PaMYB11 promotes suberin deposition in Norway spruce embryogenic tissue during cryopreservation: A novel resistance mechanism against osmosis.

The osmotic resistance mechanism has been extensively studied in whole plants or plant tissues. However, little is known about it in embryogenic tissue (ET) which is widely used in plant-based biotechnological systems. Suberin, a cell wall aliphatic and aromatic heteropolymer, plays a critical role in plant cells against osmosis stress. The suberin regulatory biosynthesis has rarely been studied in gymnosperms. Here, PaMYB11, a subgroup 11 R2R3-MYB transcription factor, plays a key role in the osmotic resistance of Norway spruce (Picea abies) ETs during cryoprotectant pretreatment. Thus, RNA-seq, histological, and analytical chemical analyses are performed on the stable transformations of PaMYB11-OE and PaMYB11-SRDX in Norway spruce ETs. DAP-seq, Y1H, and LUC are further combined to explore the PaMYB11 targets. Activation of PaMYB11 is necessary and sufficient for suberin lamellae deposition on Norway spruce embryogenic cell walls, which plays a decisive role in ET survival under osmotic stress. Transcriptome analysis shows that PaMYB11 enhances suberin lamellae monomer synthesis by promoting very long-chain fatty acid (VLCFA) synthesis. PaPOP, PaADH1, and PaTET8L, the first two (PaADH1 and PaPOP, included) involved in VLCFA synthesis, are proved to be the direct targets of PaMYB11. Our study identified a novel osmotic response directed by PaMYB11 in Norway spruce ET, which provides a new understanding of the resistance mechanism against osmosis in gymnosperms.

Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development.

BACKGROUND: Conserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms. RESULTS: To launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies. CONCLUSIONS: This study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Defense against membership inference attack in graph neural networks through graph perturbation.

Graph neural networks have demonstrated remarkable performance in learning node or graph representations for various graph-related tasks. However, learning with graph data or its embedded representations may induce privacy issues when the node representations contain sensitive or private user information. Although many machine learning models or techniques have been proposed for privacy preservation of traditional non-graph structured data, there is limited work to address graph privacy concerns. In this paper, we investigate the privacy problem of embedding representations of nodes, in which an adversary can infer the user's privacy by designing an inference attack algorithm. To address this problem, we develop a defense algorithm against white-box membership inference attacks, based on perturbation injection on the graph. In particular, we employ a graph reconstruction model and inject a certain size of noise into the intermediate output of the model, i.e., the latent representations of the nodes. The experimental results obtained on real-world datasets, along with reasonable usability and privacy metrics, demonstrate that our proposed approach can effectively resist membership inference attacks. Meanwhile, based on our method, the trade-off between usability and privacy brought by defense measures can be observed intuitively, which provides a reference for subsequent research in the field of graph privacy protection.

Integrated Lipidomic and Transcriptomic Analysis Reveals Phospholipid Changes in Somatic Embryos of Picea asperata in Response to Partial Desiccation.

Partial desiccation treatment (PDT) is an effective technology for promoting the germination and conversion of conifer somatic embryos (SEs). PDT, as a drought stress, induces intensive physiological responses in phospholipid metabolism, which are not well understood in the conifer SEs. Here, we integrated lipidomics, transcriptomics and proteomics analyses to reveal the molecular basis of lipid remodeling under PDT in Picea asperata SEs. Among the 82 lipid molecular species determined by mass spectrometry, phosphatidic acid (PA) had a significant effect after PDT and was the most critical lipid in the response to PDT. The transcriptomics results showed that multiple transcripts in the glycerolipid and glycerophospholipid metabolism pathways were differentially expressed, and these included five PLDα1 transcripts that catalyze the conversion of phosphatidylcholine (PC) to PA. Furthermore, the enzyme activity of this phospholipase D (PLD) was significantly enhanced in response to PDT, and PDT also significantly increased the protein level of PLDα1 (MA_10436582g0020). In addition, PA is a key factor in gibberellin, abscisic acid and ethylene signal transduction. One GDI1, one DELLA, three ABI1s, two SnRK2s, one CTR and 12 ERFs showed significantly differential expression between SEs before and after PDT in this study. Our data suggest that the observed increases in the PA contents might result from the activation of PLDα by PDT. PA not only affects the physical and chemical properties of the cell membrane but also participates in plant hormone signal transduction. Our work provides novel insight into the molecular mechanism through which PDT promotes the germination of SEs of coniferous tree species and fills the gap in the understanding of the mechanism of somatic embryo lipid remodeling in response to PDT.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

