Fibroblast growth factor 21 attenuates ventilator-induced lung injury by inhibiting the NLRP3/caspase-1/GSDMD pyroptotic pathway.

BACKGROUND: Ventilator-induced lung injury (VILI) is caused by overdistension of the alveoli by the repetitive recruitment and derecruitment of alveolar units. This study aims to investigate the potential role and mechanism of fibroblast growth factor 21 (FGF21), a metabolic regulator secreted by the liver, in VILI development. METHODS: Serum FGF21 concentrations were determined in patients undergoing mechanical ventilation during general anesthesia and in a mouse VILI model. Lung injury was compared between FGF21-knockout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect. RESULTS: Serum FGF21 levels in patients and mice with VILI were significantly higher than in those without VILI. Additionally, the increment of serum FGF21 in anesthesia patients was positively correlated with the duration of ventilation. VILI was aggravated in FGF21-KO mice compared with WT mice. Conversely, the administration of FGF21 alleviated VILI in both mouse and cell models. FGF21 reduced Caspase-1 activity, suppressed the mRNA levels of Nlrp3, Asc, Il-1ß, Il-18, Hmgb1 and Nf-κb, and decreased the protein levels of NLRP3, ASC, IL-1ß, IL-18, HMGB1 and the cleaved form of GSDMD. CONCLUSIONS: Our findings reveal that endogenous FGF21 signaling is triggered in response to VILI, which protects against VILI by inhibiting the NLRP3/Caspase-1/GSDMD pyroptosis pathway. These results suggest that boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for the treatment of VILI during anesthesia or critical care.

Pathogenesis of ventilator-induced lung injury: metabolomics analysis of the lung and plasma.

INTRODUCTION: Nowadays,the mechanical ventilation (MV) aims to rest the respiratory muscles while providing adequate gas exchange, and it has been a part of basic life support during general anesthesia as well as in critically ill patients with and without respiratory failure. However, MV itself has the potential to cause or worsen lung injury, which is also known as ventilator-induced lung injury (VILI). Thus, the early diagnosis of VILI is of great importance for the prevention and treatment of VILI. OBJECTIVE: This study aimed to investigate the metabolomes in the lung and plasma of mice receiving mechanical ventilation (MV). METHODS: Healthy mice were randomly assigned into control group; (2) high volume tidal (HV) group (30 ml/kg); (3) low volume tidal (LV) group (6 ml/kg). After ventilation for 4 h, mice were sacrificed and the lung tissue and plasma were collected. The lung and plasma were processed for the metabolomics analysis. We also performed histopathological examination on the lung tissue. RESULTS: We detected moderate inflammatory damage with alveolar septal thickening in the HV group compared with the normal and LV groups.The metabolomics analysis results showed MV altered the metabolism which was characterized by the dysregulation of γ-amino butyric acid (GABA) system and urea cycle (desregulations in plasma and lung guanidinosuccinic acid, argininosuccinic acid, succinic acid semialdehyde and lung GABA ), Disturbance of citric acid cycle (CAC) (increased plasma glutamine and lung phosphoenol pyruvate) and redox imbalance (desregulations in plasma and/or lung ascorbic acid, chenodeoxycholic acid, uric acid, oleic acid, stearidonic acid, palmitoleic acid and docosahexaenoic acid). Moreover, the lung and plasma metabolomes were also significantly different between LV and HV groups. CONCLUSIONS: Some lung and plasma metabolites related to the GABA system and urea cycle, citric acid cycle and redox balance were significantly altered, and they may be employed for the evaluation of VILI and serve as targets in the treatment of VILI.

Mouse Bone Marrow Mesenchymal Stem Cells Inhibit Sepsis-Induced Lung Injury in Mice via Exosomal SAA1.

Sepsis is a global disease burden, and approximately 40% of cases develop acute lung injury (ALI). Bone marrow mesenchymal stromal cells (BMSCs) and their exosomes are widely used in treating a variety of diseases including sepsis. As an acute phase protein, serum amyloid A1 (SAA1) regulates inflammation and immunity. However, the role of SAA1 in BMSCs-exosomes in septic lung injury remains to be elucidated. Exosomes derived from serum and BMSCs were isolated by ultracentrifugation. SAA1 was silenced or overexpressed in mouse BMSCs using lentiviral plasmids, containing either SAA1-targeting short interfering RNAs or SAA1 cDNA. Sepsis was induced by cecal ligation and puncture (CLP). LPS was used to induce ALI in mice. Mouse alveolar macrophages were isolated by flow cytometry. Levels of SAA1, endotoxin, TNF-α, and IL-6 were measured using commercial kits. LPS internalization was monitored by immunostaining. RT-qPCR or immunoblots were performed to test gene and protein expressions. Serum exosomes of patients with sepsis-induced lung injury had significantly higher levels of SAA1, endotoxin, TNF-α, and IL-6. Overexpression of SAA1 in BMSCs inhibited CLP- or LPS-induced lung injury and decreased CLP- or LPS-induced endotoxin, TNF-α, and IL-6 levels. Administration of the SAA1 blocking peptide was found to partially inhibit SAA1-induced LPS internalization by mouse alveolar macrophages and reverse the protective effect of SAA1. In conclusion, BMSCs inhibit sepsis-induced lung injury through exosomal SAA1. These results highlight the importance of BMSCs, exosomes, and SAA1, which may provide novel directions for the treatment of septic lung injury.

Elevated angiotensin II induces platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner during sepsis.

Thrombocytopenia is independently related with increased mortality in severe septic patients. Renin-angiotensin system (RAS) is elevated in septic subjects; accumulating studies show that angiotensin II (Ang II) stimulate the intrinsic apoptosis pathway by promoting reactive oxygen species (ROS) production. However, the mechanisms underlying the relationship of platelet apoptosis and RAS system in sepsis have not been fully elucidated. The present study aimed to elucidate whether the RAS was involved in the pathogenesis of sepsis-associated thrombocytopenia and explore the underlying mechanisms. We found that elevated plasma Ang II was associated with decreased platelet count in both patients with sepsis and experimental animals exposed to lipopolysaccharide (LPS). Besides, Ang II treatment induced platelet apoptosis in a concentration-dependent manner in primary isolated platelets, which was blocked by angiotensin II type 1 receptor (AT1R) antagonist losartan, but not by angiotensin II type 2 receptor (AT2R) antagonist PD123319. Moreover, inhibiting AT1R by losartan attenuated LPS-induced platelet apoptosis and alleviated sepsis-associated thrombocytopenia. Furthermore, Ang II treatment induced oxidative stress level in a concentration-dependent manner in primary isolated platelets, which was partially reversed by the AT1R antagonist losartan. The present study demonstrated that elevated Ang II directly stimulated platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner in sepsis-associated thrombocytopenia. The results would helpful for understanding the role of RAS system in sepsis-associated thrombocytopenia.

Construction of Axially Chiral Arylborons via Atroposelective Miyaura Borylation.

Compared with the well-developed centrally chiral boron chemistry, C-B axially chiral chemistry remains elusive and challenging. Herein we report the first atroposelective Miyaura borylation of bromoarenes with unsymmetrical diboron reagents for the direct catalytic synthesis of optically active atropisomeric arylborons. This reaction features broad substrate scope and produces axially chiral arylborons with high yields and good enantioselectivities.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot.

MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infection by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affects plant resistance to stem rot disease caused by Sclerotinia sclerotiorum. In Brassica rapa plants infected with S. sclerotiorum, miR319a levels increased while the expression levels of several BraTCP genes significantly decreased compared with those of uninfected plants. Overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas overexpression of BraTCP4-1 promoted plant resistance. RNA sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation, electrophoretic mobility shift, and reporter transaction assays showed that BraTCP4-1 could bind to the promoters of WRKY75, WRKY70, and WRKY33 and directly activate these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in plants overexpressing BraMIR319a decreased significantly, whereas those of plants overexpressing BraTCP4-1 increased significantly, relative to the wild type. These results suggest that miR319a and its target gene BraTCP4 control stem rot resistance through pathways of WRKY genes.

