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The Surface Water and Ocean Topography (SWOT) satellite mission planned for launch in 2020 will map river elevations and inundated area globally for rivers >100 m wide. In advance of this launch, we here evaluated the possibility of estimating discharge in ungauged rivers using synthetic, daily “remote sensing” measurements derived from hydraulic models corrupted with minimal observational errors. Five discharge algorithms were evaluated, as well as the median of the five, for 19 rivers spanning a range of hydraulic and geomorphic conditions. Reliance upon a priori information, and thus applicability to truly ungauged reaches, varied among algorithms: one algorithm employed only global limits on velocity and depth, while the other algorithms relied on globally available prior estimates of discharge. We found at least one algorithm able to estimate instantaneous discharge to within 35% relative root-mean-squared error (RRMSE) on 14/16 nonbraided rivers despite out-of-bank flows, multichannel planforms, and backwater effects. Moreover, we found RRMSE was often dominated by bias; the median standard deviation of relative residuals across the 16 nonbraided rivers was only 12.5%. SWOT discharge algorithm progress is therefore encouraging, yet future efforts should consider incorporating ancillary data or multialgorithm synergy to improve results.

Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis, two enzymes share overlapping functions to produce TAGs, namely acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1-1/pdat1-2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue-specific expression of either gene has not yet been identified. We transformed a dgat1-1/dgat1-1//PDAT1/pdat1-2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron. Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c. 10% of wild-type, seed oil content remains reduced (similar to dgat1-1/dgat1-1), and non-Mendelian segregation of the PDAT1/pdat1-2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild-type. This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue-specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

In this article, we examine the scientific and sustainable research capacity outcomes of the 'Congo River: user Hydraulics and Morphology' or CRuHM project, a six-year effort supported by the Royal Society's Africa Capacity Building Initiative. This project brought together a consortium of African and UK universities to undertake the first large-scale scientific expeditions to the Congo basin of the modern era in order to better understand the hydraulics and geomorphology of this understudied but globally important river. The river is essential for navigation, irrigation, drinking water and hydroelectric power generation for the 10 basin countries and is critically important for biodiversity and global nutrient, carbon and climatological cycles. This article summarizes the new scientific understanding contributed by the project and the steps taken to ensure a meaningful legacy that would continue long beyond the finite lifetime of available funding. Actions taken to achieve this include establishing a new hydrology research centre at the University of Kinshasa as well as steps to build a wider international community of Congo basin researchers. In this way, we hope to build momentum for future funding initiatives and collaboration., Competing Interests: We declare we have no competing interests., (© 2024, The Authors.)

FatPlants, an open-access, web-based database, consolidates data, annotations, analysis results, and visualizations of lipid-related genes, proteins, and metabolic pathways in plants. Serving as a minable resource, FatPlants offers a user-friendly interface for facilitating studies into the regulation of plant lipid metabolism and supporting breeding efforts aimed at increasing crop oil content. This web resource, developed using data derived from our own research, curated from public resources, and gleaned from academic literature, comprises information on known fatty-acid-related proteins, genes, and pathways in multiple plants, with an emphasis on Glycine max, Arabidopsis thaliana, and Camelina sativa. Furthermore, the platform includes machine-learning based methods and navigation tools designed to aid in characterizing metabolic pathways and protein interactions. Comprehensive gene and protein information cards, a Basic Local Alignment Search Tool search function, similar structure search capacities from AphaFold, and ChatGPT-based query for protein information are additional features. Database URL: https://www.fatplants.net/., (© The Author(s) 2024. Published by Oxford University Press.)

Background: High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL., Methods: Ex-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival., Results: Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-κb pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect., Conclusion: Allo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.

Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible. Physaria fendleri naturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils., (© 2024. The Author(s).)

