Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA.

The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR- associated protein 9) system, a highly efficient, simple, and easy genome editing technology, offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster. However, when using CRISPR/Cas9 system to edit Drosophila melanogaster gene, Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals, and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process, which has a long and complex operation cycle In this study, on the basis of the CRISPR/Cas9 system, we introduced the tRNA-sgRNA system and triplex elements, used triplex elements to link Cas9 and tRNA-sgRNA genes, stabilized the end of Cas9 mRNA after single transcript cutting, and made the expression of both Cas9 protein and sgRNA with a single transcript a reality. And as we obtained the corresponding phenotypic progeny in one hybridization, genetic manipulation was simplified. We found that conditional knockout of the white(w) gene in the Drosophila melanogaster eye and the broad(br) gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system. So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient, extendable, and easy to use.

Thermodynamic Assessment of a Solar-Driven Integrated Membrane Reactor for Ethanol Steam Reforming.

To efficiently convert and utilize intermittent solar energy, a novel solar-driven ethanol steam reforming (ESR) system integrated with a membrane reactor is proposed. It has the potential to convert low-grade solar thermal energy into high energy level chemical energy. Driven by chemical potential, hydrogen permeation membranes (HPM) can separate the generated hydrogen and shift the ESR equilibrium forward to increase conversion and thermodynamic efficiency. The thermodynamic and environmental performances are analyzed via numerical simulation under a reaction temperature range of 100-400 °C with permeate pressures of 0.01-0.75 bar. The highest theoretical conversion rate is 98.3% at 100 °C and 0.01 bar, while the highest first-law efficiency, solar-to-fuel efficiency, and exergy efficiency are 82.3%, 45.3%, and 70.4% at 215 °C and 0.20 bar. The standard coal saving rate (SCSR) and carbon dioxide reduction rate (CDRR) are maximums of 101 g·m-2·h-1 and 247 g·m-2·h-1 at 200 °C and 0.20 bar with a hydrogen generation rate of 22.4 mol·m-2·h-1. This study illustrates the feasibility of solar-driven ESR integrated with a membrane reactor and distinguishes a novel approach for distributed hydrogen generation and solar energy utilization and upgradation.

Understanding the cometabolic degradation of sulfadiazine by an enriched ammonia oxidizing bacteria culture from both extracellular and intracellular perspectives.

Antibiotics are widely used drugs in the world and pose serious threats to ecosystems and human health. Although it has been reported that ammonia oxidizing bacteria (AOB) can cometabolize antibiotics, little has been reported on how AOB would respond to the exposure of antibiotics on extracellular and enzymatic levels, as well as the impact of antibiotics on the bioactivity of AOB. Therefore, in this study, a typical antibiotic, sulfadiazine (SDZ), was selected, and a series short-term batch tests using enriched AOB sludge were conducted to investigate the intracellular and extracellular responses of AOB along the cometabolic degradation process of SDZ. The results showed the cometabolic degradation of AOB made the main contribution to SDZ removal. When the enriched AOB sludge was exposed to SDZ, ammonium oxidation rate, ammonia monooxygenase activity, adenosine triphosphate concentration and dehydrogenases activity were negatively affected. The amoA gene abundance increased 1.5 folds within 24 h, which may enhance the uptake and utilization of substrates and maintain stable metabolic activity. In the tests with and without ammonium, the concentration of total EPS increased from 264.9 to 231.1 mg/gVSS to 607.7 and 538.2 mg/gVSS, respectively, under the exposure to SDZ, which was mainly contributed by the increase of proteins in tightly bound extracellular polymeric substances (EPS) and polysacharides in tightly bound EPS and soluble microbial products. The proportion of tryptophan-like protein and humic acid-like organics in EPS also increased. Moreover, SDZ stress stimulated the secretion of three quorum sensing signal molecules, C4-HSL (from 140.3 to 164.9 ng/L), 3OC6-HSL (from 17.8 to 42.4 ng/L) and C8-HSL (from 35.8 to 95.9 ng/L) in the enriched AOB sludge. Among them, C8-HSL may be a key signal molecule that promoted the secretion of EPS. The findings of this study could shed more light on the cometabolic degradation of antibiotics by AOB.

Peroxisome Deficiency in Cochlear Hair Cells Causes Hearing Loss by Deregulating BK Channels.

