Search

Background and aims Messenger RNA (mRNA)-based therapeutics are rapidly progressing to the clinic and hold tremendous potential for benefiting millions of people worldwide. Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive aspect, however until recently, it has remained poorly explored. In this study, we examined for the first time, the therapeutic utility of mRNA delivery in liver fibrosis and cirrhosis, which contributes to millions of deaths, annually. Here, we aimed to demonstrate therapeutic efficacy of the human transcription factor hepatocyte nuclear factor alpha (HNF4A) encoding mRNA in chronically injured murine liver leading to fibrosis and cirrhosis. Methods We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNP) encapsulating HNF4A mRNA. To identify potential mechanisms, we performed microarray-based gene expression profiling, single cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Results Expression of HNF4A encoding mRNA led to restoration of metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of HNF4A mRNA encapsulated-LNP induced a robust inhibition of fibrogenesis in four independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Conclusion Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Lay summary Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality, worldwide. Herein, we take advantage of a promising, emerging mRNA therapy approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides the proof-of-concept that mRNA therapy would be a promising strategy for reversing liver fibrosis and cirrhosis.

Purple/red appearance is one of the common phenotypic variations in leaves, stems, and siliques of oilseed rape (Brassica napusL.) but very rare in flowers. In this study, the causal genes for the purple/red traits in stems and flowers in two accessions of oilseed rape (DH_PR and DH_GC001, respectively) derived from the wide hybridization were fine mapped and candidate genes were determined by methods combined with bulked segregant analysis (BSA) and RNA-seq analysis. Both purple stem trait and red flowers trait were mapped to the locus as the AtPAP2 homologous genes (BnaPAP2.C6a and BnaPAP2.A7b, respectively) belonging to the R2R3-MYB family. Sequencing of the BnaPAP2.C6a gene in purple stem accession revealed several Indels and SNPs in its promoter region, intron 1 as well as exons. However, only a 211bp insertion was identified in promoter region of BnaPAP2.A7b of DH_GC001. Our results not only contribute to a better understanding of anthocyanin inheritance in B. napus, but also provide a useful toolbox for future breeding of cultivars with purple/red traits through combination of different functional alleles and homologs.

Objectives. Tacrolimus is characterized by high pharmacokinetic variability in combination with a narrow therapeutic range. However, influence of donor CYP3A5 genotype and graft-to-recipient body weight ratio (GRWR) on tacrolimus’ pharmacokinetics after pediatric living donor liver transplantation (LDLT) remains unclear. Methods. A total of 174 LDLT recipients ( n = 40 ), SS/EX ( n = 38 ), LS/NEX ( n = 48 ), and LS/EX ( n = 48 ). Pharmacokinetics of tacrolimus and clinical outcomes were analyzed. Results. The relationships between the concentration-dose ratio and donor CYP3A5 genotypes and graft size were examined 3, 7, 14, and 30 days after the transplantation. Tacrolimus C0 levels varied greatly among groups, although recipients started with the same initial dosage. LS/EX recipients had significantly lower C0 levels in comparison with those of other groups. The use of CYP3A5-EX-grafts and a greater GRWR both resulted in significantly higher TAC dose requirements and lower C/D ratios. However, the significance of GRWR no longer exists 3 months after transplantation. The multivariate generalized linear mixed model analysis showed that donor CYP3A5 genotypes ( F = 11.876 ; P = 0.01 ) and GRWR ( F = 4.631 ; P = 0.033 ) were independent impact factors for C/D ratios 3, 7, 14, and 30 days after transplantation. Donor CYP3A5-EX genotype was associated with significantly increasing risks of infectious complications and significantly lower Cylex ATP values. However, no significant difference was observed in acute rejections among 4 groups. Conclusions. Monitoring of C0 levels alone is not reliable to guide tacrolimus administration. Donor CYP3A5 and GRWR both significantly affect tacrolimus pharmacokinetics after pediatric LDLT. The use of Cylex ATP tests would be helpful to avoid overimmunosuppression.

