Single-cell calcium monitoring of Caco-2 cell co-cultured with intestinal microbiome through carbon fiber based potentiometric microelectrode.

The Caco-2 cells were used as intestinal epithelial cell model to illustrate the hyperuricemia (HUA) mechanism under the co-culture of the imbalanced intestinal microbiome in this work. The uric acid (UA) concentration in the HUA process was monitored, and could be up to 425 µmol/L at 8 h co-cultured with the imbalanced intestinal microbiome. Single-cell potentiometry based on ion-selective microelectrode was used to study extracellular calcium change, which is hypothesized to play an important role in the UA excretion. The potential signal of the calcium in the extremely limited microenvironment around single Caco-2 cell was recorded through the single-cell analysis platform. The potential signal of sharp decrease and slow increase followed within a few seconds indicates the sudden uptake and gradually excretion process of calcium through the cell membrane. Moreover, the value of the potential decrease increases with the increase of the time co-cultured with the imbalanced intestinal microbiome ranging from 0 to 8 h. The Ca2+ concentration around the cell membrane could decrease from 1.3 mM to 0.4 mM according to the potential decrease of 27.0 mV at the co-culture time of 8 h. The apoptosis ratio of the Caco-2 cells also exhibits time dependent with the co-culture of the imbalanced intestinal microbiome, and was 39.1 ± 3.6 % at the co-culture time of 8 h, which is much higher than the Caco-2 cells without any treatment (3.9 ± 2.9 %). These results firstly provide the links between the UA excretion with the apoptosis of the intestinal epithelial cell under the interaction of the imbalanced intestinal microbiome. Moreover, the apoptosis could be triggered by the calcium signaling.

Influence of Condensed and Hydrolysable Tannins on the Bacterial Community, Protein Degradation, and Fermentation Quality of Alfalfa Silage.

This study evaluated the effects of hydrolysable tannin (HT) and condensed tannin (CT) on the bacterial community, fermentation quality, and proteolysis of alfalfa silage. Alfalfa was wilted to a dry matter (DM) of 35% fresh weight and ensiled with or without 4% HT or 4% CT. The application rates of tannins were based on fresh weight, and each treatment was ensiled in triplicate. After 60 d of fermentation, the CT-treated group had lower concentrations of ammonia nitrogen (NH3-N) and free amino acid nitrogen (AA-N), but greater lactic acid concentration, than those in the control and HT-treated silage (p < 0.05). Compared to the control group, the application of tannins increased the abundance of Pseudomonas (negatively correlated with aminopeptidases activity), and decreased the abundance of Pediococcus­which was positively correlated with aminopeptidases activity­and the concentrations of non-protein nitrogen (NPN), NH3-N, and AA-N. The application of HT decreased the abundance of Lactobacillus and increased the abundances of Enterococcus, while the opposite results were observed in the CT-treated group. The application of HT and CT reduced the proteolysis in treated silages, but the two were different in terms of their mechanism and their effects on the bacterial communities of the alfalfa silage.

Involvement of CitCHX and CitDIC in developmental-related and postharvest-hot-air driven citrate degradation in citrus fruits.

Citrate is the predominant organic acid associated with taste in citrus fruit. Although citrate metabolism has been widely studied in recent years, the potential contributions of transport proteins to citrate content remain unclear. In the present study, high-acid citrus fruit Gaocheng ('GC', Citrus sp.) and low-acid citrus fruit Satsuma mandarin ('SM', Citrus unshiu Marc.) were selected for study, and the degradation of citrate was deduced to be the main cause of the difference in acidity in fully mature fruits. RNA-seq analysis was carried out on 'GC' and 'SM' fruit samples over the same time course, and the results indicated that citrate degradation occurred mainly through the glutamine pathway, catalyzed by CitAco3-CitGS2-CitGDU1, and also two transport-related genes, CitCHX and CitDIC, were shown to be associated with citrate degradation. These results were confirmed by real-time PCR. In postharvest 'GC' fruit, the expressions of these two transport-related genes were induced by 2-fold under hot air treatment, accompanied by a reduction of 7%-9% in total acid degradation. Transient expression of CitCHX and CitDIC in tobacco leaves was performed, and the citrate content was reduced by 62%, 75% and 78% following CitCHX, CitDIC and CitCHX plus CitDIC treatments, respectively, as compared with expression of an empty vector. Overall, these data indicated that two transport proteins, CitCHX and CitDIC, are not only involved in citrate degradation during fruit development, but also involved in postharvest hot air triggered citrate reduction.

Transcriptome and metabolome analyses of sugar and organic acid metabolism in Ponkan (Citrus reticulata) fruit during fruit maturation.

Ponkan (Citrus reticulata Blanco cv. Ponkan) is an important mandarin citrus in China. However, the low ratio of sugars to organic acids makes it less acceptable for consumers. In this work, three stages (S120, early development stage; S195, commercial harvest stage; S205, delayed harvest stage) of Ponkan fruit were selected for study. Among 28 primary metabolites analyzed in fruit, sugars increased while organic acids in general decreased. RNA-Seq analysis was carried out and 19,504 genes were matched to the Citrus clementina genome, with 85 up-regulated and 59 down-regulated genes identified during fruit maturation. A sucrose phosphate synthase (SPS) gene was included in the up-regulated group, and this was supported by the transcript ratio distribution. Expression of two asparagine transferases (AST), and a specific ATP-citrate lyase (ACL) and glutamate decarboxylase (GAD) members increased during fruit maturation. It is suggested that SPS, AST, ACL and GAD coordinately contribute to sugar accumulation and organic acid degradation during Ponkan fruit maturation. Both the glycolysis pathway and TCA cycle were accelerated during later maturation, indicating the flux change from sucrose metabolism to organic acid metabolism was enhanced, with citrate degradation occurring mainly through the gamma-aminobutyric acid (GABA) and acetyl-CoA pathways.