The peroxisome is a ubiquitous organelle in rodent cells and plays important roles in a variety of cell types and tissues. It is previously indicated that peroxisomes are associated with auditory function, and patients with peroxisome biogenesis disorders (PBDs) are found to have hearing dysfunction, but the specific role of peroxisomes in hearing remains unclear. In this study, two peroxisome-deficient mouse models (Atoh1-Pex5-/- and Pax2-Pex5-/- ) are established and it is found that peroxisomes mainly function in the hair cells of cochleae. Furthermore, peroxisome deficiency-mediated negative effects on hearing do not involve mitochondrial dysfunction and oxidative damage. Although the mammalian target of rapamycin complex 1 (mTORC1) signaling is shown to function through peroxisomes, no changes are observed in the mTORC1 signaling in Atoh1-Pex5-/- mice when compared to wild-type (WT) mice. However, the expression of large-conductance, voltage-, and Ca2+ -activated K+ (BK) channels is less in Atoh1-Pex5-/- mice as compared to the WT mice, and the administration of activators of BK channels (NS-1619 and NS-11021) restores the auditory function in knockout mice. These results suggest that peroxisomes play an essential role in cochlear hair cells by regulating BK channels. Hence, BK channels appear as the probable target for treating peroxisome-related hearing diseases such as PBDs.

Enhanced removal of cephalexin and sulfadiazine in nitrifying membrane-aerated biofilm reactors.

Nitrification process has been reported to be capable of degrading various pharmaceuticals due to the cometabolism of ammonia-oxidizing bacteria (AOB). The membrane aerated biofilm reactor (MABR) is an emerging configuration in wastewater treatment with advantages of high nitrification rate and low energy consumption. However, there are very few studies investigating the degradation of antibiotics at environmentally relevant levels in nitrifying MABR systems. In this study, the removal of two widely used antibiotics, cephalexin (CFX) and sulfadiazine (SDZ), was evaluated in two independent MABRs with nitrifying biofilms. The impacts of CFX and SDZ exposure on the nitrification performance and microbial community structure within biofilms were also investigated. The results showed that nitrifying biofilms were very efficient in removing CFX (94.6%) and SDZ (75.4%) with an initial concentration of 100 µg/L when hydraulic retention time (HRT) was 4 h in the reactors. When HRT decreased from 4 h to 3 h, the removal rates of CFX and SDZ increased significantly from 23.4 ± 1.0 µg/(L·h) and 18.7 ± 1.1 µg/(L·h), respectively, to 27.7 ± 1.3 µg/(L·h) (p＜0.01) and 20.8 ± 2.4 µg/(L·h) (p＜0.05), while the removal efficiencies decreased to 86.0% and 61.5%, respectively. Despite the exposure to CFX and SDZ, the nitrification performance was not affected, and microbial community structure within biofilms also remained relatively stable. This study shows that nitrifying MABR process is a promising option for the efficient removal of antibiotics from domestic wastewater.

Thermodynamic Analysis of Methylcyclohexane Dehydrogenation and Solar Energy Storage via Solar-Driven Hydrogen Permeation Membrane Reactor.

A novel methylcyclohexane (MCH) dehydrogenation system driven by solar energy with a hydrogen permeation membrane (HPM) reactor is proposed in this study. It is a promising method, via this novel system, to generate pure hydrogen and store intermittent solar energy. In this research, the thermodynamic analysis of MCH dehydrogenation via the HPM reactor was conducted based on numerical simulation. The conversion rates and thermodynamic efficiencies under different temperatures (150-350 °C), permeate pressures from 0.001 to 0.5 bar, and solar irradiation in the four seasons were studied and analyzed. Under a hydrogen partial pressure difference, HPM can separate hydrogen and shift the reaction equilibrium forward for a higher conversion rate of MCH, which can reach nearly 99.7% in this system. The first-law of thermodynamic efficiency, the solar-to-fuel efficiency, and the exergy efficiency are up to 95.58%, 38.65%, and 94.22%, respectively. This study exhibits the feasibility and potential of MCH dehydrogenation via the HPM reactor driven by solar energy and provides a novel approach for solar energy storage.

Unravelling kinetic and microbial responses of enriched nitrifying sludge under long-term exposure of cephalexin and sulfadiazine.

Wastewater treatment plants (WWTPs) have been identified as one of the reservoirs of antibiotics. Although nitrifying bacteria have been reported to be capable of degrading various antibiotics, there are very few studies investigating long-term effects of antibiotics on kinetic and microbial responses of nitrifying bacteria. In this study, cephalexin (CFX) and sulfadiazine (SDZ) were selected to assess chronic impacts on nitrifying sludge with stepwise increasing concentrations in two independent bioreactors. The results showed that CFX and SDZ at an initial concentration of 100 µg/L could be efficiently removed by enriched nitrifying sludge, as evidenced by removal efficiencies of more than 88% and 85%, respectively. Ammonia-oxidizing bacteria (AOB) made a major contribution to the biodegradation of CFX and SDZ via cometabolism, compared to limited contributions from heterotrophic bacteria and nitrite-oxidizing bacteria. Chronic exposure to CFX (≥30 µg/L) could stimulate ammonium oxidation activity in terms of a significant enhancement of ammonium oxidation rate (p < 0.01). In contrast, the ammonium oxidation activity was inhibited due to exposure to 30 µg/L SDZ (p < 0.01), then it recovered after long-term adaption under exposure to 50 and 100 µg/L SDZ. In addition, 16S rRNA gene amplicon sequencing revealed that the relative abundance of AOB decreased distinctly from 23.8% to 28.8% in the control phase (without CFX or SDZ) to 14.2% and 10.8% under exposure to 100 µg/L CFX and SDZ, respectively. However, the expression level of amoA gene was up-regulated to overcome this adverse impact and maintain a stable and efficient removal of both ammonium and antibiotics. The findings in this study shed a light on chronic effects of antibiotic exposure on kinetic and microbial responses of enriched nitrifying sludge in WWTPs.

