Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Against Rubicon Ameliorates NAFLD by Modulating CD36 Along with Glycerophospholipid Metabolism.

Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of various chronic metabolic hepatic diseases with limited therapeutics. Rubicon, an essential regulator in lysosomal degradation, is reported to exacerbate hepatic steatosis in NAFLD mice and patients, indicating its probability of being a therapeutic target for NAFLD treatment. In this study, the therapeutic potential of Rubicon blockage is investigated. Lipid nanoparticles carrying Rubicon-specific CRISPR-Cas9 components exhibited liver accumulation, cell internalization, and Rubicon knockdown. A single administration of the nanoparticles results in attenuated lipid deposition and hepatic steatosis, with lower circulating lipid levels and decreased adipocyte size in NAFLD mice. Furthermore, the increase of phosphatidylcholine and phosphatidylethanolamine levels can be observed in the NAFLD mice livers after Rubicon silencing, along with regulatory effects on metabolism-related genes such as CD36, Gpcpd1, Chka, and Lpin2. The results indicate that knockdown of Rubicon improves glycerophospholipid metabolism and thereby ameliorates the NAFLD progression, which provides a potential strategy for NAFLD therapy via the restoration of Rubicon.

A Novel Bifunctional Fusion Protein (Anti-IL-17A-sST2) Protects against Acute Liver Failure, Modulating the TLR4/MyD88 Pathway and NLRP3 Inflammasome Activation.

Acute liver failure (ALF) is a serious inflammatory disorder with high mortality rates, which poses a significant threat to human health. The IL-33/ST2 signal is a crucial regulator in inflammation responses associated with lipopolysaccharide (LPS)-induced macrophages. The IL-17A signaling pathway promotes the release of chemokines and inflammatory cytokines, recruiting neutrophils and T cells under LPS stimulation, thus facilitating inflammatory responses. Here, the potential therapeutic benefits of neutralizing the IL-17A signal and modulating the IL-33/ST2 signal in ALF were investigated. A novel dual-functional fusion protein, anti-IL-17A-sST2, was constructed, which displayed high purity and biological activities. The administration of anti-IL-17A-sST2 resulted in significant anti-inflammatory benefits in ALF mice, amelioration of hepatocyte necrosis and interstitial congestion, and reduction in TNF-α and IL-6. Furthermore, anti-IL-17A-sST2 injection downregulated the expression of TLR4 and NLRP3 as well as important molecules such as MyD88, caspase-1, and IL-1ß. The results suggest that anti-IL-17A-sST2 reduced the secretion of inflammatory factors, attenuated the inflammatory response, and protected hepatic function by regulating the TLR4/MyD88 pathway and inhibiting the NLRP3 inflammasome, providing a new therapeutic approach for ALF.

RtNAC055 promotes drought tolerance via a stomatal closure pathway linked to methyl jasmonate/hydrogen peroxide signaling in Reaumuria trigyna.

The stomata regulate CO2 uptake and efficient water usage, thereby promoting drought stress tolerance. NAC proteins (NAM, ATAF1/2, and CUC2) participate in plant reactions following drought stress, but the molecular mechanisms underlying NAC-mediated regulation of stomatal movement are unclear. In this study, a novel NAC gene from Reaumuria trigyna, RtNAC055, was found to enhance drought tolerance via a stomatal closure pathway. It was regulated by RtMYC2 and integrated with jasmonic acid signaling and was predominantly expressed in stomata and root. The suppression of RtNAC055 could improve jasmonic acid and H2O2 production and increase the drought tolerance of transgenic R. trigyna callus. Ectopic expression of RtNAC055 in the Arabidopsis atnac055 mutant rescued its drought-sensitive phenotype by decreasing stomatal aperture. Under drought stress, overexpression of RtNAC055 in poplar promoted ROS (H2O2) accumulation in stomata, which accelerated stomatal closure and maintained a high photosynthetic rate. Drought upregulated the expression of PtRbohD/F, PtP5CS2, and PtDREB1.1, as well as antioxidant enzyme activities in heterologous expression poplars. RtNAC055 promoted H2O2 production in guard cells by directly binding to the promoter of RtRbohE, thus regulating stomatal closure. The stress-related genes RtDREB1.1/P5CS1 were directly regulated by RtNAC055. These results indicate that RtNAC055 regulates stomatal closure by maintaining the balance between the antioxidant system and H2O2 level, reducing the transpiration rate and water loss, and improving photosynthetic efficiency and drought resistance.

Effect of granulation on chlorine-release behavior during municipal solid waste incineration.

The preparation of refuse-derived fuel (RDF) is an effective and simple means of rural municipal solid waste utilization. The release of chlorine during RDF combustion is important as it causes high-temperature corrosion and pollutants emission such as HCl, dioxins, etc. In this paper, constant-temperature and increasing-temperature combustion experiments were carried out using an electrically heating furnace to analyse the effects of granulation (pressure and additives) on the release of chlorine in particles. During the constant-temperature combustion below 800 °C, only organic chlorine was released from the RDF. The increase of granulation pressure from 1 MPa to 10 MPa did not affect the total amount of chlorine release, but delayed the organic chlorine release by increasing the gas diffusion resistance. During the constant-temperature combustion above 900 °C, inorganic chlorine was released as well. The increase of granulation pressure enhanced the inorganic chlorine release significantly by promoting the reactants contact. During the increasing-temperature combustion, the increase of granulation pressure delayed the organic chlorine release as well but inhibited the inorganic chlorine release. This was mainly attributed to the slow temperature rise to 900 °C, during which the inherent calcium in the RDF reacted with silicon and aluminium, resulting in less reactants for an inorganic chlorine release reaction. Three calcium-based additives were used to inhibit chlorine release. CaCO3 showed no dechlorination effect, and CaO showed better dechlorination effect than Ca(OH)2. For the constant-temperature combustion at 900 °C, the addition of CaO with a Ca/Cl ratio of 2 achieved a dechlorination efficiency of over 90%, with little influence from the granulation pressure. For the increasing-temperature combustion, the granulation pressure had a significant influence on CaO dechlorination effectiveness. Only at a granulation pressure as high as 10 MPa, did the addition of CaO with the Ca/Cl ratio of 2.5 achieve a dechlorination efficiency of 95%.