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL., Competing Interests: Declaration of interests C.M.C. receives honorarium for advisory board membership from Bayer, Elephas, Nektar Therapeutics, Novartisn, and WiCell Research Institute, who had no input in the study design, analysis, manuscript preparation, or decision to submit for publication., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g., oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered tobacco (Nicotiana tabacum) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 tracking describe metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEC2 line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested interference of the glyoxylate cycle with photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEAFY COTYLEDON 2 (LEC2) transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

The chemical and physical properties of vegetable oils are largely dictated by the ratios of 4-6 common fatty acids contained within each oil. However, examples of plant species that accumulate from trace amounts to >90% of certain unusual fatty acids in seed triacylglycerols have been reported. Many of the general enzymatic reactions that drive both common and unusual fatty acid biosynthesis and accumulation in stored lipids are known, but which isozymes have evolved to specifically fill this role and how they coordinate in vivo is still poorly understood. Cotton (Gossypium sp.) is the very rare example of a commodity oilseed that produces biologically relevant amounts of unusual fatty acids in its seeds and other organs. In this case, unusual cyclopropyl fatty acids (named after the cyclopropane and cyclopropene moieties within the fatty acids) are found in membrane and storage glycerolipids (e.g. seed oils). Such fatty acids are useful in the synthesis of lubricants, coatings, and other types of valuable industrial feedstocks. To characterize the role of cotton acyltransferases in cyclopropyl fatty acid accumulation for bioengineering applications, we cloned and characterized type-1 and type-2 diacylglycerol acyltransferases from cotton and compared their biochemical properties to that of litchi (Litchi chinensis), another cyclopropyl fatty acid-producing plant. The results presented from transgenic microbes and plants indicate both cotton DGAT1 and DGAT2 isozymes efficiently utilize cyclopropyl fatty acid-containing substrates, which helps to alleviate biosynthetic bottlenecks and enhances total cyclopropyl fatty acid accumulation in the seed oil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Masson SAS.)

Generating new strategies to improve plant performance and yield in crop plants becomes increasingly relevant with ongoing and predicted global climate changes. E3 ligases that function as key regulators within the ubiquitin proteasome pathway often are involved in abiotic stress responses, development, and metabolism in plants. The aim of this research was to transiently downregulate an E3 ligase that uses BTB/POZ-MATH proteins as substrate adaptors in a tissue-specific manner. Interfering with the E3 ligase at the seedling stage and in developing seeds results in increased salt-stress tolerance and elevated fatty acid levels, respectively. This novel approach can help to improve specific traits in crop plants to maintain sustainable agriculture.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Physaria fendleri is a burgeoning oilseed crop that accumulates the hydroxy fatty acid (HFA), lesquerolic acid, and can be a non-toxic alternative crop to castor for production of industrially valuable HFA. Recently, P. fendleri was proposed to utilize a unique seed oil biosynthetic pathway coined "triacylglycerol (TAG) remodeling" that utilizes a TAG lipase to remove common fatty acids from TAG allowing the subsequent incorporation of HFA after initial TAG synthesis, yet the lipase involved is unknown. SUGAR DEPENDENT 1 (SDP1) has been characterized as the dominant TAG lipase involved in TAG turnover during oilseed maturation and germination. Here, we characterized the role of a putative PfeSDP1 in both TAG turnover and TAG remodeling. In vitro assays confirmed that PfeSDP1 is a TAG lipase and demonstrated a preference for HFA-containing TAG species. Seed-specific RNAi knockdown of PfeSDP1 resulted in a 12%-16% increase in seed weight and 14%-19% increase in total seed oil content with no major effect on seedling establishment. The increase in total oil content was primarily due to ~4.7% to ~14.8% increase in TAG molecular species containing two HFA (2HFA-TAG), and when combined with a smaller decrease in 1HFA-TAG content the proportion of total HFA in seed lipids increased 4%-6%. The results are consistent with PfeSDP1 involved in TAG turnover but not TAG remodeling to produce 2HFA-TAG. Interestingly, the concomitant reduction of 1HFA-TAG in PfeSDP1 knockdown lines suggests PfeSDP1 may have a role in reverse TAG remodeling during seed maturation that produces 1HFA-TAG from 2HFA-TAG. Overall, our results provide a novel strategy to enhance the total amount of industrially valuable lesquerolic acid in P. fendleri seeds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Azeez, Parchuri and Bates.)

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13 CO 2 -labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop., (Published by Elsevier Inc.)

Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight with the TAG fatty acid composition determining its nutritional value or use in the biofuel or chemical industries. Two major pathways for production of diacylglycerol (DAG), the immediate precursor to TAG, have been identified in plants: de novo DAG synthesis and conversion of the membrane lipid phosphatidylcholine (PC) to DAG, with each pathway producing distinct TAG compositions. However, neither pathway fits with previous biochemical and transcriptomic results from developing Physaria fendleri seeds for accumulation of TAG containing >60% lesquerolic acid (an unusual 20 carbon hydroxylated fatty acid), which accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that PC-derived DAG is utilized to initially produce TAG with only one lesquerolic acid. Subsequently a nonhydroxylated fatty acid is removed from TAG (transiently reproducing DAG) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. Reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering oil fatty acid composition after initial TAG synthesis; and the comparison of current results to that of related Brassicaceae species in the literature suggests the possibility of TAG remodeling involved in incorporation of very long-chain fatty acids into the TAG sn-1 position in various plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2 + NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo . Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro , but induced cytokine release syndrome (CRS) in vivo . Infusing Perforin -/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α -/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H + NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects., Competing Interests: SG is an employee of Provenance Biopharmaceuticals, and has a patent related to hu14.18-IL2. CC reports honorarium from Nektar Therapeutics and Novartis. These companies had no input in the study design, analysis, manuscript preparation or decision to submit for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bates, Rakhmilevich, Cho, Bouchlaka, Rao, Hales, Orentas, Fry, Gilles, Sondel and Capitini.)

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell-like subtype (ABC) than germinal center B cell-like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. IMPLICATIONS: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma., (©2021 American Association for Cancer Research.)

Mesenchymal stromal cells (MSCs) are being tested as a cell therapy in clinical trials for dozens of inflammatory disorders, with varying levels of efficacy reported. Suitable and robust preclinical animal models for testing the safety and efficacy of different types of MSC products before use in clinical trials are rare. We here introduce two highly robust animal models of immune pathology: 1) acute radiation syndrome (ARS) and 2) graft versus host disease (GvHD), in conjunction with studying the immunomodulatory effect of well-characterized Interferon gamma (IFNγ) primed bone marrow derived MSCs. The animal model of ARS is based on clinical grade dosimetry precision and bioluminescence imaging. We found that allogeneic MSCs exhibit lower persistence in naïve compared to irradiated animals, and that intraperitoneal infusion of IFNγ prelicensed allogeneic MSCs protected animals from radiation induced lethality by day 30. In direct comparison, we also investigated the effect of IFNγ prelicensed allogeneic MSCs in modulating acute GvHD in an animal model of MHC major mismatched bone marrow transplantation. Infusion of IFNγ prelicensed allogeneic MSCs failed to mitigate acute GvHD. Altogether our results demonstrate that infused IFNγ prelicensed allogeneic MSCs protect against lethality from ARS, but not GvHD, thus providing important insights on the dichotomy of IFNγ prelicensed allogenic MSCs in well characterized and robust animal models of acute tissue injury., Competing Interests: CC receives honorarium from Nektar Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chinnadurai, Bates, Kunugi, Nickel, DeWerd, Capitini, Galipeau and Kimple.)

Arbuscular mycorrhizal fungi (AMF) are among the most ancient, widespread and functionally important symbioses on Earth that help feed the world. Yet, mass-production of clean (i.e. in vitro produced), safe and robust inoculum at affordable costs remains a critical challenge. Very recently, Luginbuehl et al. (2017) found that plants supply lipids to the symbiotic partner, thus 'providing the AMF with a robust source of carbon for their metabolic needs'. Hence, engineering plants for enhanced delivery of lipids to AMF could represent an innovative avenue to produce a novel generation of high-quality and cost-effective bio-fortified AMF inoculants for application in agro-ecosystems., (© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Lipids are produced through a dynamic metabolic network involving branch points, cycles, reversible reactions, parallel reactions in different subcellular compartments, and distinct pools of the same lipid class involved in different parts of the network. For example, diacylglycerol (DAG) is a biosynthetic and catabolic intermediate of many different lipid classes. Triacylglycerol can be synthesized from DAG assembled de novo, or from DAG produced by catabolism of membrane lipids, most commonly phosphatidylcholine. Quantification of lipids provides a snapshot of the lipid abundance at the time they were extracted from the given tissue. However, quantification alone does not provide information on the path of carbon flux through the metabolic network to synthesize each lipid. Understanding lipid metabolic flux requires tracing lipid metabolism with isotopically labeled substrates over time in living tissue. [ 14 C]acetate and [ 14 C]glycerol are commonly utilized substrates to measure the flux of nascent fatty acids and glycerol backbones through the lipid metabolic network in vivo. When combined with mutant or transgenic plants, tracing of lipid metabolism can provide information on the molecular control of lipid metabolic flux. This chapter provides a method for tracing in vivo lipid metabolism in developing Arabidopsis thaliana seeds, including analysis of 14 C labeled lipid classes and fatty acid regiochemistry through both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) approaches.

Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids ( e.g. , vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [ 14 C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the individually labeled molecular species which is traditionally performed by thin layer chromatography. Here we present a reverse phase HPLC method for the separation of MGDG molecular species from tobacco leaves in under 35 min. The quantification of each 14 C-labeled molecular species was accomplished by an in-line flow radio detector. This method of analysis for [ 14 C]Acetate labeled MGDG molecular species by radio-HPLC provides a rapid, high throughput, and reliable analytical approach to identify changes in MGDG metabolism due to bioengineering or other perturbations of metabolism., Competing Interests: Competing interestsThe authors report no competing interests., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Seed triacylglycerol (TAG) biosynthesis involves a metabolic network containing multiple different diacylglycerol (DAG) and acyl donor substrate pools. This network of pathways overlaps with those for essential membrane lipid synthesis and utilizes multiple different classes of TAG biosynthetic enzymes. Acyl flux through this network ultimately dictates the final oil fatty acid composition. Most strategies to alter seed oil composition involve the overexpression of lipid biosynthetic enzymes, but how these enzymes are assembled into metabolons and which substrate pools are used by each is still not well understood. To understand the roles of different classes of TAG biosynthetic acyltransferases in seed oil biosynthesis, we utilized the Arabidopsis ( Arabidopsis thaliana ) diacylglycerol acyltransferase mutant dgat1-1 (in which phosphatidylcholine:diacylglycerol acyltransferase (AtPDAT1) is the major TAG biosynthetic enzyme), and enhanced TAG biosynthesis by expression of Arabidopsis acyltransferases AtDGAT1 and AtDGAT2, as well as the DGAT2 enzymes from soybean ( Glycine max ), and castor ( Ricinus communis ), followed by isotopic tracing of glycerol flux through the lipid metabolic network in developing seeds. The results indicate each acyltransferase has a unique effect on seed oil composition. AtDGAT1 produces TAG from a rapidly produced phosphatidylcholine-derived DAG pool. However, AtPDAT1 and plant DGAT2 enzymes utilize a different and larger bulk phosphatidylcholine-derived DAG pool that is more slowly turned over for TAG biosynthesis. Based on metabolic fluxes and protein:protein interactions, our model of TAG synthesis suggests that substrate channeling to select enzymes and spatial separation of different acyltransferases into separate metabolons affect efficient TAG production and oil fatty acid composition., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ye et al.)

Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2-4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0-3, 0-2, and 0-1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to 14 C based metabolic tracing of lipid metabolism in leaves and seeds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Improvements in modelling power and input data have vastly improved the precision of physical flood models, but translation into economic outputs requires depth-damage functions that are inadequately verified. In particular, flood damage is widely assumed to increase monotonically with water depth. Here, we assess flood vulnerability in the US using >2 million claims from the National Flood Insurance Program (NFIP). NFIP claims data are messy, but the size of the dataset provides powerful empirical tests of damage patterns and modelling approaches. We show that current depth-damage functions consist of disparate relationships that match poorly with observations. Observed flood losses are not monotonic functions of depth, but instead better follow a beta function, with bimodal distributions for different water depths. Uncertainty in flood losses has been called the main bottleneck in flood risk studies, an obstacle that may be remedied using large-scale empirical flood damage data.