Achieving simultaneous nitrogen and antibiotic removal in one-stage partial nitritation-Anammox (PN/A) process.

Partial nitritation-Anammox (PN/A) process has been recognized as a sustainable process for biological nitrogen removal. Although various antibiotics have been ubiquitously detected in influent of wastewater treatment plants, little is known whether functional microorganisms in the PN/A process are capable of biodegrading antibiotics. This study aimed to investigate simultaneous nitrogen and antibiotic removal in a lab-scale one-stage PN/A system treating synthetic wastewater containing a widely-used antibiotic, sulfadiazine (SDZ). Results showed that maximum total nitrogen (TN) removal efficiency of 86.1% and SDZ removal efficiency of 95.1% could be achieved when treating 5 mg/L SDZ under DO conditions of 0.5-0.6 mg/L. Compared to anammox bacteria, ammonia-oxidizing bacteria (AOB) made a major contribution to SDZ degradation through their cometabolic pathway. A strong correlation between amoA gene and SDZ removal efficiency was found (p < 0.01). In addition, the degradation products of SDZ did not exhibit any inhibitory effects on Escherichia coli. The findings suggest that it is promising to apply the PN/A process to simultaneously remove antibiotics and nitrogen from contaminated wastewater.

Insight into the nitrification kinetics and microbial response of an enriched nitrifying sludge in the biodegradation of sulfadiazine.

The intensive use of antibiotics results in the continuous release of antibiotics into wastewater treatment systems, leading to the spread of antibiotic resistance. Nitrifying system is reported to be capable of degrading antibiotics, yet few studies have systematically investigated the inherent correlation among ammonium oxidation rate, antibiotic degradation and genetic expression of nitrifying bacteria along the process. This study selected a widely used sulfonamide antibiotic, sulfadiazine (SDZ), to investigate its biodegradation potential by an enriched nitrifying culture and the response of nitrifying bacteria against antibiotic exposure. Our results demonstrated that SDZ degradation was mainly contributed by cometabolism of ammonia-oxidizing bacteria (AOB), rather than biomass adsorption. The quantitative reverse transcription PCR (RT-qPCR) analysis revealed that the expression level of amoA gene was down-regulated due to the SDZ exposure. In addition, the degradation products of SDZ did not exhibit inhibitory effect on Escherichia coli K12, indicating the biotoxicity of SDZ could be mitigated after biodegradation. The findings offer insights regarding the biodegradation process of sulfonamide antibiotics via cometabolism by AOB.

Cometabolic biodegradation of cephalexin by enriched nitrifying sludge: Process characteristics, gene expression and product biotoxicity.

The nitrifying systems have been reported to be able to biodegrade micropollutants, yet it is still unclear about the cometabolism of ammonia-oxidizing bacteria (AOB) towards micropollutants, in particular their enzyme and transcriptional responses under exposure of micropollutants. This study investigated cometabolic biodegradation of a selected antibiotic, cephalexin (CFX), by an enriched nitrifying culture through a series of batch experiments, together with the assessments of enzymatic activity, key gene expression, and biotoxicity of the degradation products. More than 99% CFX with an initial concentration of 50 µg/L could be removed with the presence of ammonium, while <44% of CFX removal was observed in the absence of ammonium, suggesting the cometabolic degradation of CFX by ammonia-oxidizing bacteria (AOB). After the addition of 50 µg/L CFX, the ammonia oxidizing rate (AOR) decreased from 36.6 to 11.0 mg N/(L·h·g VSS), followed by a slight recovery when CFX concentration decreased to below 8 µg/L. Ammonia monooxygenase (AMO) activity showed a similar trend with that of AOR. The quantitative reverse transcription PCR assay indicated that the expression level of amoA gene was significantly upregulated (up to 3-fold, p < 0.05) due to the addition of CFX, while decreased to the normal level once CFX was degraded, suggesting a mechanism of AOB to neutralize the toxicity of CFX by metabolizing ammonia more effectively. Meanwhile, the biotoxicity test showed the degradation products of CFX did not exhibit any antibacterial impacts in terms of cell viability, compared to the parent compounds. Our finding shed a light on AMO-mediated cometabolic biodegradation of antibiotics in nitrifying cultures.