Identification and reactivity of pigments in prominent vegetable leaves of Basella alba L. var. 'Rubra' (Malabar spinach).

The unique profiles of betacyanins as well as their stability and antioxidant activity in purple leaf extracts of the fast-growing, soft-stemmed vine Basella alba L. var. 'Rubra', known as Malabar spinach, are partly characterized for the first time. The distribution of gomphrenin and its acylated derivatives in the leaves is completely different from the profiles of the pigments in the fruits. The most abundant acylated pigment in leaves (24%) turned out 6'-O-E-sinapoyl-gomphrenin (gandolin), however, the most significant difference in the pigment profiles is a presence of two novel pigments tentatively identified as highly abundant 6'-O-(3,4-dimethoxy-E-cinnamoyl)-gomphrenin and 6'-O-(3,4,5-trimethoxy-E-cinnamoyl)-gomphrenin as well as their isoforms. Significant degradation of the pigments in the fruit extracts under the impact of selected metal cations and UV-Vis irradiation as well as high protective activity of the leaf extract matrix were observed. Partial chromatographic purification of the leaf extract resulted in an increase of the pigment concentration which was correlated positively with the increased antioxidant activity of obtained fractions.

Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

Structural Studies on Diverse Betacyanin Classes in Matured Pigment-Rich Fruits of Basella alba L. and Basella alba L. var. 'Rubra' (Malabar Spinach).

Identification of betacyanins in Basella alba L. and Basella alba L. var. 'Rubra' fruits was performed by low- and high-resolution mass spectrometry (LRMS and HRMS) as well as 1H, 13C and two-dimensional NMR which revealed hitherto completely not known betacyanin classes in the plant kingdom. Especially, the presence of unique nitrogenous acyl moieties in the structures of the pigments was ascertained by the HRMS Orbitrap detection. Except for detected polar betacyanin glycosylated derivatives, presence of a series of previously not reported pigments such as malonylated betanidin 6-O-ß-glusosides with their acyl migration isomers along with the evidence of the 3''-hydroxy-butyrylated betacyanins is reported. The first complete NMR data were obtained for novel and principal acylated gomphrenins with hydroxycinnamic acids: 6'-O-E-caffeoyl-gomphrenin (malabarin), 6'-O-E-sinapoyl-gomphrenin (gandolin), 6'-O-E-4-coumaroyl-gomphrenin (globosin) and 6'-O-E-feruloyl-gomphrenin (basellin).

Phytochemical Molecules from the Decarboxylation of Gomphrenins in Violet Gomphrena globosa L.-Floral Infusions from Functional Food.

Herein, the generation of decarboxylated derivatives of gomphrenin pigments exhibiting potential health-promoting properties and the kinetics of their extraction during tea brewing from the purple flowers of Gomphrena globosa L. in aqueous and aqueous citric acid solutions were investigated. Time-dependent concentration monitoring of natural gomphrenins and their tentative identification was carried out by LC-DAD-ESI-MS/MS. The high content of acylated gomphrenins and their principal decarboxylation products, 2-, 15-, 17-decarboxy-gomphrenins, along with minor levels of their bidecarboxylated derivatives, were reported in the infusions. The identification was supported by the determination of molecular formulas of the extracted pigments by liquid chromatography coupled with high-resolution mass spectrometry (LCMS-IT-TOF). The influence of plant matrix on gomphrenins' stability and generation of their derivatives, including the extraction kinetics, was determined by studying the concentration profiles in the primary and diluted infusions. Isolated and purified acylated gomphrenins from the same plant material were used for the preliminary determination of their decarboxylated derivatives. The acylated gomphrenins were found to be more stable than nonacylated ones. Citric acid addition had a degradative influence on natural gomphrenins mainly during the longer tea brewing process (above 15 min); however, the presence of plant matrix significantly increased the stability for betacyanins' identification.

Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia: data from the randomised UK LRF CLL4 trial.

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

Micropropagation of Rubus and Ribes spp.

Micropropagation is the most appropriate method for large-scale production of Rubus and Ribes spp. The proliferation rate of Rubus spp. differs in shoot tips and nodal segments. The culture media used for raspberry and blackberry propagation are MS-based supplemented with different combination and ratio of plant growth regulators, depending on the stage of culture. The initiation medium containing 0.4 mg L(-1) BA and 0.1 mg L(-1) IBA is used to stabilize shoot cultures. In multiplication media, concentration of cytokinin is doubled. In vitro rooting of shoots is achieved on media supplemented with 1.0 mg L(-1) IBA. Ribes spp. cultures are initiated from shoot tips, meristem, or dormant buds on MS medium supplemented with 2.0 mg L(-1) BA, 0.5 mg L(-1) IBA, and 0.1 mg L(-1) GA(3.) After stabilization of shoot cultures in 3-4-week time, shoot multiplication is carried out on MS medium containing 1.0 mg L(-1) BA and 0.1 mg L(-1) IBA. Shoots 2 cm long are cultured to rooting on a medium amended with 2.0 mg L(-1) IBA and 5.0 mg L(-1) IAA. Rooted plantlets are transferred to universal peat substrate and acclimatized in the greenhouse.

Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein.

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ(70), σ(S), σ(54), σ(32), σ(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σ(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σ(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency.