Differentially expressed circRNA and functional pathways in the hippocampus of epileptic mice based on next-generation sequencing.

Epilepsy is a clinical syndrome caused by the highly synchronized abnormal discharge of brain neurons. It has the characteristics of paroxysmal, transient, repetitive, and stereotyped. Circular RNAs (circRNAs) are a recently discovered type of noncoding RNA with diverse cellular functions related to their excellent stability; additionally, some circRNAs can bind and regulate microRNAs (miRNAs). The present study was designed to screen the differentially expressed circRNA in an acute seizure model of epilepsy in mice, analyze the related miRNA and mRNA, and study their participating functions and enrichment pathways. In order to obtain the differential expression of circRNA in epilepsy and infer their function, we used next-generation sequencing and found significantly different transcripts. CIRI (circRNA identifier) software was used to predict circRNA from the hippocampus cDNA, EdgeR was applied for the differential circRNA analysis between samples, and Cytoscape 3.7.2 software was used to draw the network diagram. A total of 10,388 differentially expressed circRNAs were identified, of which 34 were upregulated and 66 were downregulated. Among them, mm9_circ_008777 and mm9_circ_004424 were the key upregulated genes, and their expression in the epilepsy group was verified using Quantitative real-time PCR (QPCR). The analysis indicated that the extracted gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were closely related to several epilepsy-associated processes. This study determined that mm9_circ_008777 and mm9_circ_004424 are potential biomarkers of epilepsy, which play important roles in epilepsy-related pathways. These results could help improve the understanding of the biological mechanisms of circRNAs and epilepsy treatments.

Warming Shapes nirS- and nosZ-Type Denitrifier Communities and Stimulates N2O Emission in Acidic Paddy Soil.

Warming strongly stimulates soil nitrous oxide (N2O) emission, contributing to the global warming trend. Submerged paddy soils exhibit huge N2O emission potential; however, the N2O emission pathway and underlying mechanisms for warming are not clearly understood. We conducted an incubation experiment using 15N to investigate the dynamics of N2O emission at controlled temperatures (5, 15, 25, and 35°C) in 125% water-filled pore space. The community structures of nitrifiers and denitrifiers were determined via high-throughput sequencing of functional genes. Our results showed that elevated temperature sharply enhanced soil N2O emission from submerged paddy soil. Denitrification was the main contributor, accounting for more than 90% of total N2O emission at all treatment temperatures. N2O flux was coordinatively regulated by nirK-, nirS-, and nosZ-containing denitrifiers but not ammonia-oxidizing archaea or ammonia-oxidizing bacteria. The nirS-containing denitrifiers were more sensitive to temperature shifts, especially at a lower temperature range (5 to 25°C), and showed a stronger correlation with N2O flux than that of nirK-containing denitrifiers. In contrast, nosZ-containing denitrifiers exhibited substantial variation at higher temperatures (15 to 35°C), thereby playing an important role in N2O consumption. Certain taxa of nirS- and nosZ-containing denitrifiers regulated N2O flux, including nirS-containing denitrifiers affiliated with Rhodanobacter and Cupriavidus as well as nosZ-containing denitrifiers affiliated with Azoarcus and Azospirillum. Together, these findings suggest that elevated temperature can significantly increase N2O emission from denitrification in submerged paddy soils by shifting the overall community structures and enriching some indigenous taxa of nirS- and nosZ-containing denitrifiers. IMPORTANCE The interdependence between global warming and greenhouse gas N2O has always been the hot spot. However, information on factors contributing to N2O and temperature-dependent community structure changes is scarce. This study demonstrated high-temperature-induced N2O emission from submerged paddy soils, mainly via stimulating denitrification. Further, we speculate that key functional denitrifiers drive N2O emission. This study showed that denitrifiers were more sensitive to temperature rise than nitrifiers, and the temperature sensitivity differed among denitrifier communities. N2O-consuming denitrifiers (nosZ-containing denitrifiers) were more sensitive at a higher temperature range than N2O-producing denitrifiers (nirS-containing denitrifiers). This study's findings help predict N2O fluxes under different degrees of warming and develop strategies to mitigate N2O emissions from paddy fields based on microbial community regulation.

[Characteristics of soil nitrogen cycle and mechanisms underlying the increase in rice yield with partial substitution of mineral fertilizers with organic manure in a paddy ecosystem: A review]. / 稻作系统有机肥替代部分化肥的土壤氮循环特征及增产机制.

Partial substitution of mineral fertilizers with organic manure is a key strategy for stable and increase crop yield accompanying with zero growth of mineral fertilizers. Based on recent stu-dies, we reviewed the effects of partial substitution of mineral fertilizers with organic manure on rice yield, nitrogen utilization efficiency, soil nitrogen fractions, and microbial nitrogen fixation, ammonification, nitrification, and denitrification in rice paddy ecosystems. We further compared the cha-racteristics of soil nitrogen cycle of mineral fertilizers alone and partial substitution of mineral fertili-zers with organic manure. The partial substitution altered key processes of nitrogen cycling, including enhancement of ammonification, mediation of nitrification and denitrification, reduction of ammonia volatilization and nitrogen loss, improved the status of nitrogen supplements (enriching the supplement of low-molecular-weight organic nitrogen, adjusting the distribution of inorganic nitrogen components, increasing the amount of microbial biomass nitrogen, and decreasing the loss of total nitrogen), improved soil nitrogen supply (increasing supply of small molecule organic nitrogen, coordinating inorganic nitrogen components and proportions, and increasing soil microbial biomass nitrogen and total nitrogen fixation), which promoted nitrogen uptake and regulated nitrogen allocation in rice plant to realize stability and enhancement of rice yield.

[Differential Responses of Rhizospheric nirK- and nirS-type Denitrifier Communities to Different Phosphorus Levels in Paddy Soil].

Phosphorus is an essential life element, which can affect the activities and functions of denitrifiers. Both nirK and nirS genes can code nitrite reductase; however, it remains unclear whether nirK- and nirS-containing denitrifers respond differentially to changes in the availability of phosphorus in paddy soil. In this study, P-deficient paddy soil was used to grow rice plants. Three phosphorus levels established by applying P fertilizer at a rate of 0 mg·kg-1 (CK), 15 mg·kg-1 (P1), and 30 mg·kg-1(P2), respectively. The abundance and community structure of nirK- and nirS- containing denitrifers were determined using quantitative PCR and high-throughput sequencing techniques. Results indicated that nirK- and nirS-containing communities responded differentially to changes in the P levels. The nirS-containing communities are more sensitive to the changes in P in both rhizosphere and bulk soil samples. In addition, the abundance of nirS genes was 2-3 times higher in the P2 treatment than in the CK treatment. Furthermore, the nirS community structure is also clearly differed from the CK treatment. However, P addition only induced partial modification of the community structure and abundance of nirK-containing denitrifiers. Moreover, compared to the bulk soil with each phosphorus level, the nirS community structure in the rhizosphere soil changed significantly; however, only the P2 treatment induced significant increases in the abundance of the nirS gene. In contrast, no significant differences in the abundance and composition of nirK-containing denitrifers were detected between rhizosphere and bulk soils under different phosphorus levels. Collectively, application of phosphate fertilizer in P-deficient paddy soil could significantly increase the abundance of nirK- and nirS-containing denitrifiers, changing their community structures, with nirS-type showing a greater sensitivity than nirK-type denitrifiers. In comparison, the denitrifying communities in the rhizosphere were more sensitive to variable P levels than that in the bulk soil. Compared to bulk soils, rice growth shifted the community structure of nirS- and nirK-containing denitrifiers in rhizosphere soils at each level of P, but failed to induce significant changes in their abundance (except for P2) that could cause a significant increase in nirS abundance. These results could provide a theoretical basis for exploring the effects of fertilization on soil denitrification.

[Effect of Dicyandiamide on N2O Emission in Fallow Paddy Field and Rape Cropping].

The emissions of greenhouse gas in winter are often neglected, and the latest research results showed that N2O emissions in fallow paddy field and winter oilseed rape are still large, research on mitigating the N2O flux and the mechanism behind them is of significance for mitigating N2O emissions from agricultural soil. By using static chamber techniques and molecular biology techniques, the N2O emission as well as the community composition and abundance of ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) from fallow paddy field, rape cropping with and without DCD treatment in Taoyuan agricultural ecological experiment station of the Chinese Academy of sciences were measured. The results showed that the addition of DCD significantly inhibited N2O emissions in fallow paddy field and rape cropping by 36.7% and 23.6%, respectively. The application of DCD in fallow paddy field inhibited the abundance of AOA and AOB by 59.3% and 73.7%, respectively, but only changed the community structure of AOA. The addition of DCD in rape cropping only changed the community structure and inhibited the abundance of AOB. This research showed that DCD application could effectively mitigate the N2O emissions in fallow paddy field and winter rape cropping under different mitigation mechanisms.

