Overexpression of the Phosphatidylcholine:DiacylglycerolCholinephosphotransferase (PDCT) gene increases carbon flux toward triacylglycerol (TAG) synthesis in Camelinasativa seeds.

Camelinasativa has considerable promise as a dedicated industrial oilseed crop. Its oil-based blends have been tested and approved as liquid transportation fuels. Previously, we utilized metabolomic and transcriptomic profiling approaches and identified metabolic bottlenecks that control oil production and accumulation in seeds. Accordingly, we selected candidate genes for the metabolic engineering of Camelina. Here we targeted the overexpression of Camelina PDCT gene, which encodes the phosphatidylcholine: diacylglycerol cholinephosphotransferase enzyme. PDCT is proposed as a gatekeeper responsible for the interconversions of diacylglycerol (DAG) and phosphatidylcholine (PC) pools and has the potential to increase the levels of TAG in seeds. To confirm whether increased CsPDCT activity in developing Camelina seeds would enhance carbon flux toward increased levels of TAG and alter oil composition, we overexpressed the CsPDCT gene under the control of the seed-specific phaseolin promoter. Camelina transgenics exhibited significant increases in seed yield (19-56%), seed oil content (9-13%), oil yields per plant (32-76%), and altered polyunsaturated fatty acid (PUFA) content compared to their parental wild-type (WT) plants. Results from [14C] acetate labeling of Camelina developing embryos expressing CsPDCT in culture indicated increased rates of radiolabeled fatty acid incorporation into glycerolipids (up to 64%, 59%, and 43% higher in TAG, DAG, and PC, respectively), relative to WT embryos. We conclude that overexpression of PDCT appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, thereby further increasing oil yields in Camelina.

Exploring Camelina sativa lipid metabolism regulation by combining gene co-expression and DNA affinity purification analyses.

Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa.

To ensure global food security under the changing climate, there is a strong need for developing 'climate resilient crops' that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate.

Relationship Between Progression-Free Survival, Objective Response Rate, and Overall Survival in Clinical Trials of PD-1/PD-L1 Immune Checkpoint Blockade: A Meta-Analysis.

PD-1/PD-L1 immune checkpoint blockade (ICB) has improved overall survival (OS) in solid tumor trials; however, parallel improvements in Response Evaluation Criteria in Solid Tumors (RECIST)-based surrogate end points, progression-free survival (PFS), and objective response rate (ORR), are not always observed. Here, we assess the surrogacy of PFS/ORR for OS with ICB therapy across advanced/metastatic tumors. In a trial-level analysis (N = 40 randomized trials), PFS, ORR, and OS treatment effects were correlated (Spearman's rho). In a patient-level analysis, data were extracted from available trials of durvalumab; the correlation of PFS and OS was evaluated (Bayesian normal-induced-copula-estimation model) and the ordinal association between objective response and OS hazard ratio (HR) were assessed with concordance index measures. High correlation was observed between PFS HR and OS HR in intention-to-treat (ITT; rho = 0.76) and PD-L1-enriched populations (0.74); modest (or limited) benefit in PFS was associated with meaningful improvement in OS. Moderate correlations were observed between ΔORR and OS HR: ITT, -0.63; PD-L1-enriched, -0.53. At the patient level, a positive association was observed between PFS and OS in non-small cell lung cancer (Kendall's Tau = 0.793; 95% confidence interval, 0.789-0.797), head and neck squamous cell carcinoma (0.794; 0.789-0.798), and bladder cancer (0.872; 0.869-0.875). Objective responders had significantly better OS (concordance index > 0.9) than nonresponders across these tumor types. Modest (or limited) improvement in RECIST-based end points did not rule out meaningful OS benefit, indicating they are imperfect surrogates and do not fully capture ICB clinical benefit. Therefore, caution is advised when basing early discontinuation of novel ICB agents on these end points.

Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase.

