Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy.

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.

Impact of uncertainty on regional carbon peak paths: an analysis based on carbon emissions accounting, modeling, and driving factors.

Regional carbon emission paths have an important impact on the realization of China's carbon emission peak target. Due to the uncertainty of future development model, the change of carbon emissions will also face uncertainty, which will make achieving the peak target challenging. Taking Shandong, Henan, and Guangdong, three of China's most populous provinces, as examples, this study analyzed the impacts of uncertainties in carbon accounting principles, driving factors, and simulation mechanism on achieving the peak target. The results show that (1) under the baseline scenario, the accounting principles based on primary energy consumption and IPCC sector consumption will make the peaking time of Guangdong be evaluated as 2018 and 2030, respectively, and the simulation based on IPCC sector accounting will advance the peaking time of Shandong by at least 5 years, while Henan will be less affected. (2) When considering the impact of the energy structure, Guangdong and Henan are estimated to peak in 2011 and 2018, while without considering the impact of the energy structure, the peak in the two provinces may be after 2035. Energy structure has no effect on the estimation of peaking time for Shandong. In addition, the k value in the ridge regression method also has no effect on the peaking time for the three provinces; it only affects the simulations of annual carbon emissions. This study also presented the carbon emission trajectory under different scenarios; from the simulation results, environmental regulation measures such as accelerating industrial structure transformation and increasing energy consumption intensity may help to achieve the peak carbon emission target as soon as possible. It also suggests that uncertainty should be included in future carbon assessments to present a more complete carbon emission trajectory.

Exploring the Dependence and Influencing Factors of Carbon Emissions from the Perspective of Population Development.

Working towards sustainable population development is an important part of carbon mitigation efforts, and decoupling carbon emissions from population development has great significance for carbon mitigation. Based on the construction of a comprehensive population development index (PDI), this study adopts a decoupling model to explore the dependence between carbon emissions and PDI across 30 Chinese provinces from 2001 to 2017. Then, the stochastic impacts by regression on population, affluence and technology (STIRPAT) model is used to investigate the impact of population factors on carbon emissions. The results show that the decoupling relationship between carbon emissions and PDI has experienced a transformation from expansive negative coupling to expansive coupling and then to weak decoupling at the national level, while some provinces have experienced the same evolutionary process, but the decoupling state in most provinces is not ideal. Sending talent to western provinces and developing low-carbon supporting industries will accelerate carbon decoupling. At the national level, incorporating environmental protection into the existing education system as part of classroom teaching could contribute to carbon decoupling.

Data Mining and Validation of AMPK Pathway as a Novel Candidate Role Affecting Intramuscular Fat Content in Pigs.

Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathway on IMF deposition in the longissimus dorsi (LD) muscles of pigs. Potential signaling pathways were screened by mining data from three gene expression profiles in the Gene Expression Omnibus (GEO) database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of two pig breeds with different IMF contents (Large White and Min). Western blot analysis was used to detect the expression levels of several candidate proteins. Our results showed that the AMPK signaling pathway was screened via bioinformatics analysis. Ten key hub genes of this signaling pathway (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKß, CPT1A, CPT1B, PGC-1α, CD36, and ACC1) were differentially expressed between the Large White and Min pigs. Western blot analysis further confirmed that LKB1/CaMKK2-AMPK-ACC1-CPT1A axis dominates the activity of AMPK signaling pathway. Statistical analyses revealed that AMPK signaling pathway activity clearly varied among the two pig breeds. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.

CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine miR-17-92 Cluster.

Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.

The Possible Role of Complete Loss of Myostatin in Limiting Excessive Proliferation of Muscle Cells (C2C12) via Activation of MicroRNAs.

Myostatin (MSTN) is a member of the TGF-ß superfamily that negatively regulates skeletal muscle growth and differentiation. However, the mechanism by which complete MSTN deletion limits excessive proliferation of muscle cells remains unclear. In this study, we knocked out MSTN in mouse myoblast lines using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system and sequenced the mRNA and miRNA transcriptomes. The results show that complete loss of MSTN upregulates seven miRNAs targeting an interaction network composed of 28 downregulated genes, including TGFB1, FOS and RB1. These genes are closely associated with tumorigenesis and cell proliferation. Our study suggests that complete loss of MSTN may limit excessive cell proliferation via activation of miRNAs. These data will contribute to the treatment of rhabdomyosarcoma (RMS).

Precise spatial control of cavitation erosion in a vessel phantom by using an ultrasonic standing wave.

In atherosclerotic inducement in animal models, the conventionally used balloon injury is invasive, produces excessive vessel injuries at unpredictable locations and is inconvenient in arterioles. Fortunately, cavitation erosion, which plays an important role in therapeutic ultrasound in blood vessels, has the potential to induce atherosclerosis noninvasively at predictable sites. In this study, precise spatial control of cavitation erosion for superficial lesions in a vessel phantom was realised by using an ultrasonic standing wave (USW) with the participation of cavitation nuclei and medium-intensity ultrasound pulses. The superficial vessel erosions were restricted between adjacent pressure nodes, which were 0.87 mm apart in the USW field of 1 MHz. The erosion positions could be shifted along the vessel by nodal modulation under a submillimetre-scale accuracy without moving the ultrasound transducers. Moreover, the cavitation erosion of the proximal or distal wall could be determined by the types of cavitation nuclei and their corresponding cavitation pulses, i.e., phase-change microbubbles with cavitation pulses of 5 MHz and SonoVue microbubbles with cavitation pulses of 1 MHz. Effects of acoustic parameters of the cavitation pulses on the cavitation erosions were investigated. The flow conditions in the experiments were considered and discussed. Compared to only using travelling waves, the proposed method in this paper improves the controllability of the cavitation erosion and reduces the erosion depth, providing a more suitable approach for vessel endothelial injury while avoiding haemorrhage.

[Study on the difference between components in volatile oil of Citrus reticulata before and after being processed].

OBJECTIVE: To analyze the change of components in volatile oil of Citrus reticulata before and after being processed. METHODS: The volatile oil of Citrus reticulata was extracted by steam distillation and comparatively studied by GC-MS. RESULTS: The content of volatile of Citrus reticulata after being steamed decreased from 1.13% to 1.06%. 34 components was detected in Citrus reticulata, 30 components was detected in the processed Citrus reticulata, 24 components in volatile oil of Citrus reticulata were identified. 15 components in volatile oil of Citrus reticulata before and after being processed were the same, 9 components were not detected after steamed, but there were 9 new kinds being detected. The relative contents of 4 components increased, 10 components decreased. CONCLUSION: There are many differences between the components in the volatile oil of Citrus reticulata before and after being processed and also the content of the components may offer some theoretical evidence for drug property transform and different clinical application.