A new approach towards highly sensitive detection of endogenous N-acetylaspartic acid, N-acetylglutamic acid, and N-acetylaspartylglutamic acid in brain tissues based on strong anion exchange monolith microextraction coupled with UHPLC-MS/MS.

A novel in-tube solid-phase microextraction coupled with an ultra-high performance liquid chromatography-mass spectrometry method has been established for simultaneous quantification of three crucial brain biomarkers N-acetylaspartic acid (NAA), N-acetylglutamic acid (NAG), and N-acetylaspartylglutamic acid (NAAG). A polymer monolith with quaternary ammonium as the functional group was designed and exhibited efficient enrichment of target analytes through strong anion exchange interaction. Under the optimized conditions, the proposed method displayed wide linear ranges (0.1-80 nM for NAA and NAG, 0.2-160 nM for NAAG) with good precision (RSDs were lower than 15%) and low limits of detection (0.019-0.052 nM), which is by far the most sensitive approach for NAA, NAG, and NAAG determination. Furthermore, this approach has been applied to measure the target analytes in mouse brain samples, and endogenous NAA, NAG, and NAAG were successfully detected and quantified from only around 5 mg of cerebral cortex, cerebellum, and hippocampus. Compared with existing methods, the newly developed method in the current study provides highest sensitivity and lowest sample consumption for NAA, NAG, and NAAG measurements, which would potentially be utilized in determining and tracking these meaningful brain biomarkers in diseases or treatment processes, benefiting the investigations of pathophysiology and treatment of brain disorders.

Shifts of soil archaeal nitrification and methanogenesis with elevation in water level fluctuation zone of the three Gorges Reservoir, China.

The water level fluctuation zone is a unique ecological zone exposed to long-term drying and flooding and plays a critical role in the transport and transformation of carbon and nitrogen materials in reservoir-river systems. Archaea are a vital component of soil ecosystems in the water level fluctuation zones, however, the distribution and function of archaeal communities in responde to long-term wet and dry alternations are still unclear. The community structure of archaea in the drawdown areas at various elevations of the Three Gorges Reservoir was investigated by selecting surface soils (0-5 cm) of different inundation durations at three sites from upstream to downstream according to the flooding pattern. The results revealed that prolonged flooding and drying increased the community diversity of soil archaea, with ammonia-oxidizing archaea being the dominant species in non-flooded regions, while methanogenic archaea were abundant in soils that had been flooded for an extended period of time. Long-term alternation of wetting and drying increases methanogenesis but decreases nitrification. It was determined that soil pH, NO3--N, TOC and TN are significant environmental factors affecting the composition of soil archaeal communities (P = 0.02). Long-term flooding and drying changed the community composition of soil archaea by altering environmental factors, which in turn influenced nitrification and methanogenesis in soils at different elevations. These findings contribute to our understanding of soil carbon and nitrogen transport transformation processes in the water level fluctuation zone as well as the effects of long-term wet and dry alternation on soil carbon and nitrogen cycles. The results of this study can provide a basis for ecological management, environmental management, and long-term operation of reservoirs in water level fluctuation zones.

Development of SSR markers and genetic diversity analysis based on RAD-seq technology among Chinese populations of Daphnia magna.

BACKGROUND: Daphnia magna belongs to the Cladocera order and plays an important role in the aquatic ecosystem. With the intensification of water pollution, the wild population of D. magna has declined rapidly in recent years, and insufficient molecular markers have limited effective research and conservation of this species. METHODS AND RESULTS: 26 novel microsatellite (SSR) markers were developed in an artificially domesticated D. magna and 12 wild D. magna populations using restriction site-associated DNA sequencing (RAD-seq). The results showed that the observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.083 to 0.999 and 0.085 to 0.862, respectively. The PIC ranged from 0.368 to 0.805. These results indicate that the developed SSR marker is highly polymorphic. Nei's genetic identity (H) ranged from 0.0926 to 0.3462. Shannon's Information index (I) ranged from 0.1333 to 0.4799. Genetic distance and Nei's genetic identity analysis, NJ tree diagram analysis, and PCoA analysis were conducted on populations of D. magna from different regions. The results show that the D. magna genetic relationship between Liaoning and Shanxi, Hunan and Anhui, and Beijing and Hainan are relatively close, while the genetic structure of D. magna in Guangdong, Jiangsu, and Sichuan is quite different from other sampling sites. An analysis of population genetic structure divided the D. magna samples into two major groups. CONCLUSIONS: These results indicate that the genetic structure of D. magna differs considerably in different regions. Our research results and the newly developed polymorphic SSR markers for D. magna are of great significance in terms of the genetic breeding of D. magna, identification of wild and artificially domesticated populations and conservation genetics research.

Qualitative and quantitative determination of the primary active components and metabolites in human plasma after oral administration of Shuanghuanglian liquid.

Shuanghuanglian oral liquid is a common traditional Chinese medicine used to treat respiratory tract infections. Its major components are baicalin, chlorogenic acid, and forsythin. In this study, the main drug-related components in human plasma after oral administration of Shuanghuanglian were initially identified using ultra-performance liquid chromatography-ultraviolet detector/quadrupole time-of-flight mass spectrometry. Thirteen components from baicalin were identified, including the parent drug baicalin and aglycone baicalein. Only one metabolite related to chlorogenic acid, a sulfate conjugate formed after hydrolysis, and one metabolite related to forsythin, a sulfate conjugate of forsythin aglycone, were detected. Subsequently, a liquid chromatography-tandem mass spectrometry method was established and validated to simultaneously determine baicalin and baicalein, the primary active components. After simple protein precipitation, the analytes were separated on a BEH C18 column using a 5 min-gradient elution to avoid interference from baicalin isomers and their in-source dissociation. Excellent linearity was observed over the concentration ranges of 5.00-2000 ng/ml for baicalin and 1.00-100 ng/ml for baicalein. The validated method was successfully applied to a pharmacokinetic study of an oral administration of 60 ml Shuanghuanglian in healthy subjects. This study provided a foundation to investigate the clinical efficacy and safety of Shuanghuanglian further.