Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Identification and characterization of reaction products of 5-hydroxytryptamine with methylglyoxal and glyoxal by liquid chromatography/tandem mass spectrometry.

RATIONALE: Methylglyoxal (MGO) and glyoxal (GO) are known to be at high levels in humans with diabetes. They react with amine-containing proteins and amino acids to form advanced glycation end products, however, their reactivity with other amine-containing metabolites, such as neurotransmitters, has not been explored. In this study, we aimed at studying the reactivity of 5-hydroxytryptamine (5-HT) with MGO or GO, which may alter the metabolic function of 5-HT. METHODS: Stock solutions of 5-HT, MGO and GO were made in PBS buffer at pH 7.4 and 5-HT was incubated with MGO or GO at different concentrations. The reactions were also performed at physiological concentrations. The reaction mixtures collected at different incubation times were analyzed by direct ESI-HRMS, LC/MS and LC/MS/MS to detect/characterize the products. Agilent 6545 Q-TOF and Agilent 6420 triple quadrupole mass spectrometers were used for the study, and LC separations were performed on a C18 column. RESULTS: The direct ESI-HRMS data of the reaction mixtures showed formation of three and four reaction products when 5-HT was reacted with MGO and GO, respectively. All the products showed dominant [M + H]+ ions. The products were characterized by HRMS, LC/MS/MS and literature reports on similar compounds. The products can easily be identified by LC/MS based on the accurate mass values together with retention time information. The MS/MS of the reaction products showed structure-indicative fragment ions. CONCLUSIONS: 5-HT reacts with one or two MGO/GO to form a set of reaction products. The reaction between 5-HT and MGO or GO was faster at higher concentrations of MGO/GO (<10 min), and the same products were found even at physiological concentrations (<48 h). The LC/MS/MS (SRM) method can be used to screen the reaction products when present at low levels.

Differential Cationization of Fatty Acids with Monovalent Cations Studied by ESI-MS/MS and Computational Approach.

RATIONALE: The intercellular and intracellular transport of charged species (Na+ /K+ ) entail interaction of the ions with neutral organic molecules and formation of adduct ions. The rate of transport of the ions across the cell membrane(s) may depend on the stability of the adduct ions, which in turn rely on structural aspects of the organic molecules that interact with the ions. METHODS: Positive ion ESI mass spectra were recorded for the solutions containing fatty acids (FAs) and monovalent cations (X=Li+ , Na+ , K+ , Rb+ and Cs+ ). Product ion spectra of the [FA+X]+ ions were recorded at different collision energies. Theoretical studies were exploited under both gas phase and solvent phase to investigate the structural effects of the fatty acids during cationization. Stability of [FA+X]+ adduct ions were further estimated by means of AIM topological analyses and interaction energy (IE) values. RESULTS: Positive ion ESI-MS analyses of the solution of FAs and X+ ions showed preferential binding of the K+ ions to FAs. The K+ ion binding increased with the increase in number of double bonds of FAs, while decreased with increase in the number of carbons of FAs. Dissociation curves of [FA+X]+ ions indicated the relative stability order of the [FA+X]+ ions and it was in line with the observed trends in ESI-MS. The solvent phase computational studies divulged the mode of binding and the binding efficiencies of different FAs with monovalent cations. CONCLUSIONS: Among the studied monovalent cations, the cationization of FAs follow the order K+ >>Na+ >Li+ >Rb+ >Cs+ . The docosahexaenoic acid showed high efficiency in binding with K+ ion. The K+ ion binding efficiency of FAs depends on the number of double bonds in unsaturated FAs and the carbon chain length in saturated FAs. The cationization trends of FAs obtained from the ESI-MS, ESI-MS/MS analyses were in good agreement with solvent phase computational studies.

Characterization of N-methylated amino acids by GC-MS after ethyl chloroformate derivatization.

Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α-amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N-methyl and N,N-dimethyl amino acids were synthesized by the methylation of α-amino acids and characterized by a GC-MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC-MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N-methyl (1-18) and N,N-dimethyl amino acids (19-35) showed abundant [M-COOC2 H5 ]+ ions. The fragment ions due to loss of C2 H4 , CO2 , (CO2 + C2 H4 ) from [M-COOC2 H5 ]+ were of structure indicative for 1-18. The EI spectra of 19-35 showed less number of fragment ions when compared with those of 1-18. The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H]+ ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds (1-35) were confirmed by high-resolution mass spectra data and further substantiated by the data obtained from 13 C2 -labeled glycines and N-ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.

Closed mitral valvotomy during pregnancy. A 20-year experience.

Closed mitral valvotomy for rheumatic mitral stenosis was performed on 126 pregnant women (average duration of pregnancy c. 21 weeks), 91% of whom were in NYHA functional class III or IV. Associated functional tricuspid regurgitation was present in 47 (37%) of the women, and 102 (81%) had critical mitral stenosis (digitally assessed valve area less than 1 cm2). There was no surgical mortality. Postoperatively 84% of the women were in NYHA class I. Clinical evidence of pulmonary artery hypertension and tricuspid regurgitation regressed postoperatively in most patients. Full-term normal delivery was achieved in 82% of the pregnancies, with total fetal mortality 6%. There were no congenital abnormalities and the infants' progress was normal. At 5-year follow-up 86% of the women were in NYHA class I or II and at 10 years the figure was 60%. The restenosis rate was 2%/year and the late mortality 3.3%. Closed mitral valvotomy during pregnancy thus was safe and reliable, giving significant functional and clinical improvement without adversely affecting the fetus.

Pregnancy in patients with prosthetic cardiac valve. A 10-year experience.

Pregnancy after valve replacement has been considered hazardous because of maternal and fetal complications secondary to anticoagulant medication, in addition to basic myocardial problems. Of 229 females aged 15-45 years with prosthetic valve replacement, 37 (including 34 with Björk-Shiley valve and anticoagulants) subsequently had a total of 47 pregnancies. Fullterm delivery of a normal infant was achieved in 40 cases. There were three premature births, two spontaneous abortions, one stillbirth and one ectopic pregnancy. The fetal mortality was 8.5%. Valve thrombosis developed in two cases, but surgical treatment was successful. Oral anticoagulants (acenocoumarin and dipyridamole) were continued throughout pregnancy. Heparin was substituted before labour began, but discontinued after delivery, when effective oral anticoagulation was resumed. Our experience showed that pregnancy in women with mechanical heart valve prosthesis and continued oral intake of anticoagulants is safe and successful in most cases.