OBJECTIVE: Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS: Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

Suppression of Vascular Macrophage Activation by Nitro-Oleic Acid and its Implication for Abdominal Aortic Aneurysm Therapy.

PURPOSE: Abdominal aortic aneurysm (AAA) is one of the leading causes of death in the developed world and is currently undertreated due to the complicated nature of the disease. Herein, we aimed to address the therapeutic potential of a novel class of pleiotropic mediators, specifically a new drug candidate, nitro-oleic acid (NO2-OA), on AAA, in a well-characterized murine AAA model. METHODS: We generated AAA using a mouse model combining AAV.PCSK9-D377Y induced hypercholesterolemia with angiotensin II given by chronic infusion. Vehicle control (PEG-400), oleic acid (OA), or NO2-OA were subcutaneously delivered to mice using an osmotic minipump. We characterized the effects of NO2-OA on pathophysiological responses and dissected the underlying molecular mechanisms through various in vitro and ex vivo strategies. RESULTS: Subcutaneous administration of NO2-OA significantly decreased the AAA incidence (8/28 mice) and supra-renal aorta diameters compared to mice infused with either PEG-400 (13/19, p = 0.0117) or OA (16/23, p = 0.0078). In parallel, the infusion of NO2-OA in the AAA model drastically decreased extracellular matrix degradation, inflammatory cytokine levels, and leucocyte/macrophage infiltration in the vasculature. Administration of NO2-OA reduced inflammation, cytokine secretion, and cell migration triggered by various biological stimuli in primary and macrophage cell lines partially through activation of the peroxisome proliferator-activated receptor-gamma (PPARγ). Moreover, the protective effect of NO2-OA relies on the inhibition of macrophage prostaglandin E2 (PGE2)-induced PGE2 receptor 4 (EP4) cAMP signaling, known to participate in the development of AAA. CONCLUSION: Administration of NO2-OA protects against AAA formation and multifactorial macrophage activation. With NO2-OA currently undergoing FDA approved phase II clinical trials, these findings may expedite the use of this nitro-fatty acid for AAA therapy.

Multi-omics sequencing provides insight into floral transition in Catalpa bungei. C.A. Mey.

BACKGROUND: Floral transition plays an important role in development, and proper time is necessary to improve the value of valuable ornamental trees. The molecular mechanisms of floral transition remain unknown in perennial woody plants. "Bairihua" is a type of C. bungei that can undergo floral transition in the first planting year. RESULTS: Here, we combined short-read next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to provide a more complete view of transcriptome regulation during floral transition in C. bungei. The circadian rhythm-plant pathway may be the critical pathway during floral transition in early flowering (EF) C. bungei, according to horizontal and vertical analysis in EF and normal flowering (NF) C. bungei. SBP and MIKC-MADS-box were seemingly involved in EF during floral transition. A total of 61 hub genes were associated with floral transition in the MEturquoise model with Weighted Gene Co-expression Network Analysis (WGCNA). The results reveal that ten hub genes had a close connection with the GASA homologue gene (Cbu.gene.18280), and the ten co-expressed genes belong to five flowering-related pathways. Furthermore, our study provides new insights into the complexity and regulation of alternative splicing (AS). The ratio or number of isoforms of some floral transition-related genes is different in different periods or in different sub-genomes. CONCLUSIONS: Our results will be a useful reference for the study of floral transition in other perennial woody plants. Further molecular investigations are needed to verify our sequencing data.

