Characterization of the Key Compounds of Bell Pepper by Spectrophotometry and Gas Chromatography on the Effects of Induced Stress on the Concentration of Secondary Metabolite.

Sweet peppers are consumed worldwide, and traditional uses have sparked interest in their applications as dietary antioxidants, which can be enhanced in plants using elicitors. These are endowed with phytochemicals with potential health benefits such as antioxidants, bioavailability, and bioaccessibility. The trend in metabolomics shows us chemical fingerprints linking metabolomics, innovative analytical form, and bioinformatics tools. The objective was to evaluate the impact of multiple stress interactions, elicitor concentrations, and electrical conductivity on the concentration of secondary metabolites to relate their response to metabolic pathways through the foliar application of a cocktail of said elicitors in pepper crops under greenhouse conditions. The extracts were analyzed by spectrophotometry and gas chromatography, and it was shown that the PCA analysis identified phenolic compounds and low molecular weight metabolites, confirming this as a metabolomic fingerprint in the hierarchical analysis. These compounds were also integrated by simultaneous gene and metabolite simulants to obtain effect information on different metabolic pathways. Showing changes in metabolite levels at T6 (36 mM H2O2 and 3.6 dS/m) and T7 (0.1 mM SA and 3.6 dS/m) but showing statistically significant changes at T5 (3.6 dS/m) and T8 (0.1 mM SA, 36 mM H2O2, and 3.6 dS/m) compared to T1 (32 dS/m) or control. Six pathways changed significantly (p < 0.05) in stress-induced treatments: aminoacyl t-RNA and valine-leucine-isoleucine biosynthesis, and alanine-aspartate-glutamate metabolism, glycoxylate-dicarboxylate cycle, arginine-proline, and citrate. This research provided a complete profile for the characterization of metabolomic fingerprint of bell pepper under multiple stress conditions.

Pitahaya Peel: A By-Product with Great Phytochemical Potential, Biological Activity, and Functional Application.

Hylocereus spp. present two varieties of commercial interest due to their color, organoleptic characteristics, and nutritional contribution, such as Hylocerous polyrhizus and Selenicerus undatus. The fruit recognized as dragon fruit or Pitahaya is an exotic fruit whose pulp is consumed, while the peel is discarded during the process. Studies indicate that the pulp has vitamin C and betalains, and seeds are rich in essential fatty acids, compounds that can contribute to the prevention of chronic non-communicable diseases (cancer, hypertension, and diabetes). In the present study, polyphenolic compounds, biological activity, and fatty acids present in the peel of the two varieties of pitahaya peel were evaluated, showing as a result that the variety S. undatus had higher antioxidant activity with 51% related to the presence of flavonoids 357 mgRE/g sample and fatty acids (hexadecanoic acid and linoleate) with 0.310 and 0.248 mg AG/g sample, respectively. On the other hand, H. polyrhizuun showed a significant difference in the inhibitory activity of amylase and glucosidase enzymes with 68% and 67%, respectively. We conclude that pitahaya peel has potential health effects and demonstrate that methylated fatty acids could be precursors to betalain formation, as well as showing effects against senescence and as a biological control against insects; in the same way, the peel can be reused as a by-product for the extraction of important enzymes in the pharmaceutical and food industry.

Changes in the Content of Phenolic Compounds and Biological Activity in Traditional Mexican Herbal Infusions with Different Drying Methods.

Mexican spices are used in the supplementation of the human diet and as medicinal herbs for the particularly high amounts of compounds capable of deactivating free radicals. In addition, these spices can have beneficial effects on chronic, no-transmissible diseases such as type II diabetes and hypertension arterial. The objective of this study is to determine the content of phenolic compounds on the antioxidant activity and inhibitory enzymes of α-amylase, α-glucosidase and angiotensin-converting enzyme in melissa, peppermint, thyme and mint, which are subjected to microwave drying, conventional and freeze-drying to be used as alternative treatments. Spices were evaluated to determine total phenols, flavonoids, tannins, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), (2,2'-azino-bis- (3-ethyl benzothiazolin-6-ammonium sulphonate) (ABTS) and Ferric Reducing/Antioxidant Power (FRAP), enzymatic activity. The investigation showed that conventional drying caused a decrease in antioxidant properties and inhibitory activity, in some species, while remained preserved in microwave drying and freeze-drying. The activity of polyphenol oxides and peroxidase decreases with high temperatures and these increase with the use of cold temperatures. This study aims to determine the extent of optimal drying required to preserve phenolic compounds, and the positive effect on antioxidant activity and enzymatic activity in in vitro models, which will produce benefits for the infusion processing industry and the pharmaceutical industry.

Real-time cellular analysis as a novel approach for in vitro cytotoxicity testing of medical device extracts.

Cytotoxicity measurements are often performed to evaluate the biocompatibility of medical device materials. Here, we describe the use of a real-time cell analyzer (RTCA) system for the investigation of biocompatibility of medical devices by comparing RTCA results to two distinct methods described in the International Organization for Standardization (ISO) guidelines. Mouse L-929 fibroblast proliferation was assessed every 15 min from 24 to 100 h during the pretreatment and postextract addition period. Simultaneously, we performed quantitative cytotoxicity analyses using water-soluble tetrazolium salt (WST-1) and qualitatively scored cytotoxicity by examining changes in morphology at 24-h intervals. The RTCA uses electrical impedance to measure cell viability quantified as a normalized cellular index (CI) which was converted in this study to a reactivity grade. Results from microscopic analyses were expressed as a reactivity grade, based on morphology as defined by the ISO 10993-5:2009. There was a clear correlation between addition of cytotoxic agents and, both, decreased normalized CI and concomitant cell layer destruction observed by microscopy. Results obtained from the colorimetric WST-1 assays also correlated with normalized CI at various time points tested. The results indicate that RTCA allows for automated and accurate assessment of biocompatibility of medical devices and biomaterials.