Meg3-DMR, not the Meg3 gene, regulates imprinting of the Dlk1-Dio3 locus.

The imprinted delta like 1 homolog (DLK1) - thyroxine deiodinase type III (DIO3) locus regulates development and growth. Its imprinting regulation involves two differentially methylated regions (DMRs), intergenic-DMR (IG-DMR) and maternally expressed gene 3-DMR (Meg3-DMR). In mice, a maternal deletion of the IG-DMR leads to LOI in the locus, proving that the IG-DMR is a cis-acting imprinting control region of the locus. However, the Meg3-DMR overlaps with the promoter, exon 1 and intron 1 of the Meg3 gene. Because deletion of the Meg3-DMR inactivates the Meg3 gene, their roles in imprinting regulation of Meg3-DMR mice is unknown. Therefore, we generated two mouse models: Meg3Δ(1-4) and Meg3Δ(2-4), respectively targeting exons 1-4 and exons 2-4 of the Meg3 gene. A maternal deletion of Meg3Δ(1-4) caused embryonic death and LOI in both embryos and placentas, but did not affect methylation status of the IG-DMR. In contrast, mice carrying a maternal deletion of Meg3Δ(2-4) were born normally and did not have LOI. These data indicate that it is the Meg3-DMR, not the Meg3 gene, which regulates imprinting of the Dlk1-Dio3 locus.

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits.

Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.

Downregulation of R-Spondin1 Contributes to Mechanical Stretch-Induced Lung Injury.

OBJECTIVES: The R-spondin family attenuates tissue damage via tightening endothelium and preventing vascular leakage. This study aims to investigate whether R-spondins protect against mechanical stretch-induced endothelial dysfunction and lung injury and to reveal the underlying mechanisms. DESIGN: Randomized controlled study. SETTING: University research laboratory. SUBJECTS: Patients scheduled to undergo surgery with mechanical ventilation support. Adult male Institute of Cancer Research mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Patients underwent a surgical procedure with mechanical ventilation support of 3 hours or more. Mice were subjected to mechanical ventilation (6 or 30 mL/kg) for 0.5-4 hours. Another group of mice were intraperitoneally injected with 1 mg/kg lipopolysaccharide, and 12 hours later subjected to mechanical ventilation (10 mL/kg) for 4 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 4 hours. MEASUREMENTS AND MAIN RESULTS: R-spondin1 were downregulated in both surgical patients and experimental animals exposed to mechanical ventilation. Intratracheal instillation of R-spondin1 attenuated, whereas knockdown of pulmonary R-spondin1 exacerbated ventilator-induced lung injury and mechanical stretch-induced lung vascular endothelial cell apoptosis. The antiapoptotic effect of R-spondin1 was mediated through the leucine-rich repeat containing G-protein coupled receptor 5 in cyclic stretched mouse lung vascular endothelial cells. We identified apoptosis-stimulating protein of p53 2 as the intracellular signaling protein interacted with leucine-rich repeat containing G-protein coupled receptor 5. R-spondin1 treatment decreased the interaction of apoptosis-stimulating protein of p53 2 with p53 while increased the binding of apoptosis-stimulating protein of p53 2 to leucine-rich repeat containing G-protein coupled receptor 5, therefore resulting in inactivation of p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. CONCLUSIONS: Mechanical ventilation leads to down-regulation of R-spondin1. R-spondin1 may enhance the interaction of leucine-rich repeat containing G-protein coupled receptor 5 and apoptosis-stimulating protein of p53 2, thus inactivating p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. R-spondin1 may have clinical benefit in alleviating mechanical ventilator-induced lung injury.

MiR-135a Protects Vascular Endothelial Cells Against Ventilator-Induced Lung Injury by Inhibiting PHLPP2 to Activate PI3K/Akt Pathway.

