Glutamine metabolism in fluorosis: Integrated metabolomics and transcriptomics analysis.

OBJECTIVE: To identify the potential metabolic biomarkers of fluorosis and the pathogenesis of fluorosis. METHODS: Sprague Dawley rats in this study were randomly divided into fluoride exposure and control groups. In the fluoride exposure group, six offspring rats without dental fluorosis were defined as group A, and six offspring rats with dental fluorosis were defined as group C. Eight offspring rats in the control group were defined as group B. The metabolites in plasma were determined using GC-MS, with differential metabolites (DMs) identified using VIP > 1, and P < 0.05. Cluster analysis, KEGG pathway enrichment analysis and Receiver Operating Characteristic (ROC) analysis were subsequently performed. The DMs which were caused by fluoride exposure in the previous study were used to verify our results. The GSE70719 from GEO database were used to support this research at the mRNA level and in vitro experiment were selected to verify above results. RESULTS: The 13 up-regulated and 4 down-regulated DMs were identified in the group A + C, the 18 up-regulated and 4 down-regulated DMs were identified in group A, and the 12 up-regulated and 2 down-regulated DMs were identified in group C. All groups showed enrichment in Aminoacyl-tRNA synthesis, D-glutamine and D-glutamate metabolism, Nitrogen metabolism, and Purine metabolism pathways. ROC analysis revealed that L-glutamine had excellent diagnostic ability for fluorosis (AUC > 0.85, P < 0.05). Changes in major DMs (L-glutamine, 4-hydroxyproline and L-alanine) were consistent with previous findings. Transcriptomic results showed the significant alteration of GLS gene in the fluoride exposure group. In vitro experiments confirmed decreased GLS and SLC1A5 genes expression. CONCLUSION: L-glutamine emerges as a potential biomarker for fluorosis. Glutamine metabolism was involved in the pathogenesis of fluorosis.

Mediation of mitochondrial DNA copy number and oxidative stress in fluoride-related bone mineral density alteration in Chinese farmers.

Excessive fluoride can adversely affect bone mineral density (BMD). Oxidative stress and mitochondrial dysfunction are crucial mechanisms of health damage induced by fluoride. Here, a cross-sectional survey involving 907 Chinese farmers (aged 18-60) was carried out in Tongxu County in 2017, aiming to investigate the significance of mitochondrial DNA copy number (mtDNAcn) and oxidative stress in fluoride-related BMD change. Concentrations of urinary fluoride (UF), serum oxidative stress biomarkers, including total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), as well as relative mtDNAcn in peripheral blood were determined. The multivariable linear model and mediation analysis were performed to assess associations between UF, oxidative stress, and relative mtDNAcn with BMD. Results showed that GSH-Px levels increased by 6.98 U/mL [95% confidence interval (CI) 3.41-10.56)] with each 1.0 mg/L increment of UF. After stratification, the T-AOC, relative mtDNAcn, and BMD decreased by 0.04 mmol/L (-0.08 ~ -0.01), 0.29-unit (-0.55 ~ -0.04), and 0.18-unit (-0.33 ~ -0.03) with every 1.0 mg/L elevation of UF in the excessive fluoride group (EFG, adults with UF > 1.6 mg/L), respectively. Furthermore, T-AOC and relative mtDNAcn were favorably related to the BMD in the EFG (ß = 0.82, 95%CI 0.16-1.48 for T-AOC; ß = 0.11, 95%CI 0.02-0.19 for relative mtDNAcn). Mediation analysis showed that relative mtDNAcn and T-AOC mediated 15.4% and 17.1% of the connection between excessive fluoride and reduced BMD, respectively. Findings suggested that excessive fluoride was related to lower BMD in adults, and the decrement of T-AOC and relative mtDNAcn partially mediate this relationship.

Correlations between bone metabolism biomarkers and fluoride exposure in adults and children.

Increased exposure to fluoride, which notably affects bone metabolism, is a global concern. However, the correlations and sensitivity of bone metabolism to fluoride remain controversial. In this cross-sectional study, 549 children (aged 7-12 years) and 504 adults (≥ 18 years old) were recruited in the high-fluoride areas of the Henan Province. Urinary fluoride (UF) level was determined using a fluoride electrode. Fasting venous blood serum was collected to measure bone metabolism biomarkers. The selected bone metabolism biomarkers for children included bone alkaline phosphatase (BALP), serum alkaline phosphatase (ALP), osteocalcin (OCN), calcitonin (CT), parathyroid hormone (PTH), phosphorus (P5+), and calcium (Ca2+). For adults, the biomarkers included ALP, CT, PTH, ß-CrossLaps (ß-CTX), P5+, and Ca2+. The correlations between UF and bone metabolism biomarkers were analyzed using binary logistic regression, a trend test, a generalized additive model, and threshold effect analysis. Regression analysis indicated a significant correlation between serum OCN, PTH, and UF levels in children aged 7-9 years. Serum OCN, PTH, and BALP contents were significantly correlated with UF in boys (P < 0.05). Furthermore, the interaction between age and UF affected serum P5+ and PTH (P < 0.05). The generalized additive model revealed nonlinear dose-response relationships between P5+, BALP, and UF contents in children (P < 0.05). Serum OCN level was linearly correlated with the UF concentration (P < 0.05). Similarly, a significant correlation was observed between ß-CTX and UF levels in adults. In addition, significant correlations were observed between UF-age and serum Ca2+, ß-CTX, and PTH contents. There was a non-linear correlation between serum Ca2+, P5+, and ß- CTX and UF levels (P < 0.05). Overall, serum OCN, BALP, and P5+ levels can serve as sensitive bone metabolism biomarkers in children, while ß-CTX, P5+, and Ca2+ can be considered fluoride-sensitive bone metabolism biomarkers in adults.

RcMYB8 enhances salt and drought tolerance in rose (Rosa chinensis) by modulating RcPR5/1 and RcP5CS1.

Plant Myeloblastosis (MYB) proteins function crucially roles upon variegated abiotic stresses. Nonetheless, their effects and mechanisms in rose (Rosa chinensis) are not fully clarified. In this study, we characterized the effects of rose RcMYB8 under salt and drought tolerances. For induction of the RcMYB8 expression, NaCl and drought stress treatment were adopted. Rose plants overexpressing RcMYB8 displayed enhanced tolerance to salinity and drought stress, while silencing RcMYB8 resulted in decreased tolerance, as evidenced by lowered intra-leaf electrolyte leakage and callose deposition, as well as photosynthetic sustainment under stressed conditions. Here, we further show that RcMYB8 binds similarly to the promoters of RcPR5/1 and RcP5C51 in vivo and in vitro. Inhibiting RcP5CS1 by virus-induced gene silencing led to decreased drought tolerance through the reactive oxygen species (ROS) homeostatic regulation. RcP5CS1-silenced plants showed an increase in ion leakage and reduce of proline content, together with the content of malondialdehyde (MDA) increased, lowered activities of Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Our study highlights the transcriptional modulator role of RcMYB8 in drought and salinity tolerances, which bridges RcPR5/1 and RcP5CS1 by promoting ROS scavenging. Besides, it is probably applicable to the rose plant engineering for enhancing their abiotic stress tolerances.

Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening method coupled with an aldehyde dehydrogenase.

A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERAT29L and LbDERAF163Y, were identified and combined, giving a double mutant LbDERAT29L/F163Y which showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERAT29L/163Y obtained by X-ray diffraction with 1.77 Å resolution revealed the structural changes responsible for the significant activity improvement.