[N2O Consumption Ability of Submerged Paddy Soil and the Regulatory Mechanism].

A large number of researches showed that the N2O negative emissions from flooding paddy fields, peatlands and other wetlands ecosystem were frequent and considerable, which is of great significance on alleviating the greenhouse gas effect. However, there are few reports about the transformation and microbial mechanism of N2O between atmosphere and paddy soil. The slurry of surface paddy soil (0-5 cm) was incubated in laboratory conditions, and the effect of enhanced N2O concentrations in headspace on the N2O consumption capacity of submerged paddy soil and the response of nosZ gene abundance were explored. The results showed that, paddy soil under flooding and anaerobic conditions harbored very strong potential of N2O reduction along with a relatively high nosZ gene abundance (108 copies·g-1 dry soil at DNA level). Regression analysis presented the N2O concentrations in headspace were positively correlated (r2=1, P<0.001) to the N2O consumption rates of paddy soil slurry, indicating the high N2O concentration could stimulate the N2O consumption power, to a very high rate of 4567.99 µg·(m2·h)-1. Meanwhile, there were no significant differences in the high abundance of nosZ gene among N2O treatments, demonstrating the nosZ gene abundance at DNA level might not be the main controller of N2O consumption ability in submerged paddy soil and further study on the key microbial factor is needed.

[Effects of Fertilization on Soil Microbial Abundance and Community Structure at DNA and cDNA Levels in Paddy Soils].

Fertilizer applications have important effects on soil microbial abundance and community structure. In this study, total soil microbial DNA and RNA were directly extracted from paddy soils of N0 (control treatment, no nitrogen fertilizer), NPK (balanced fertilization), NPK+LS (balanced fertilization with additional 3.0 t·hm-2 rice straw incorporation) and NPK+HS (balanced fertilization with additional 6.0 t·hm-2 rice straw incorporation) treatments in a long-term fertilization experiment of double rice cropping system in Changsha County, Hunan Province. Soil bacteria community structures were evaluated by analyzing the 16S rRNA gene fragments at DNA and cDNA levels with Terminal Restriction Fragment Length Polymorphism (T-RFLP) and quantitative PCR techniques. Balancing fertilization with chemical fertilizers and rice straw incorporation significantly changed the composition of bulk (DNA-based) and potentially active (mRNA-based) soil bacterial community as shown in T-RFLP profiles, and also reduced the bulk soil microbial diversity, but not the potentially active ones, as compared with the control treatment. The DNA-based abundance of 16S rRNA gene was on average 377 times as many as the m-RNA based population size. Compared to N0,balanced fertilization with rice straw incorporation (NPK+LS and NPK+HS) increased the bulk and active copy numbers of 16S rRNA gene, but not for balanced fertilization (NPK). The abundance and microbial community structure were not significantly different between the NPK+LS and NPK+HS treatments. Redundancy analysis (RDA) showed that soil ammonium was the key environmental factor determining the bulk and active soil microbial community structure among the treatments. In conclusion, the effect of fertilization on soil microbial abundance and community structure could be indicated at both DNA and cDNA levels; the cDNA information could better reflect the adaptability of bacterial community to the environmental stress.

[Effects of combined applications of pig manure and chemical fertilizers on CH4 and N2O emissions and their global warming potentials in paddy fields with double-rice cropping].

A field experiment was carried out to study the effects of combined applications of pig manure and chemical fertilizers on CH4 and N2O emissions, which were measured using the static chamber/gas chromatography method, and their global warming potentials in typical paddy fields with double-rice cropping in Hunan province. The results showed that the combined applications of pig manure and chemical fertilizers did not change the seasonal patterns of CH4 and N2O emissions from paddy soils, but significantly changed the magnitudes of CH4 and N2O fluxes in rice growing seasons as compared with sole application of chemical fertilizers. During the two rice growing seasons, the cumulative CH4 emissions for the pig manure and chemical nitrogen (N) fertilizer each contributing to 50% of the total applied N (1/2N + PM) treatment were higher than those for the treatments of no N fertilizer (ON), half amount of chemical N fertilizer (1/2N) and 100% chemical N fertilizer (N) by 54.83%, 33.85% and 43.30%, respectively (P < 0.05), whilst the cumulative N2O emissions for the 1/2N + PM treatment were decreased by 67.50% compared with N treatment, but increased by 129.43% and 119.23% compared with ON and 1/2N treatments, respectively (P < 0.05). CH4 was the dominant contributor to the global warming potential (GWP) in both rice growing seasons, which contributed more than 99% to the integrated GWP of CH4 and N2O emissions for all the four treatments. Both GWP and yield-scaled GWP for the treatment of 1/2N + PM were significantly higher than the other three treatments. The yield-scaled GWP for the treatment of 1/2N + PM was higher than those for the N, 1/2N and ON treatments by 58.21%, 26.82% and 20. 63%, respectively. Therefore, combined applications of pig manure and chemical fertilizers in paddy fields would increase the GWP of CH4 and N2O emissions during rice growing seasons and this effect should be considered in regional greenhouse gases emissions inventory.