MRGing chromatin dynamics and cellular senescence.

Normal primary cells have a finite ability to divide in culture and after a number of population doublings enter a state of irreversible cell cycle arrest known as replicative senescence. Several cellular stresses have been shown to induce a senescence-like growth arrest including shortened telomeres, DNA-damaging stresses, and drastic changes in chromatin structure, for example, through histone deacetylase (HDAC) induction. Histones are core components of chromatin which are subject to a number of chemical modifications that influence the dynamic state of chromatin structure. Proper chromatin structure formation is crucial for most DNA-dependent processes including transcription, replication, and repair which have a profound impact on cellular proliferation and senescence. Several genes important for chromatin remodeling such as the tumor suppressors p53 and retinoblastoma (Rb) affect cellular senescence by mediating changes in chromatin structure and gene expression. The Morf4-Related Gene (MRG) family of transcription factors forms stable interactions with chromatin-modifying complexes including histone acetyltransferase (HAT) and HDAC complexes and interact with Rb. Further, the MRG family was founded by a gene, Mortality Factor on Chromosome 4, capable of inducing senescence in immortalized cell lines. In this paper, we review the role of the MRG family of proteins in chromatin dynamics and cellular senescence.

Slx5 promotes transcriptional silencing and is required for robust growth in the absence of Sir2.

The broadly conserved Sir2 NAD(+)-dependent deacetylase is required for chromatin silencing. Here we report the discovery of physical and functional links between Sir2 and Slx5 (Hex3), a RING domain protein and subunit of the Slx5/8 complex, [corrected] which is a ubiquitin E3 ligase that targets sumoylated proteins. Slx5 interacted with Sir2 by two-hybrid and glutathione S-transferase-binding assays and was found to promote silencing of genes at telomeric or ribosomal DNA (rDNA) loci. However, deletion of SLX5 had no detectable effect on the distribution of silent chromatin components and only slightly altered the deacetylation of histone H4 lysine 16 at the telomere. In vivo assays indicated that Sir2-dependent silencing was functionally intact in the absence of Slx5. Although no previous reports suggest that Sir2 contributes to the fitness of yeast populations, we found that Sir2 was required for maximal growth in slx5Delta mutant cells. A similar requirement was observed for mutants of the SUMO isopeptidase Ulp2/Smt4. The contribution of Sir2 to optimal growth was not due to known Sir2 roles in mating-type determination or rDNA maintenance but was connected to a role of sumoylation in transcriptional silencing. These results indicate that Sir2 and Slx5 jointly contribute to transcriptional silencing and robust cellular growth.

Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit DNA-repair defects post exposure to gamma ionizing radiation.

MORF4-related gene on chromosome 15 (MRG15) is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA-damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA-repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA-damage repair in mammalian cells.

Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing. To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant Drosophila Sir2 (dSir2). First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues. We then carried out in vitro transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin assembled from native (and mostly unacetylated) histones. It was possible, however, that repression by dSir2 requires an acetylated histone substrate. We therefore tested the transcriptional effects of dSir2 with native histones that were hyperacetylated by treatment with acetic anhydride. Assembly of the hyperacetylated histones onto DNA yields a soluble histone-DNA complex that differs from canonical nucleosomal chromatin. With this hyperacetylated histone-DNA complex, we observed potent (50- to 100-fold) NAD-dependent transcriptional repression by purified dSir2. In contrast, repression by dSir2 was not observed in parallel experiments in which histones were hyperpropionylated with propionic anhydride. We also found that dSir2 mediates the formation of a nuclease-resistant fast-sedimenting histone-DNA complex in an NAD-dependent manner. Unlike dSir2, the dHDAC1 deacetylase does not strongly repress transcription or generate a nuclease-resistant histone-DNA complex. Furthermore, with yeast Sir2, the transcriptional repression we observe correlates with deacetylation activity in vitro and silencing activity in vivo. These findings suggest that deacetylation by Sir2 causes a conformational change or rearrangement of histones into a transcriptionally repressive chromatin structure.

A unique class of conditional sir2 mutants displays distinct silencing defects in Saccharomyces cerevisiae.

Silencing provides a critical means of repressing transcription through the assembly and modification of chromatin proteins. The NAD(+)-dependent deacetylation of histones by the Sir2p family of proteins lends mechanistic insight into how SIR2 contributes to silencing. Here we describe three locus-specific sir2 mutants that have a spectrum of silencing phenotypes in yeast. These mutants are dependent on SIR1 for silencing function at the HM silent mating-type loci, display distinct phenotypes at the rDNA, and have dominant silencing defects at the telomeres. Telomeric silencing is restored if the mutant proteins are directly tethered to subtelomeric regions, via a Gal4p DNA-binding domain (GBD), or are recruited by tethered GBD-Sir1p. These sir2 mutations are found within conserved residues of the SIR2 family and lead to defects in catalytic activity. Since one of the mutations lies outside the previously defined minimal catalytic core, our results show that additional regions of Sir2p can be important for enzymatic activity and that differences in levels of activity may have distinct effects at the silenced loci.