Verification of DNA motifs in Arabidopsis using CRISPR/Cas9-mediated mutagenesis.

Transcription factors (TFs) and chromatin-modifying factors (CMFs) access chromatin by recognizing specific DNA motifs in their target genes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has been widely used to discover the potential DNA-binding motifs for both TFs and CMFs. Yet, an in vivo method for verifying DNA motifs captured by ChIP-seq is lacking in plants. Here, we describe the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to verify DNA motifs in their native genomic context in Arabidopsis. Using a single-guide RNA (sgRNA) targeting the DNA motif bound by REF6, a DNA sequence-specific H3K27 demethylase in plants, we generated stable transgenic plants where the motif was disrupted in a REF6 target gene. We also deleted a cluster of multiple motifs from another REF6 target gene using a pair of sgRNAs, targeting upstream and downstream regions of the cluster, respectively. We demonstrated that endogenous genes with motifs disrupted and/or deleted become inaccessible to REF6. This strategy should be widely applicable for in vivo verification of DNA motifs identified by ChIP-seq in plants

Association of a single nucleotide polymorphism of the Leptin gene with growth traits in Takifugu rubripes

Leptin (LEP) plays a role in animal growth, appetite regulation, energy partition, and body composition. In this study, 296 cultivated Takifugu rubripes individuals were screened for LEP gene polymorphisms using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. Two single nucleotide polymorphism (SNP) mutations were detected in T. rubripes LEP gene: C/G at position 232 nucleotide (nt) and C/T at position 310 nt. One-way ANOVA of the SNPs and growth traits showed that genotype CC of primer L2 had a significantly higher body length (BL), body weight (BW), and absolute growth rate of BL (ABL) and BW (ABW) than genotypes CG and GG (P<0.05). The genotype frequencies of CC, CG, and GG were 61.15%, 35.14%, and 3.17%, respectively. Genotype CC at the C232G site was advantageous and significantly associated with growth traits. This site would be an excellent molecular marker to select and cultivate through statistical analysis. Additionally, the second unmutated and mutated mRNA structures were compared and analyzed. These results indicate that the T. rubripes LEP gene may be a candidate for these growth traits

How Consumers' Need for Variety and Social Consumption Influences Festival Patronage and Spending

This paper investigates the influence of motivational goals such as variety seeking and social consumption on consumers’ patronage and spending at craft beer festivals. In doing so, we develop and test a number of hypotheses by examining information collected via means of a survey questionnaire proposed in 2017 to visitors of a large beer festival in the UK. Findings of our analysis unveil how cognitive engagement affects individuals’ behavior with regard to responding to and financially engage with beer festivals. Results also identify cognitive engagement as an important mediator of the effects related to variety-seeking and social consumption. From a managerial perspective, findings reveal important attributes affecting consumers’ drivers towards craft beers, contributing to understand which dimension of consumer engagement influence their behaviors. Overall, the study provides fresh empirical evidence in terms of identifying and recognizing consumers’ behaviors with regard to defining future trends in the craft beer sector

Driving forces and impacts of food system nitrogen flows in China, 1990 to 2012

Food nitrogen (N), which includes animal-food (AN) and plant-food N (PN), has been driven by population growth (PG), dietary changes associated with incomegrowth (DC) andrural-urban migration (M) over the past three decades, andthese changes combined with theirNcost, have caused some effect onNuse in China's food system. Although there is an increasing literature on food N and its environmental impacts in China, the relative magnitude of these driving forces are notwell understood. Here we first quantify the differences in per capita AN and PNconsumption in urban and rural areas and their impacts on N input to the food system during 1990–2012, and then quantify the relative contributions of DC, PG andMin the overall N change during this period. Our results show that a resident registered as living in city required 0.5 kg more AN yr−1 and 0.5 kg less PN yr−1 than one living in a rural area, in 2012. DC, PG and M accounted for 52%, 31% and 17% of the total AN increase, respectively. These three factors caused 46% of the increased N use for food production over the past two decades. Another 54% was mainly caused by the declining in N use efficiencies of the food system. Foodsourced N loss intensity in urban and rural areas were 502 and 162 kg N hm−2 in 2012, a three-fold difference due to the increasing amount and a linear rural-urbanflowofNinput, and inadequateNrecovery via solid waste and wastewater treatment in cities. Our study highlights China is facing higher risks of environmental N pollution with urbanization, because of the high demand for AN and higher food-sourced N loss intensity in urban than in rural areasThis manuscript is based on the several projects, sponsored by the National Basic Research Program of China (2014CB953801), the Young Talents Projects of the Institute of Urban Environment, the Chinese Academy of Sciences (IUEMS201402) and the National Natural Science Foundation of China (31500391)

