrol and a 620-fold increase (6.2 mg/g DW) in viniferin, which to date is the highest viniferin accumulation reported in plant cultures. We suggest that this strategy of combining higher Phe availability and STS expression generates grape cell cultures as potential factories for sustainable production of stilbenes with a minor effect on the levels of flavonoids.

Global climate change and the expected increase in temperature are altering the relationship between geography and grapevine ( V. vinifera ) varietal performance, and the implications of which are yet to be fully understood. We investigated berry phenology and biochemistry of 30 cultivars, 20 red and 10 white, across three seasons (2017-2019) in response to a consistent average temperature difference of 1.5°C during the growing season between two experimental sites. The experiments were conducted at Ramat Negev (RN) and Ramon (MR) vineyards, located in the Negev desert, Israel. A significant interaction between vineyard location, season, and variety affected phenology and berry indices. The warmer RN site was generally associated with an advanced phenological course for the white cultivars, which reached harvest up to 2 weeks earlier than at the MR site. The white cultivars also showed stronger correlation between non-consecutive phenological stages than did the red ones. In contrast, harvest time of red cultivars considerably varied according to seasons and sites. Warmer conditions extended fruit developmental phases, causing berry shriveling and cluster collapse in a few cultivars such as Pinot Noir, Ruby Cabernet, and Tempranillo. Analyses of organic acid content suggested differences between red and white cultivars in the content of malate, tartrate, and citrate in response to the temperature difference between sites. However, generally, cultivars at lower temperatures exhibited lower concentrations of pulp organic acids at véraison, but acid degradation until harvest was reduced, compared to the significant pace of acid decline at the warmer site. Sugars showed the greatest differences between sites in both white and red berries at véraison, but differences were seasonal dependent. At harvest, cultivars of both groups exhibited significant variation in hexose/sucrose ratio, and the averages of which varied from 1.6 to 2.9. Hexose/sucrose ratio was significantly higher among the red cultivars at the warmer RN, while this tendency was very slight among white cultivars. White cultivars seem to harbor a considerable degree of resilience due to a combination of earlier and shorter ripening phase, which avoids most of the summer heat. Taken together, our study demonstrates that the extensive genetic capacity of V. vinifera bears significant potential and plasticity to withstand the temperature increase associated with climate change., Competing Interests: AB was employed by Ramat Negev Desert Agro-Desert Research Center, a public research station, which operates under the financial umbrella of the company Ramat Negev Works Ltd., which belongs to the public entity Ramat Negev Regional Authority. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Gashu, Sikron Persi, Drori, Harcavi, Agam, Bustan and Fait.)

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

The aim of this study was to determine the level of clenbuterol residues in muscle tissue of pigs after repeat administration in a growth-promoting dose. An anabolic dose of clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group (n=12) for 28 days, whereas control animals (n=3) were left untreated. Clenbuterol treated pigs were randomly sacrificed (n=3) on days 0, 3, 7 and 14 of treatment discontinuation and clenbuterol residues determined in muscle tissue. Determination of residual clenbuterol was by enzyme-linked immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmation method. The highest clenbuterol content in the muscle of treated animals was recorded on day 0 of treatment cessation (4.40±0.37 ng/g) and significantly (p<0.05) exceeded the maximum residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49±0.22 ng/g and on day 7 0.10±0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content was below the limit of detection (<0.1 ng/g) in all samples. Administration of clenbuterol as a growth promoter in pig production could lead to residues in meat for human consumption up to 7 days after treatment discontinuation., (Copyright © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