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei.

BACKGROUND: "Bairihua", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. RESULTS: SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in "Bairihua" and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in "Bairihua". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. CONCLUSIONS: Our results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

Endothelial TFEB (Transcription Factor EB) Positively Regulates Postischemic Angiogenesis.

RATIONALE: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. OBJECTIVE: We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. METHODS AND RESULTS: In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). CONCLUSIONS: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

KLF11 (Krüppel-Like Factor 11) Inhibits Arterial Thrombosis via Suppression of Tissue Factor in the Vascular Wall.

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Construction of a high-density genetic map and QTL mapping of leaf traits and plant growth in an interspecific F1 population of Catalpa bungei × Catalpa duclouxii Dode.

BACKGROUND: Catalpa bungei is an important tree species used for timber in China and widely cultivated for economic and ornamental purposes. A high-density linkage map of C. bungei would be an efficient tool not only for identifying key quantitative trait loci (QTLs) that affect important traits, such as plant growth and leaf traits, but also for other genetic studies. RESULTS: Restriction site-associated DNA sequencing (RAD-seq) was used to identify molecular markers and construct a genetic map. Approximately 280.77 Gb of clean data were obtained after sequencing, and in total, 25,614,295 single nucleotide polymorphisms (SNPs) and 2,871,647 insertions-deletions (InDels) were initially identified in the genomes of 200 individuals of a C. bungei (7080) × Catalpa duclouxii (16-PJ-3) F1 population and their parents. Finally, 9072 SNP and 521 InDel markers that satisfied the requirements for constructing a genetic map were obtained. The integrated genetic map contained 9593 pleomorphic markers in 20 linkage groups and spanned 3151.63 cM, with an average distance between adjacent markers of 0.32 cM. Twenty QTLs for seven leaf traits and 13 QTLs for plant height at five successive time points were identified using our genetic map by inclusive composite interval mapping (ICIM). Q16-60 was identified as a QTL for five leaf traits, and three significant QTLs (Q9-1, Q18-66 and Q18-73) associated with plant growth were detected at least twice. Genome annotation suggested that a cyclin gene participates in leaf trait development, while the growth of C. bungei may be influenced by CDC48C and genes associated with phytohormone synthesis. CONCLUSIONS: This is the first genetic map constructed in C. bungei and will be a useful tool for further genetic study, molecular marker-assisted breeding and genome assembly.

Brown Adipocyte-Specific PPARγ (Peroxisome Proliferator-Activated Receptor γ) Deletion Impairs Perivascular Adipose Tissue Development and Enhances Atherosclerosis in Mice.

Objective- Perivascular adipose tissue (PVAT) contributes to vascular homeostasis by producing paracrine factors. Previously, we reported that selective deletion of PPARγ (peroxisome proliferator-activated receptor γ) in vascular smooth muscle cells resulted in concurrent loss of PVAT and enhanced atherosclerosis in mice. To address the causal relationship between loss of PVAT and atherosclerosis, we used BA-PPARγ-KO (brown adipocyte-specific PPARγ knockout) mice. Approach and Results- Deletion of PPARγ in brown adipocytes did not affect PPARγ in white adipocytes or vascular smooth muscle cells or PPARα and PPARδ expression in brown adipocytes. However, development of PVAT and interscapular brown adipose tissue was remarkably impaired, associated with reduced expression of genes encoding lipogenic enzymes in the BA-PPARγ-KO mice. Thermogenesis in brown adipose tissue was significantly impaired with reduced expression of thermogenesis genes in brown adipose tissue and compensatory increase in subcutaneous and gonadal white adipose tissues. Remarkably, basal expression of inflammatory genes and macrophage infiltration in PVAT and brown adipose tissue were significantly increased in the BA-PPARγ-KO mice. BA-PPARγ-KO mice were crossbred with ApoE KO (apolipoprotein E knockout) mice to investigate the development of atherosclerosis. Flow cytometry analysis confirmed increased systemic and PVAT inflammation. Consequently, atherosclerotic lesions were significantly increased in mice with impaired PVAT development, thus indicating that the lack of normal PVAT is sufficient to drive increased atherosclerosis. Conclusions- PPARγ is required for functional PVAT development. PPARγ deficiency in PVAT, while still expressed in vascular smooth muscle cell, enhances atherosclerosis and results in vascular and systemic inflammation, providing new insights on the specific roles of PVAT in atherosclerosis and cardiovascular disease at large.