BACKGROUND/AIMS: Loss of endothelial barrier function plays an important role in the development of ventilator-induced lung injury (VILI). This study aimed to investigate the effects of miR135a on VILI in a model of mechanical stretch (MS)-induced human umbilical vein endothelial cell (HUVEC) injury. METHODS: HUVECs were randomly assigned to 7 groups: blank, negative control (NC), NC+MS, miR135a over-expression (mi-miR135a), mi-miR135a + MS, miR135a silencing (si-miR135a) and si-miR135a + MS groups. MS was induced by subjecting cells to cyclic stretch at 20% stretch for 4 h. After 24 h, levels of reactive oxygen species (ROS) were measured by DCFH-DA fluorescence intensity. Apoptosis was measured using annexin V-FITC/propidium iodide assay with flow cytometry. Inflammatory cytokine levels were determined by ELISA. Barrier integrity was determined using FITC-conjugated dextran assay. Expression levels of PI3K, p-PI3K, Akt, p-Akt, Bcl-2 and Bax were examined using western blotting. The interaction between miR135a and PHLPP2 was evaluated by dual-luciferase reporter assay. RESULTS: Our results showed that MS reduced cell numbers, increased the number of apoptotic cells, increased ROS, barrier dysfunction and inflammatory cytokines in HUVECs, and reduced p-PI3K and p-Akt expression; silencing of miR135a worsened MS-induced HUVEC injury. However, miR135a over-expression protected HUVECs against MS-induced increases in apoptotic cells, ROS, barrier dysfunction and inflammatory cytokines, which were accompanied by activation of the PI3K/Akt signaling pathway. Simultaneous silencing of miR135a and PHLPP2 partially salvaged the effects of miR135a silencing, and miR135a was found to interact with PHLPP2. CONCLUSION: miR135a may protect HUVECs from MS-induced injury by inhibiting PHLPP2 to activate PI3k/Akt signaling pathway.

NLRP3 Inflammasome Activation Contributes to Mechanical Stretch-Induced Endothelial-Mesenchymal Transition and Pulmonary Fibrosis.

OBJECTIVES: Mechanical ventilation can induce lung fibrosis. This study aimed to investigate whether ventilator-induced lung fibrosis was associated with endothelial-mesenchymal transition and to uncover the underlying mechanisms. DESIGN: Randomized, controlled animal study and cell culture study. SETTING: University research laboratory. SUBJECTS: Adult male Institute of Cancer Research, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) knockout and wild-type mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Institute of Cancer Research, NLRP3 knockout and wild-type mice were subjected to mechanical ventilation (20 mL/kg) for 2 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 24 hours. MEASUREMENTS AND MAIN RESULTS: Mice subjected to mechanical ventilation exhibited increases in collagen deposition, hydroxyproline and type I collagen contents, and transforming growth factor-ß1 in lung tissues. Ventilation-induced lung fibrosis was associated with increased expression of mesenchymal markers (α smooth muscle actin and vimentin), as well as decreased expression of endothelial markers (vascular endothelial-cadherin and CD31). Double immunofluorescence staining showed the colocalization of CD31/α smooth muscle actin, CD31/vimentin, and CD31/fibroblast-specific protein-1 in lung tissues, indicating endothelial-mesenchymal transition formation. Mechanical ventilation also induced NLRP3 inflammasome activation in lung tissues. In vitro direct mechanical stretch of primary mouse lung vascular endothelial cells resulted in similar NLRP3 activation and endothelial-mesenchymal transition formation, which were prevented by NLRP3 knockdown. Furthermore, mechanical stretch-induced endothelial-mesenchymal transition and pulmonary fibrosis were ameliorated in NLRP3-deficient mice as compared to wild-type littermates. CONCLUSIONS: Mechanical stretch may promote endothelial-mesenchymal transition and pulmonary fibrosis through a NLRP3-dependent pathway. The inhibition of endothelial-mesenchymal transition by NLRP3 inactivation may be a viable therapeutic strategy against pulmonary fibrosis associated with mechanical ventilation.