RtNAC100 involved in the regulation of ROS, Na+ accumulation and induced salt-related PCD through MeJA signal pathways in recretohalophyte Reaumuria trigyna.

NAM, ATAF1/2, and CUC2 (NAC) proteins regulate plant responses to salt stress. However, the molecular mechanisms by which NAC proteins regulate salt-induced programmed cell death (PCD) are unclear. We identified 56 NAC genes, 35 of which had complete open reading frames with complete NAM domain, in the R. trigyna transcriptome. Salt stress and methyl jasmonate (MeJA) mediated PCD-induced leaf senescence in R. trigyna seedlings. Salt stress accelerated endogenous JA biosynthesis, upregulating RtNAC100 expression. This promoted salt-induced leaf senescence in R. trigyna by regulating RtRbohE and RtSAG12/20 and enhancing ROS accumulation. Transgenic assays showed that RtNAC100 overexpression aggravated salt-induced PCD in transgenic lines by promoting ROS and Na+ accumulation, ROS-Ca2+ hub activation, and PCD-related gene expression. Therefore, RtNAC100 induces PCD via the MeJA signaling pathway in R. trigyna under salt stress.

Optimization of Enzymatic Cell Disruption for Improving Lipid Extraction from Schizochytrium sp. through Response Surface Methodology.

This study is aimed to explore the optimal conditions of cell disruption in the extraction algae oil process, using alkaline protease to disrupt cell of Schizochytrium sp. to extract oil in this paper. The effects of enzymatic lysis temperature, enzymatic lysis time, enzyme dosage and pH value on oil yield and DHA yield were studied. Through the combination of single factor test and response surface design, the optimal cell disruption conditions were screened out. The fatty acid composition of algal oil was analyzed by gas chromatography-massspectrometry (GC-MS). The results showed that when the conditions were: enzymatic lysis temperature 55°C, enzymatic lysis time 9 h, enzyme dosage 3% of biomass and pH 8,oil yield and DHA yield reached the highest 14.52 g/L and 7.12 g/L, respectively. When the strains were cultured in 50 L fermentor, oil yield reached 26.27 g/L and DHA yield reached 12.89 g/L. They were 1.81 times higher than that in shake-flask cultivation. The optimization experiment provides the basis for the industrial production of Schizochytrium sp.

Enhancement of docosahexaenoic acid production by low-energy ion implantation coupled with screening method based on Sudan black B staining in Schizochytrium sp.

Schizochytrium sp. is a hopeful docosahexaenoic acid (DHA) producing candidate due to its rapid growth rate and high DHA proportion in total lipid content. In this study, low-energy ion implantation was applied to Schizochytrium sp. to induce high DHA-producing mutants. Screening these mutants by Sudan black B staining, a mutant strain S1 which showed a 61% improvement in DHA production than that of the parent strain was successfully selected. Subsequently, parameters of DHA production of mutant strain S1 were optimized in a 500-mL Erlenmeyer flask. Under the optimum fermentation conditions, the production of DHA and the percentage of DHA in total lipid of mutant strain S1 were 6.52g/L and 46.2%, respectively. This study provides an effective breeding strategy for improved DHA production of Schizochytrium sp. through combination of the novel mutagenesis technology, the effective screening method and fermentation optimization.

Multiple superoxide dismutase 1/splicing factor serine alanine 15 variants are associated with the development and progression of diabetic nephropathy: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Genetics study.

BACKGROUND: Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS: We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS: We observed association between rs17880135 in the 3' region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10(-3)) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS: Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

Multiple variants in vascular endothelial growth factor (VEGFA) are risk factors for time to severe retinopathy in type 1 diabetes: the DCCT/EDIC genetics study.

OBJECTIVE: We sought to determine if any common variants in the gene for vascular endothelial growth factor (VEGFA) are associated with long-term renal and retinal complications in type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 1,369 Caucasian subjects with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) Study had an average of 17 retinal photographs and 10 renal measures over 15 years. In the DCCT/EDIC, we studied 18 single nucleotide polymorphisms (SNPs) in VEGFA that represent all linkage disequilibrium bins (pairwise r(2) > or = 0.64) and tested them for association with time to development of severe retinopathy, three or more step progression of retinopathy, clinically significant macular edema, persistent microalbuminuria, and severe nephropathy. RESULTS: In a global multi-SNP test, there was a highly significant association of VEGFA SNPs with severe retinopathy (P = 6.8 x 10(-5))-the four other outcomes were all nonsignificant. In survival analyses controlling for covariate risk factors, eight SNPs showed significant association with severe retinopathy (P < 0.05). The most significant single SNP association was rs3025021 (hazard ratio 1.37 [95% CI 1.13-1.66], P = 0.0017). Family-based analyses of severe retinopathy provide evidence of excess transmission of C at rs699947 (P = 0.029), T at rs3025021 (P = 0.013), and the C-T haplotype from both SNPs (P = 0.035). Multi-SNP regression analysis including 15 SNPs, and allowing for pairwise interactions, independently selected 6 significant SNPs (P < 0.05). CONCLUSIONS: These data demonstrate that multiple VEGFA variants are associated with the development of severe retinopathy in type 1 diabetes.