BACKGROUND: Camelina sativa has attracted much interest as alternative renewable resources for biodiesel, other oil-based industrial products and a source for edible oils. Its unique oil attributes attract research to engineering new varieties of improved oil quantity and quality. The overexpression of enzymes catalyzing the synthesis of the glycerol backbone and the sequential conjugation of fatty acids into this backbone is a promising approach for increasing the levels of triacylglycerol (TAG). In a previous study, we co-expressed the diacylglycerol acyltransferase (DGAT1) and glycerol-3-phosphate dehydrogenase (GPD1), involved in TAG metabolism, in Camelina seeds. Transgenic plants exhibited a higher-percentage seed oil content, a greater seed mass, and overall improved seed and oil yields relative to wild-type plants. To further increase seed oil content in Camelina, we utilized metabolite profiling, in conjunction with transcriptome profiling during seed development to examine potential rate-limiting step(s) in the production of building blocks for TAG biosynthesis. RESULTS: Transcriptomic analysis revealed approximately 2518 and 3136 transcripts differentially regulated at significant levels in DGAT1 and GPD1 transgenics, respectively. These transcripts were found to be involved in various functional categories, including alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. We quantified the relative contents of over 240 metabolites. Our results indicate major metabolic switches in transgenic seeds associated with significant changes in the levels of glycerolipids, amino acids, sugars, and organic acids, especially the TCA cycle and glycolysis intermediates. CONCLUSIONS: From the transcriptomic and metabolomic analysis of DGAT1, GPD1 and DGAT1 + GPD1 expressing lines of C. sativa, we conclude that TAG production is limited by (1) utilization of fixed carbon from the source tissues supported by the increase in glycolysis pathway metabolites and decreased transcripts levels of transcription factors controlling fatty acids synthesis; (2) TAG accumulation is limited by the activity of lipases/hydrolases that hydrolyze TAG pool supported by the increase in free fatty acids and monoacylglycerols. This comparative transcriptomics and metabolomics approach is useful in understanding the regulation of TAG biosynthesis, identifying bottlenecks, and the corresponding genes controlling these pathways identified as limitations, for generating Camelina varieties with improved seed and oil yields.

A stress-associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses.

Members of Stress-Associated Protein (SAP) family in plants have been shown to impart tolerance to multiple abiotic stresses, however, their mode of action in providing tolerance to multiple abiotic stresses is largely unknown. There are 14 SAP genes in Arabidopsis thaliana containing A20, AN1, and Cys2-His2 zinc finger domains. AtSAP13, a member of the SAP family, carries two AN1 zinc finger domains and an additional Cys2-His2 domain. AtSAP13 transcripts showed upregulation in response to Cd, ABA, and salt stresses. AtSAP13 overexpression lines showed strong tolerance to toxic metals (AsIII, Cd, and Zn), drought, and salt stress. Further, transgenic lines accumulated significantly higher amounts of Zn, but less As and Cd accumulation in shoots and roots. AtSAP13 promoter-GUS fusion studies showed GUS expression predominantly in the vascular tissue, hydathodes, and the apical meristem and region of root maturation and elongation as well as the root hairs. At the subcellular level, the AtSAP13-eGFP fusion protein was found to localize in both nucleus and cytoplasm. Through yeast one-hybrid assay, we identified several AP2/EREBP family transcription factors that interacted with the AtSAP13 promoter. AtSAP13 and its homologues will be highly useful for developing climate resilient crops.

Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression.

Plant seed oil-based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1-co-expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild-type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1- and GPD1-co-expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.

Pulmonary vein occlusion and lung infarction complicating non-treated moderate single pulmonary vein stenosis after radiofrequency ablation of atrial fibrillation.

Pulmonary vein (PV) radiofrequency ablation (RFA) is an effective, curative technique for selected group of patients with atrial fibrillation (AF) refractory to antiarrhythmic drugs. However, pulmonary vein stenosis (PVS) is a potential complication which may present clinically as non-specific respiratory symptoms that often under-recognized or misdiagnosed leading to progression of low-grade stenosis to complete occlusion if not treated with timely intervention.

Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds.

BACKGROUND: Camelina sativa is an emerging dedicated oilseed crop designed for biofuel and biodiesel applications as well as a source for edible and general-purpose oils. Such valuable oilseed crop is subjected to plant breeding programs and is suggested for large-scale production of better seed and oil quality. To accomplish this objective and to further enhance its oil content, a better understanding of lipid metabolism at the molecular level in this plant is critical. Here, we applied tissue transcriptomics and lipid composition analysis to identify and profile the genes and gene networks associated with triacylglycerol (TAG) biosynthesis, and to investigate how those genes are interacting to determine the quantity and quality of Camelina oil during seed development. RESULTS: Our Camelina transcriptome data analysis revealed an approximate of 57,854 and 57,973 genes actively expressing in developing seeds (RPKM ≥ 0.1) at 10-15 (Cs-14) and 16-21 (Cs-21) days after flowering (DAF), respectively. Of these, 7932 genes showed temporal and differential gene expression during the seed development (log2 fold change ≥1.5 or ≤-1.5; P ≤ 0.05). The differentially expressed genes (DEGs) were annotated and were found to be involved in distinct functional categories and metabolic pathways. Furthermore, performing quantitative real-time PCR for selected candidate genes associated with TAG biosynthesis validated RNA-seq data. Our results showed strong positive correlations between the expression abundance measured using both qPCR and RNA-Seq technologies. Furthermore, the analysis of fatty-acid content and composition revealed major changes throughout seed development, with the amount of oil accumulate rapidly at early mid seed development stages (from 16-28 DAF onwards), while no important changes were observed in the fatty-acid profile between seeds at 28 DAF and mature seeds. CONCLUSIONS: This study is highly useful for understanding the regulation of TAG biosynthesis and identifying the rate-limiting steps in TAG pathways at seed development stages, providing a precise selection of candidate genes for developing Camelina varieties with improved seed and oil yields.

Platelet FcgammaRIIA receptor surface expression is increased in patients with ESRD and is associated with atherosclerotic cardiovascular events.

BACKGROUND: Accelerated atherogenesis is a prominent feature of end-stage renal disease (ESRD). METHODS: Expression of platelet FcgammaRIIA immunoglobulin G receptor (FcgammaR) was measured in 2 populations: (1) 48 patients with ESRD with a mean age of 50.7 +/- 2.3 (SEM) years and (2) 48 healthy age- and sex-matched controls aged 53.6 +/- 1.6 years. RESULTS: Platelet FcgammaR expression was enhanced significantly in patients with ESRD (mean fluorescence intensity [MFI], 7.88 +/- 0.42 [SEM]; 95% confidence interval [CI], 7.03 to 8.72 versus 5.07 +/- 0.21; 95% CI, 4.64 to 5.49; P < 0.0001). In patients with ESRD, multivariate analysis showed that hemoglobin level and diastolic blood pressure were related inversely to FCgammaR expression (P = 0.01 and P = 0.03, respectively). Atherosclerotic cardiovascular events (ACVEs) were recorded prospectively for the ESRD cohort. A total of 22 ACVEs, defined as the occurrence of myocardial infarction (10 events), cerebrovascular accident (7 events), and/or a peripheral vascular event (5 events), were observed. In the group with greater receptor expression (MFI > 7.785; n = 24), 13 of 17 patients (76.5%) experienced at least 1 ACVE, whereas only 4 of 17 patients (23.5%) with low FcgammaR expression (MFI < 7.785; n = 24) experienced an event (P = 0.007). CONCLUSION: (1) Uremia is associated with enhanced platelet FcgammaR expression, and (2) patients with greater platelet FcgammaR expression are significantly more likely to experience ACVEs; however, (3) the small sample size is a limitation and our work therefore is a hypothesis-generating pilot study that remains to be confirmed.