Heritability of targeted gene modifications induced by plant-optimized CRISPR systems.

The Streptococcus-derived CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system has emerged as a very powerful tool for targeted gene modifications in many living organisms including plants. Since the first application of this system for plant gene modification in 2013, this RNA-guided DNA endonuclease system has been extensively engineered to meet the requirements of functional genomics and crop trait improvement in a number of plant species. Given its short history, the emphasis of many studies has been the optimization of the technology to improve its reliability and efficiency to generate heritable gene modifications in plants. Here we review and analyze the features of customized CRISPR/Cas9 systems developed for plant genetic studies and crop breeding. We focus on two essential aspects: the heritability of gene modifications induced by CRISPR/Cas9 and the factors affecting its efficiency, and we provide strategies for future design of systems with improved activity and heritability in plants.

Proteomic Lung Analysis of Mice with Ventilator-Induced Lung Injury (VILI) Using iTRAQ-Based Quantitative Proteomics.

Ventilator-induced lung injury (VILI) has implications for mortality from acute lung injury (ALI) and for acute respiratory distress syndrome (ARDS) patients; the complicated mechanisms of VILI have not been well defined. To discover new biomarkers and mechanisms of VILI, isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-based quantitative proteomics were applied to identify differentially expressed proteins in mice treated with high tidal volume ventilation (HV), low tidal volume ventilation (LV) and lipopolysaccharide (LPS). A total of 14 dysregulated proteins showed the same change trend both in the LV and HV group and no change in the LPS group, and most importantly, the fold change of these proteins increased with the increase of volume ventilation, which indicates these proteins may be considered as potential markers specific for VILI. Ingenuity pathway analysis (IPA) canonical pathways analysis identified the top 4 canonical pathways, including the extrinsic prothrombin activation pathway, coagulation systems, the intrinsic prothrombin activation pathway and the acute phase response, suggesting that these pathways, as associated with these proteins' expression, may be important therapeutic targets for reducing VILI. These findings will provide a new perspective for understanding the pathogenesis of VILI in the future.

A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis.

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4⁻10%) and T2 (32.5⁻46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.

Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems.

We developed simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are co-expressed from a single Pol II promoter, without any additional processing machinery. Our SSTU systems are easy to construct and effective in mediating multiplex genome editing.

Salidroside Attenuates Ventilation Induced Lung Injury via SIRT1-Dependent Inhibition of NLRP3 Inflammasome.

BACKGROUND: Salidroside (SDS) is the main effective ingredient of Rhodiola rosea L with a variety of pharmacologic properties. We aim to investigate the effects of SDS on ventilation induced lung injury (VILI) and explore the possible underlying molecular mechanism. METHODS: Lung injury was induced in male ICR mice via mechanical ventilation (30 ml/kg) for 4h. The mice were divided in four groups:(1) Control group; (2) Ventilation group; (3) SDS group; (4) Ventilation with SDS group. SDS (50 mg/kg) was injected intraperitoneally 1h before operation. Mouse lung vascular endothelial cells (MLVECs) were subjected to cyclic stretch for 4h. RESULTS: It was found that SDS attenuated VILI as shown in HE staining, cell count and protein content levels in BAL fluid, W/D and Evans blue dye leakage into the lung tissue. SDS treatment inhibited the activation of NLRP3 inflammasome and subsequent caspase-1 cleavage as well as interleukin (IL)-1ß secretion both in vivo and in vitro. Moreover, SDS administration up-regulated SIRT1 expression. Importantly, knockdown of SIRT1 reversed the inhibitory effect of SDS on NLRP3 inflammasome activation. CONCLUSIONS: Taken together, these findings indicate that SDS may confer protection against ventilation induced lung injury via SIRT1-de-pendent inhibition of NLRP3 inflammasome activation.

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-ß1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-ß1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.