Nitrous oxide emissions and microbial communities variation in low dissolved oxygen and low carbon-to-nitrogen ratio anoxic-oxic wastewater treatment plant.

Dissolved oxygen (DO) levels and carbon-to-nitrogen (C/N) ratio affect nitrous oxide (N2O) emissions by influencing the physiological and ecological dynamics of nitrifying and denitrifying microbial communities in activated sludge systems. For example, Nitrosomonas is a common N2O producing nitrifying bacteria in wastewater treatment plants (WWTPs), and DO conditions can affect the N2O production capacity. Previous studies have reported N2O emission characteristics under adequate DO and C/N conditions in A/O WWTPs. However, in actual operation, owing to economic and managerial factors, some WWTPs have a long-term state of low DO levels in oxic tanks and low influent C/N. Research on N2O emission characteristics in low DO-limited and low C/N ratio WWTPs is limited. This study investigated N2O emissions and the corresponding shifts in microorganisms within an anoxic-oxic (A/O) WWTP over 9-month. Quantitative PCR was used to assess the abundance of ten functional genes related to nitrification and denitrification processes, and high-throughput sequencing of the 16S rRNA gene was employed to determine the composition change of microorganisms. The findings revealed that 1) the average N2O emission factor was 1.07% in the studied WWTP; 2) the DO-limited oxic tank primarily contributed to N2O; 3) NO2-, TOC, and C/N ratios were key factors for dissolved N2O in the aerobic tank; and 4) Nitrosomonas and Terrimonas exhibited a robust correlation with N2O emissions. This research provides data references for estimating N2O emission factors and developing N2O reduction policies in WWTPs with DO-limited and low C/N ratios.

A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants.

CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.

Protective effects of crimson snapper scales peptides against oxidative stress on Drosophila melanogaster and the action mechanism.

Peptides derived from crimson snapper scales (CSSPs) were reported to possess excellent free radical scavenging activities in vitro. In present study, the anti-aging and anti-oxidative stress effects of CSSPs were evaluated in Drosophila melanogaster models. Results showed that the addition of CSSPs in the diets of normal Drosophila could effectively extend their lifespan and improve the motor ability of aged Drosophila. Moreover, CSSPs could protect Drosophila from oxidative damage induced by H2O2, paraquat and UV irradiation. The extension of lifespan was found to be associated with the effects of CSSPs in improving the antioxidant defense system of Drosophila, manifesting as the reduction of oxidation products MDA and PCO, the elevated activities of T-SOD, CAT and GSH-Px, and the upregulated expression of antioxidant related genes after CSSPs supplemented. Furthermore, CSSPs at 6 mg/mL significantly downregulated mTOR signaling pathway and activated autophagy in aged male Drosophila, and the inhibition on mTOR activation was probably mediated by the antioxidant effects of CSSPs. Our findings suggest that CSSPs have the potential in making dietary supplements against natural aging and oxidative stress in organisms.

Multiplex and optimization of dCas9-TV-mediated gene activation in plants.

Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.

Gene disruption through base editing-induced messenger RNA missplicing in plants.

Gene knockout tools are highly desirable for basic and applied plant research. Here, we leverage the Cas9-derived cytosine base editor to introduce precise C-to-T mutations to disrupt the highly conserved intron donor site GT or acceptor site AG, thereby inducing messenger RNA (mRNA) missplicing and gene disruption. As proof of concept, we successfully obtained Arabidopsis null mutant of MTA gene in the T2 generation and rice double null mutant of GL1-1 and NAL1 genes in the T0 generation by this strategy. Elimination of the original intron donor site or acceptor site could trigger aberrant splicing at a new specific exonic site, but not at the closest GT or AG site, suggesting cryptic rules governing splice site recognition. The strategy presented expands the applications of base editing technologies in plants by providing a new means for gene inactivation without generating DNA double-strand breaks, and it can potentially serve as a useful tool for studying the biology of mRNA splicing.

Core bioactive components promoting blood circulation in the traditional Chinese medicine compound xueshuantong capsule (CXC) based on the relevance analysis between chemical HPLC fingerprint and in vivo biological effects.

Compound xueshuantong capsule (CXC) is an oral traditional Chinese herbal formula (CHF) comprised of Panax notoginseng (PN), Radix astragali (RA), Salvia miltiorrhizae (SM), and Radix scrophulariaceae (RS). The present investigation was designed to explore the core bioactive components promoting blood circulation in CXC using high-performance liquid chromatography (HPLC) and animal studies. CXC samples were prepared with different proportions of the 4 herbs according to a four-factor, nine-level uniform design. CXC samples were assessed with HPLC, which identified 21 components. For the animal experiments, rats were soaked in ice water during the time interval between two adrenaline hydrochloride injections to reduce blood circulation. We assessed whole-blood viscosity (WBV), erythrocyte aggregation and red corpuscle electrophoresis indices (EAI and RCEI, respectively), plasma viscosity (PV), maximum platelet aggregation rate (MPAR), activated partial thromboplastin time (APTT), and prothrombin time (PT). Based on the hypothesis that CXC sample effects varied with differences in components, we performed grey relational analysis (GRA), principal component analysis (PCA), ridge regression (RR), and radial basis function (RBF) to evaluate the contribution of each identified component. Our results indicate that panaxytriol, ginsenoside Rb1, angoroside C, protocatechualdehyde, ginsenoside Rd, and calycosin-7-O-ß-D-glucoside are the core bioactive components, and that they might play different roles in the alleviation of circulation dysfunction. Panaxytriol and ginsenoside Rb1 had close relevance to red blood cell (RBC) aggregation, angoroside C was related to platelet aggregation, protocatechualdehyde was involved in intrinsic clotting activity, ginsenoside Rd affected RBC deformability and plasma proteins, and calycosin-7-O-ß-D-glucoside influenced extrinsic clotting activity. This study indicates that angoroside C, calycosin-7-O-ß-D-glucoside, panaxytriol, and protocatechualdehyde may have novel therapeutic uses.

[HPLC fingerprint of compound xueshuantong capsule].

OBJECTIVE: To establish the HPLC fingerprint of Compound Xueshuantong Capsule. METHODS: Dionex Acclaim 120 C18 column (4.6 mm x 150 mm, 3 microm) was used with acetonitrile (A) and 0.05% phosphoric acid (B) in gradient elution mode. The elution profile was:0-50 min (15%-->34% A), 50-95 min (34%-->75% A); The detective wavelength was 203 nm and 270 nm. The column temperature was set at 25 degrees C and the flow rate was 1.0 mL/min. RESULTS: In the established fingerprint,42 common peaks covering 4 medicinal materials were detected, and 23 chemical compounds were identified by RRLC/MS/MS and DAD spetra. CONCLUSION: The method can be used for quality control of Compound Xueshuantong Capsule with great precision, accuracy and good reproducibility.