Zygophyllum dumosum Boiss. is a perennial Saharo-Arabian phytogeographical element and a dominant shrub on the rocky limestone southeast-facing slopes of the Negev desert. The plant is highly active during the winter, and semideciduous during the dry summer, i.e., it sheds its leaflets, while leaving the thick, fleshy petiole green and rather active during the dry season. Being resistant to extreme perennial drought, Z. dumosum appears to provide an intriguing model plant for studying epigenetic mechanisms associated with drought tolerance in natural habitats. The transition from the wet to the dry season was accompanied by a significant decrease in nuclear size and with posttranslational modifications of histone H3 N-terminal tail. Dimethylation of H3 at lysine 4 (H3K4)--a modification associated with active gene expression--was found to be high during the wet season but gradually diminished on progression to the dry season. Unexpectedly, H3K9 di- and trimethylation as well as H3K27 di- and trimethylation could not be detected in Z. dumosum; H3K9 monomethylation appears to be prominent in Z. dumosum during the wet but not during the dry season. Contrary to Z. dumosum, H3K9 dimethylation was detected in other desert plants, including Artemisia sieberi, Anabasis articulata and Haloxylon scoparium. Taken together, our results demonstrate dynamic genome organization and unique pattern of histone H3 methylation displayed by Z. dumosum, which could have an adaptive value in variable environments of the Negev desert.

The aim of the present study was to evaluate the suitability of dark hair as a matrix for determination of the beta(2)-adrenergic agonist clenbuterol residues using previously validated enzyme-linked immunosorbent assay (ELISA) as a screening method for its quantitative determination. The experimental group of mice (n = 60) were treated with two different anabolic dosages of clenbuterol for 15 days, whereas the control group of animals (n = 30) was left completely untreated. Hair samples were collected on days 0, 5, 10, and 15 of treatment. Validation of the ELISA analytical procedure showed good recovery (mean recovery 74%) with an acceptable intra-assay variation in individual measurements for all hair samples to which 5, 10, and 50 ng/g clenbuterol were added (CV < 10%). Low blank levels of clenbuterol (2.4 +/- 0.6 ng/g) were measured in hair of untreated mice, whereas significantly higher clenbuterol concentrations rising proportionally with the time of treatment were recorded in hair of mice treated with lower (6.5 mg/kg body mass) and higher (12.5 mg/kg body mass) dose of clenbuterol. The peak hair concentration of clenbuterol measured on the last day of treatment (day 15) was 1553.9 +/- 140.1 ng/g and 6248.3 +/- 589.4 ng/g in the lower and higher dose group, respectively. Study results clearly indicated dark hair as a pigmented tissue to have a high accumulation potential for clenbuterol residues, thus being the target matrix of choice for detection of clenbuterol abuse as an anabolic in meat production.

Serine 10 phosphorylation of histone H3 (H3S10ph) has long been considered a mitotic marker, which is often associated with chromosome condensation both in plants and animals. Yet, in animal cells, H3S10ph was found associated with transcriptional activation of genes. Here we extend this view to plant cells showing that H3S10ph not only occurs in dividing cells during mitosis, but also in differentiated mesophyll cells. In these cells H3S10ph displayed a peculiar localization within the nucleolus where it was restricted to specific domains reminiscent of fibrillar centers. Chromatin immunoprecipitation analysis showed that H3S10ph is associated with ribosomal DNAs. Thus, in plants H3S10ph appears to be associated with two structurally differing nuclear sites engaged in gene silencing (mitotic centromeres) and in gene transcription (nucleolus).

The incidence of transitional cell carcinoma (TCC), the fourth most common neoplasm diagnosed in men, is rising. Despite the development of several noninvasive diagnostic tests, none have gained full recognition by the clinicians. Gene expression profiling of tumors can identify new molecular markers for early diagnosis and disease follow-up. It also allows the classification of tumors into subclasses assisting in disease diagnosis and prognosis, as well as in treatment selection. In this paper, we employed expression profiling for molecular analysis of TCC. A TCC-derived cDNA microarray was constructed and hybridized with 19 probes from normal urothelium and TCC tissues. Hierarchical clustering analysis identified all normal urothelium samples to be tightly clustered and separated from the TCC samples, with 29 of the genes significantly induced (t-test, P<10(-5)) in noninvasive TCC compared to normal urothelium. The identified genes are involved in epithelial cells' functions, tumorigenesis or apoptosis, and could become molecular tools for noninvasive TCC diagnosis. Principal components analysis of the noninvasive and invasive TCC expression profiles further revealed sets of genes that are specifically induced in different tumor subsets, thus providing molecular fingerprints that expand the information gained from classical staging and grading.