Determination of cinnamic acid in human urine by flow injection chemiluminescence

It was found that cinnamic acid can react with potassium permanganate in the acidic medium and produce chemiluminescence, which was greatly enhanced by glyoxal. Under the optimum conditions, the linear range for the determination of cinnamic acid was 1.0×10-8 to 1.0×10-4 mol L-1 with a detection limit of 8.0×10-9 mol L-1, the relative standard deviation was 1.7% for 2.0×10-6 mol L-1 cinnamic acid solution in nine repeated measurements. This method was found to be novel0simple0fast and sensitive, it was successfully applied to the determination of cinnamic acid in human urine. Furthermore, the possible reaction mechanism was also discussed

Role of intestinal flora and 5-HT in depression- and anxiety-like behaviors in mice exposed to PM2.5

BackgroundSome studies have shown that PM2.5 exposure is closely related to central nervous system diseases that lead to cognitive dysfunction and change the composition of intestinal flora. However, there are few studies on the role of intestinal flora in PM2.5-induced depression- and anxiety-like behaviors in mice. ObjectiveTo observe the effects of PM2.5 exposure on depression- and anxiety-like behaviors and the composition of intestinal flora in mice, and to explore the role of intestinal flora in regulating 5-hydroxytryptamine (5-HT) in depression- and anxiety-like behaviors in mice exposed to PM2.5. MethodsEight-week-old male SPF C57BL/6J mice were randomly divided into control group (NS group), probiotic group (LGG group), PM2.5 group (PM group), and combined exposure group (PML group), 6 mice in each group. Mice in the PM group and the PML group were exposed to PM2.5 in a dynamic exposure cabinet for 6 h per day, 6 d a week for 7 consecutive weeks, and the PM2.5 concentrations were approximately 8 times higher than the outdoor concentration. The LGG group and the PML group were orally administered with Lactobacillus rhamnosus while the NS group and the PM group were orally administered with the same amount of saline. Elevated plus maze test and open field test were used to detect depression and anxiety in mice. Fecal samples of mice were collected to evaluate intestinal flora abundance, diversity, and structure between groups using high-throughput sequencing of 16S rRNA. ELISA was employed to detect the levels of 5-HT in serum and hippocampus. Spearman correlation was used to analyze the correlations of differential intestinal flora with 5-HT level in hippocampus and depression- and anxiety-like behavior indicators in mice. ResultsThe percentage of open-arm entry [M(P25, P75)] in the PM group was 0.0% (0.0%, 33.3%), lower than those in the NS group [47.7% (25.0%, 50.8%) ] and the PML group [46.9% (40.0%, 50.0%)], and the differences were statistically significant (P<0.05). The total travelled distance and the time spent in central area (\begin{document}$\bar x \pm s$\end{document}) in the PM group were (2.01±0.90) m and (10.31±1.99) s respectively, shorter than those of the NS group [(3.80±0.89) m, (14.47±3.07) s], the total travelled distance in the PML group [(2.73±1.12) m] was shorter than those of the NS group and the LGG group [(4.21±1.08) m], and the differences were statistically significant (P<0.05). Compared to the NS group, the Simpson index of the PM group significantly increased (P<0.05). Compared to the LGG group, the Simpson index of the PML group significantly decreased (P<0.05). The results of Beta diversity analysis showed that there were differences in the composition of intestinal flora among the four groups of mice. Compared with the NS group and the LGG group, the abundances of Erysipelotrichaceae and Dubosiella in the PM group and the PML group increased, while the abundances of Prevotellaceae_UCG-001 decreased, and the differences were statistically significant (P<0.05). In hippocampus, the level of 5-HT in the PM group [(135.02±10.31) μg·g−1] was lower than those in the NS group [(178.77±43.15) μg·g−1] and the LGG group [(224.85±22.98) μg·g−1], and the level of 5-HT in the PML group [(161.27±15.81) μg·g−1] was lower than that in the LGG group (P<0.05). 5-HT level in hippocampus was significantly positively correlated with the relative abundance of Prevotellaceae_UCG-001 (r=0.6090, P=0.012). The percentage of open-arm entry was significantly negatively correlated with the relative abundance of Dubosiella (r=−0.4630, P=0.023). ConclusionAtmospheric PM2.5 exposure may cause depression- and anxiety-like behaviors in mice. The observed behavior dysfunction may be associated with the changes in diversity and relative abundance of intestinal flora as well as the decrease of 5-HT level. Such depression- and anxiety-like behaviors are alleviated after adding probiotics