Orychophragmus violaceus is a Brassicaceae species widely cultivated in China, particularly as a winter cover crop in northern China because of its low-temperature tolerance and low water demand. Recently, O. violaceus has also been cultivated as a potential industrial oilseed crop because of its abundant 24-carbon dihydroxy fatty acids (diOH-FAs), which contribute to superior high-temperature lubricant properties. In this study, we performed de novo assembly of the O. violaceus genome. Whole-genome synteny analysis of the genomes of its relatives demonstrated that O. violaceus is a diploid that has undergone an extra whole-genome duplication (WGD) after the Brassicaceae-specific α-WGD event, with a basic chromosome number of x = 12. Formation of diOH-FAs is hypothesized to have occurred after the WGD event. Based on the genome and the transcriptome data from multiple stages of seed development, we predicted that OvDGAT1-1 and OvDGAT1-2 are candidate genes for the regulation of diOH-FA storage in O. violaceus seeds. These results may greatly facilitate the development of heat-tolerant and eco-friendly plant-based lubricants using O. violaceus seed oil and improve our understanding of the genomic evolution of Brassicaceae.

ObjectiveLiver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases.DesignWe analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter.ResultsWe showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver.ConclusionCollectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.

The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life‐threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)‐mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well‐established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno‐associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR‐125b‐5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV‐mediated miR‐125b‐5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR‐125b‐5p in primary mouse and human hepatocytes and contributes to the pro‐proliferative activity of miR‐125b‐5p by forkhead box G1 (FOXG1) and the cyclin‐dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR‐125b‐5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.

Summary Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement., Graphical Abstract, Highlights • Single AAV vector mediates efficient large fragment replacement in vivo and ex vivo • Fah-corrected iHeps repopulate the liver of recipient mice • Self-linearizing donor template enhances integration rate • AAV2 and AAV8 reveal differences in packaging the oversized AIO-SL vector genome, Genetics; Techniques in Genetics; Genetic Engineering

The power grid construction project is a complex system which is composed of many kinds of structures and sub projects, and affected by many factors, and its safety vulnerability is quite obvious to cause the occurrence of accidents. In order to provide a decision making to prevent and control the safety risk, the vulnerability of the power grid construction project would have been identified by both the technical and management factors based, and proposes a mathematical measurement model and the calculation steps of the safety risk value. By the case analysis, it proves that the model is practical and can be as a theoretical basis for the power infrastructure to prevent and reduce the disaster.

In the construction of hydraulic electric power, softening and slaking of swelling rock is a natural phenomenon, which is a complex process and affects the stability of rock engineering. It is difficult to describe and predict quantitatively the dynamical properties of the soft rock making use of a common method. Grey theory has proven successful in research such a complex system. This paper proposed GM(1,1) model based on remanent error analysis to the experimental fitting value of gargillite mechanical parameters, which is regarded as a new way of describing the nonlinear mechanical properties of soft rock and applied to predict the instability of engineering rockmass.

The power cable is one of the most important infrastructures, which safety and reliability is of an important significance to the stability and development of urban. To the safety risk features of urban power cable infrastructure, this paper proposed the safety risk evaluation index system and quantitative identification method based on analyzing the composition and construction features, and established the fuzzy comprehensive evaluation model of underground cable infrastructure safety risk. A case study shows that this method is feasible, and consistent with the practical construction, and proposed the measures to prevent the occurrence of major hazards.

The unstable slope rock is one of the geological disasters of mountains along the transmission lines, which includes the falling style and the slumping type, and the former is very dangerous, and threat to the security of transmission line. By means of a typical example study about the falling dangerous rock mass along the 10kV transmission lines, this paper analyzes qualitatively the basic characteristics, failure mechanism, influencing factors of the dangerous rock, et al., and calculated quantitatively the maximum depth affected by the impact of the dangerous rock to the transmission lines. The paper proposes the response measures of dangerous rocks risk, which is of a certain reference in other similar projects.