Quantitative Phosphoproteomic and Physiological Analyses Provide Insights into the Formation of the Variegated Leaf in Catalpa fargesii.

Variegated plants are valuable materials for investigating leaf color regulated mechanisms. To unveil the role of posttranslational modification in the variegated phenotype, we conducted global quantitative phosphoproteomic analysis on different leaf color sectors of Maiyuanjinqiu and the corresponding of Catalpa fargesii using Ti4+-IMAC phosphopeptide enrichment. A total of 3778 phosphorylated sites assigned to 1646 phosphoproteins were identified, and 3221 in 1434 proteins were quantified. Differential phosphoproteins (above 1.5 or below 1/1.5) in various leaf color sectors were selected for functional enrichment analyses. Gene ontology (GO) enrichment revealed that processes of photosynthesis, regulation of the generation of precursor metabolites, response to stress, homeostasis, amino acid metabolism, transport-related processes, and most of the energy metabolisms might contribute to leaf color. KEGG pathway enrichment analysis was performed based on differential phosphoproteins (DPs) in different organelles. The result showed that most enriched pathways were located in the chloroplasts and cytosol. The phosphorylation levels of glycometabolism enzymes might greatly affect leaf variegation. Measurements of fluorescence parameters and enzyme activities confirmed that protein phosphorylation could affect plant physiology by regulating enzyme activity. These results provide new clues for further study the formation mechanisms of naturally variegated phenotype.

Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid.

Nitro-conjugated linoleic acid (NO2-CLA) is formed by metabolic and inflammatory reactions of nitric oxide and nitrite, and represents the most abundant nitro-fatty acid species in humans. These electrophilic fatty acid nitroalkene derivatives mediate pleiotropic cell signaling responses. Here, we report a systematic approach to investigate the effect of NO2-CLA on human coronary artery smooth muscle cells (hCASMC), based on the RNA-Seq and bioinformatics analysis. There were extensive differentially expressed genes in NO2-CLA vs. control (510) and NO2-CLA vs. CLA (272) treatment groups, respectively. Notably, only minimal alterations were observed in CLA vs. control conditions, indicating that the electrophilic character of NO2-CLA is requited to induce differential gene expression responses independently from native CLA. Functional enrichment analysis of differentially expressed genes reveals multiple cellular processes to be affected under NO2-CLA treatment, including cell proliferation, lipid metabolism, antioxidant and inflammatory-related gene expression responses. These findings reveal that nitro-fatty acid derivatives such as NO2-CLA regulate a broad array of adaptive gene expression responses by hCASMC.

Yes-Associated Protein Inhibits Transcription of Myocardin and Attenuates Differentiation of Vascular Smooth Muscle Cell from Cardiovascular Progenitor Cell Lineage.