[N2O flux in winter and its affecting factors under different land use patterns].

Due to the low temperature in winter, the emissions of greenhouse gas are often neglected. And the latest research results showed that there is continuous N2O emission in winter, therefore, research on understanding the No2O flux regulation is important for evaluating agricultural soil N2O emission. By using static chamber techniques, the N2O emission from soils under different land use patterns including fallow paddy field, rape cropping, honey pomelo orchard and abandon land in Taoyuan agricultural ecological experimental station of Chinese Academy of Sciences was measured. The results showed that fallow paddy field and rape cropping N2O emissions were obviously higher than those of the honey pomelo orchard and abandon land, and the total N2O flux in winter decreased in the order of rape cropping > fallow paddy field > honey pomelo orchard > abandon land. Cumulative N2O emission was 0.502, 0.392, 0.162 and 0.075 kg x hm(-2), respectively. Fallow paddy field and rape cropping N2O emissions accounted for large proportions of the annual N2O emissions, while honey pomelo orchard and abandon land had small contribution to the annual N2O emissions. The correlation analysis results showed that for different land use patterns, when the soil temperature > 5 degrees C, N2O emissions in winter and soil temperature had significant positive exponential correlation, and had little to do with moisture. This research showed that: when the soil temperature > 5 degrees C, the soil temperature was the leading factor in N2O emissions in winter under different land patterns; When the soil temperature < 5 degrees C, other environmental factors had comprehensive influences on the N2O emissions.

[Effects of rice straw returning on the community structure and diversity of nitrogen-fixing gene (nifH) in paddy soil].

Taking a long-term fertilization experiment in Taoyuan Agro-ecosystem Research Station under Chinese Academy of Sciences as the platform, and selecting four treatments (no fertilization, CK; rice straw returning, C; nitrogen, phosphorus and potassium fertilization, NPK; and NPK+C) as the objects, soil samples were collected at the tillering, booting and maturing stages of rice, and the abundance, composition and diversity of nifH-containing bacterial community were measured by real-time quantitative PCR and terminal restriction fragment length polymorphism (T-RFLP), aimed to understand the effects of rice straw returning on the nifH-containing bacterial community in paddy soil. Compared with CK, treatments NPK+C and NPK increased the abundance of nifH-containing microorganisms significantly (except at tillering stage), and NPK+C had the highest abundance of nifH-containing microorganisms. Under the effects of long-term fertilization, the composition of nifH gene community in CK differed obviously from that in the other three treatments. The nifH composition had definite difference between C and NPK, but less difference between NPK and NPK+C. Long-term fertilization did not induce significant changes in nifH diversity. Therefore, long-term rice straw returning not only induced the changes of nifH gene community composition, but also resulted in a significant increase in the abundance of nifH-containing community, and hence, the increase of soil nitrogen fixing capacity.

[Effects of continuous cropping of vegetables on ammonia oxidizers community structure].

Investigations were conducted on the effects of intensive application of chemical fertilizers in crop production on soil nitrifier communities and the relationship between nitrifier communities and soil nitrification ability. Two series of vegetable soils were selected from Huangxing, Changsha, reflecting continuous vegetable cropping with about 20 years and new vegetable field with only about 2 years vegetable growing history. In each series five independent topsoils (0-20 cm) were sampled and each soil was a mixture of 10 cores randomly taken in the same field. Terminal restriction fragment length polymorphism (T-RFLP) and quantity PCR (Q-PCR) were used to determine the composition and abundance of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities. Results indicated that long-term and continuous vegetable cropping obviously changed the compositions of both AOB and AOA amoA gene, soil pH and Olsen-P content were the dominant factors affecting the composition of AOB amoA. In the vegetable soils, although the copy number of AOA amoA gene was about 5 times higher than AOB amoA gene, no significant correlation was detected between AOA amoA gene abundance and soil nitrification rate. It was not sure whether long-term and continuous vegetable cropping could shift the abundance of AOB and AOA, but it resulted in the enrichment of some dominant AOB species and increase of soil nitrification potential (PNF).