The power cable infrastructure is a high risk system in a city, and various risk factors are in the mutual influence, which are distributed throughout the whole process of project management. Based on the composition and construction features of power cable project, this paper proposed the process of security risk identification, and the safety risk evaluation index of power cable system has established, and the quantitative risk assessment and classification. By identifying the main risk of power cable system, this paper proposed strengthening the monitoring and management of risk, and the effective risk countermeasures to avoid, transfer and control risk.

All risk events of power grid usually show the complex logic relation of the factors caused by the disaster. This paper analyzed the fault reasons based on the systematology, and proposed the fuzzy fault tree model of power project in the smart construction, and distinguished the safety risk of the transmission tower collapses. It realized that the fault probability caused by the strong storm is undefined, and this paper regards the probability of the basic event as fuzzy datum, and calculated the occurrence probability of transmission tower collapses based on the fault tree logic structure analysis of intermediate and top events. The applied example shows that it is possible applicable to using the fuzzy fault tree method to analyze the safety risk of the tower collapse accidents of power project.

Sinapis alba and S. arvensis are mustard crops within the Brassiceae tribe of the Brassicaceae family, and represent an important genetic resource for crop improvement. We performed de novo assembly of Brassica nigra, S. alba and S. arvensis, and conducted comparative genomics to investigate the pattern of genomic evolution since an ancient whole-genome triplication (WGT) event. Both Sinapis species retained evidence of the Brassiceae WGT ~20.5 million years ago (Mya), with subgenome dominance observed in gene density, gene expression and selective constraint. While S. alba diverged from the ancestor of Brassica and Raphanus at ~ 12.5 Mya, the divergence time of S. arvensis and B. nigra was ~6.5 Mya. S. arvensis and B. nigra had greater collinearity compared with their relationship to either B. rapa or B. oleracea. Two chromosome of S. alba (Sal03 and Sal08) were completely collinear with two ancestral chromosomes proposed in the Ancestral Crucifer Karyotype (ACK) genomic block model, the first time this has been observed in the Brassiceae. These results are consistent with S. alba representing a relatively ancient lineage of the species evolved from the common ancestor of tribe Brassiceae, and suggest that the phylogeny of the Brassica and Sinapis genera requires some revision. Our study provides new insights into the genome evolution and phylogenetic relationships of Brassiceae and provides genomic information for genetic improvement of these plants.

We identified two new transposon insertions within the promoter of BnaFT.A2 in addition to an existing 288 bp MITE within the second intron. Each insertion event corresponds to a distinct BnaFT.A2 haplotype and is closely associated with established crop seasonal ecotypes. Florigen, encoded by FLOWERING LOCUS T (FT), plays key roles not only as a flowering hormone, but also a universal growth factor affecting several aspects of plant architecture. In rapeseed, BnaFT.A2 has been revealed as one of the major loci associated with flowering time and different ecotypes. However, it is unclear how allelic variations of BnaFT.A2 affect its function in flowering time regulation and beyond. In this study, we confirmed an existing 288 bp miniature inverted-repeat transposable element (MITE) insertion within the second intron and identified two new insertions within the promoter of BnaFT.A2-a 3971 bp CACTA and a 1079 bp Helitron. Each insertion event corresponds to a distinct BnaFT.A2 haplotype and is closely associated with established crop seasonal ecotypes. These alleles have similar tissue-specific expression patterns but discrete transcriptional patterns tightly associated with rapeseed flowering time and ecotype. RNAi lines and mutants of BnaFT.A2 flowered significantly later than controls. Differentially expressed genes (DEGs), identified in transcriptomic profiling of seedling leaves from two loss-of-function mutants (Bnaft.a2-L1 and Bnaft.a2-L2) compared with controls, indicated significant enrichment for hormone metabolic genes and roles related to plant cell wall synthesis and photosynthesis. Plants with loss-of-function BnaFT.A2 had smaller leaves and lower net photosynthetic rate compared to controls. These findings not only further clarify the genetic basis of flowering time variation and ecotype formation in B. napus, but also provide an additional toolbox for genetic improvement of seasonal adaptation and production.