Vascular smooth muscle cells (VSMCs) derived from cardiovascular progenitor cell (CVPC) lineage populate the tunica media of the aortic root. Understanding differentiation of VSMCs from CVPC will further our understanding of the molecular mechanisms contributing to aortic root aneurysms, and thus, facilitate the development of novel therapeutic agents to prevent this devastating complication. It is established that the yes-associated protein (YAP) and Hippo pathway is important for VSMC proliferation and phenotype switch. To determine the role of YAP in differentiation of VSMCs from CVPCs, we utilized the in vitro monolayer lineage specific differentiation method by differentiating human embryonic stem cells into CVPCs, and then, into VSMCs. We found that expression of YAP decreased during differentiation of VSMC from CVPCs. Overexpression of YAP attenuated expression of VSMC contractile markers and impaired VSMC function. Knockdown of YAP increased expression of contractile proteins during CVPC-VSMCs differentiation. Importantly, expression of YAP decreased transcription of myocardin during this process. Overexpression of YAP in PAC1 SMC cell line inhibited luciferase activity of myocardin proximal promoter in a dose dependent and NKX2.5 dependent manners. YAP protein interacted with NKX2.5 protein and inhibited binding of NKX2.5 to the 5'-proximal promoter region of myocardin in CVPC-derived VSMCs. In conclusion, YAP negatively regulates differentiation of VSMCs from CVPCs by decreasing transcription of myocardin in a NKX2.5-dependent manner. Stem Cells 2017;35:351-361.

Genome-wide analysis of long non-coding RNAs in Catalpa bungei and their potential function in floral transition using high-throughput sequencing.

BACKGROUND: Long non-coding RNAs (lncRNAs) have crucial roles in various biological regulatory processes. However, the study of lncRNAs is limited in woody plants. Catalpa bungei is a valuable ornamental tree with a long cultivation history in China, and a deeper understanding of the floral transition mechanism in C. bungei would be interesting from both economic and scientific perspectives. RESULTS: In this study, we categorized C. bungei buds from early flowering (EF) and normal flowering (NF) varieties into three consecutive developmental stages. These buds were used to systematically study lncRNAs during floral transition using high-throughput sequencing to identify molecular regulatory networks. Quantitative real-time PCR was performed to study RNA expression changes in different stages. In total, 12,532 lncRNAs and 26,936 messenger RNAs (mRNAs) were detected. Moreover, 680 differentially expressed genes and 817 differentially expressed lncRNAs were detected during the initiation of floral transition. The results highlight the mRNAs and lncRNAs that may be involved in floral transition, as well as the many lncRNAs serving as microRNA precursors. We predicted the functions of lncRNAs by analysing the relationships between lncRNAs and mRNAs. Seven lncRNA-mRNA interaction pairs may participate in floral transition. CONCLUSIONS: This study is the first to identify lncRNAs and their potential functions in floral transition, providing a starting point for detailed determination of the functions of lncRNAs in C. bungei.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

OBJECTIVE: CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. APPROACH AND RESULTS: We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. CONCLUSIONS: These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits.

Hepatic Transmembrane 6 Superfamily Member 2 Regulates Cholesterol Metabolism in Mice.

BACKGROUND & AIMS: The rs58542926 C>T variant of the transmembrane 6 superfamily member 2 gene (TM6SF2), encoding an E167K amino acid substitution, has been correlated with reduced total cholesterol (TC) and cardiovascular disease. However, little is known about the role of TM6SF2 in metabolism. We investigated the long-term effects of altered TM6SF2 levels in cholesterol metabolism. METHODS: C57BL/6 mice (controls), mice that expressed TM6SF2 specifically in the liver, and mice with CRISPR/Cas9-mediated knockout of Tm6sf2 were fed chow or high-fat diets. Blood samples were collected from all mice and plasma levels of TC, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol, and triglycerides were measured. Liver tissues were collected and analyzed by histology, real-time polymerase chain reaction, and immunoblot assays. Adenovirus vectors were used to express transgenes in cultured Hep3B hepatocytes. RESULTS: Liver-specific expression of TM6SF2 increased plasma levels of TC and LDL-c, compared with controls, and altered liver expression of genes that regulate cholesterol metabolism. Tm6sf2-knockout mice had decreased plasma levels of TC and LDL-c, compared with controls, and consistent changes in expression of genes that regulate cholesterol metabolism. Expression of TM6SF2 promoted cholesterol biosynthesis in hepatocytes. CONCLUSIONS: TM6SF2 regulates cholesterol metabolism in mice and might be a therapeutic target for cardiovascular disease.

WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce.

BACKGROUND: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). RESULTS: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. CONCLUSIONS: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.