[Change characteristics of soil available nitrogen and phosphorus and heavy metal contents after long-term cultivation of vegetables].

Soil samples were collected from three vegetable fields under different years of cultivation in Changsha suburbs of Hunan Province, South-central China to study the accumulation characteristics, risks, and sources of soil available nitrogen and phosphorus and heavy metals in the fields. With the increasing year of vegetable cultivation, the soil NO3(-)-N, Olsen-P, and heavy metals contents in the fields increased significantly. The average contents of soil NO3(-)-N, Olsen-P, and Cd in the vegetable fields having been cultivated for 1-2 years in Ningxiang County, 10-15 years in Changsha County, and 30 years in Kaifu District were 21.1, 31.9 and 0.33 mg x kg(-1), 42.0, 146.9 and 0.52 mg x kg(-1), and 49.5, 219.9 and 1.40 mg x kg(-1), respectively. The cumulative index (CI) of soil heavy metals generally followed the sequence of Cd >> Cu > Pb > Ni > Zn. Principal component analysis and cluster analysis showed that compared with soil NH4 OAc-extracted potassium, pH, organic matter and NH4(+)-N, that were dominated by natural factors, the soil Olsen-P and NO3(-)-N had the similar accumulation characteristics with the soil heavy metals, being mainly controlled by fertilization. It was considered that the soil environment and health quality of the vegetable fields in Changsha suburbs were not optimistic. The longer the cultivation year of vegetables, the more the soil NO3(-)-N, Olsen-P, and heavy metals accumulated in the fields. The accumulation of these elements in the fields could be primarily due to the long-term fertilization.

[Effects of rice straw on the diversity of nitrifying genes (amoA and hao) in paddy soil].

The effects of long-term (16 years) fertilization on the diversity and community structure of soil ammonia-oxidizing gene (amoA) and hydroxylamine-oxidizing gene (hao) in paddy soil were evaluated using the methods of polymerase chain reaction, cloning and sequencing. The soil samples were collected from the treatments of NPK (CK) and NPK plus rice straw (SR) of the long-term field fertilization experiment in Taoyuan Agro-ecological Experimental Station. The Shannon Indices showed that the diversity of amoA and hao in SR treatment was lower than that in CK, and LUBSHUFF statistical analyses demonstrated that the sequence compositions of both amoA and huo libraries were significantly different between CK and SR. The phylogenetic trees indicated that some clusters appeared in SR treatment but were not detected in CK treatment. As to amoA, only Nitrosospira besides the uncultured amoA sequences were cloned from the two treatments, while no Nitrosomonas species were detected. As to hao, the strains from Silicibacter and Methylococcus were dominant in CK, while in SR the strains from Nitrosospira and Nitrosomonas were dominant. Sum up, the long-term rice straw application has caused a remarkable impact on the diversity and community structure on Nitrosobacteria.

[Effect of long-term fertilization on the diversity of nitrite reductase genes (nirK and nirS) in paddy soil].

In order to investigate the effects of long-term application of nitrogen fertilizer on soil denitrifying communities, the diversities of nir genes (nirK and nirS) were studied using molecular approaches in the long-term paddy field experiment (started in 1990) located in Taoyuan. Analysis of clone sequences indicated that the nirK fragments from paddy soil showed close similarity (90.7%) to the nirK sequences registered in GenBank database, but were not related to any known strain. Whereas, most of the airS clones showed low similarity (74.7%) to the nirS gene fragments registered in GenBank. The Chao1 estimates showed that the diversity of nirK gene 13) OTUs] than in N treatment [(49 +/- 9) OTUs], but the difference was not significant. However, application of nitrogen fertilizer resulted in significant difference of nirS-community compared to CK. Nitrogen fertilizer had obvious effect on tbe community structure of nirK-denitrifiers (p < 0.022), but the nirS-containing community was not affected. Based on phylogenetic analysis, nirK clones grouped into three clusters with aggregations of some OTUs cloned from N treatment. Although nirS clones grouped into four clusters, the majority of the clones were attributed in one cluster. The results suggested that application of nitrogen fertilizer had a greater influence on the diversity of nirS-containing bacterial community than that of the nirK. However, the community structure of nirK-containing denitrifiers was more sensitive to nitrogen fertilization than that of the mrS.