The triacylglycerols (TAGs; i.e. oils) that accumulate in plants represent the most energy-dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco ( Nicotiana tabacum ) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis)-and-pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through nonengineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected, as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. In diffuse large B cell lymphoma (DLBCL), the mechanisms of PRMT5 dysregulation and its role in lymphomagenesis remain largely unknown. Here we demonstrate that B cell receptor (BCR) signaling regulates PRMT5 expression in DLBCL cells. Immunohistochemical analysis reveals elevated levels of PRMT5 expression in DLBCL cases and in germinal center (GC) B cells when compared to naive B cells. PRMT5 can be induced in naive B cells by BCR stimulation. We discovered that BTK-NF-κB signaling induces PRMT5 transcription in activated B cell-like (ABC) DLBCL cells while BCR downstream PI3K-AKT-MYC signaling upregulates PRMT5 expression in both ABC and GCB DLBCL cells. PRMT5 inhibition inhibits the growth of DLBCL cells in vitro and patient derived xenografts. Genomic and biochemical analysis demonstrate that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a feedback regulatory mechanism to enhance cell survival and proliferation. Co-targeting PRMT5 and AKT by their specific inhibitors is lethal to DLBCL cell lines and primary cancer cells. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate PRMT5 and AKT inhibitors for DLBCL.

In the setting of radiation-induced trauma, exposure to high levels of radiation can cause an acute radiation syndrome (ARS) causing bone marrow (BM) failure, leading to life-threatening infections, anemia, and thrombocytopenia. We have previously shown that human macrophages educated with human mesenchymal stem cells (MSCs) by coculture can significantly enhance survival of mice exposed to lethal irradiation. In this study, we investigated whether exosomes isolated from MSCs could replace direct coculture with MSCs to generate exosome educated macrophages (EEMs). Functionally unique phenotypes were observed by educating macrophages with exosomes from MSCs (EEMs) primed with bacterial lipopolysaccharide (LPS) at different concentrations (LPS-low EEMs or LPS-high EEMs). LPS-high EEMs were significantly more effective than uneducated macrophages, MSCs, EEMs, or LPS-low EEMs in extending survival after lethal ARS in vivo. Moreover, LPS-high EEMs significantly reduced clinical signs of radiation injury and restored hematopoietic tissue in the BM and spleen as determined by complete blood counts and histology. LPS-high EEMs showed significant increases in gene expression of STAT3, secretion of cytokines like IL-10 and IL-15, and production of growth factors like FLT-3L. LPS-EEMs also showed increased phagocytic activity, which may aid with tissue remodeling. LPS-high EEMs have the potential to be an effective cellular therapy for the management of ARS., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The eukaryotic pathway of galactolipid synthesis involves fatty acid synthesis in the chloroplast, followed by assembly of phosphatidylcholine (PC) in the endoplasmic reticulum (ER), and then turnover of PC to provide a substrate for chloroplast galactolipid synthesis. However, the mechanisms and classes of lipids transported between the chloroplast and the ER are unclear. PC, PC-derived diacylglycerol, phosphatidic acid, and lyso-phosphatidylcholine (LPC) have all been implicated in ER-to-chloroplast lipid transfer. LPC transport requires lysophosphatidylcholine acyltransferase (LPCAT) activity at the chloroplast to form PC before conversion to galactolipids. However, LPCAT has also been implicated in the opposite chloroplast-to-ER trafficking of newly synthesized fatty acids through PC acyl editing. To understand the role of LPC and LPCAT in acyl trafficking we produced and analyzed the Arabidopsis ( Arabidopsis thaliana ) act1 lpcat1 lpcat2 triple mutant. LPCAT1 and LPCAT2 encode the major lysophospholipid acyltransferase activity of the chloroplast, and it is predominantly for incorporation of nascent fatty acids exported form the chloroplast into PC by acyl editing. In vivo acyl flux analysis revealed eukaryotic galactolipid synthesis is not impaired in act1 lpcat1 lpcat2 and uses a PC pool distinct from that of PC acyl editing. We present a model for the eukaryotic pathway with metabolically distinct pools of PC, suggesting an underlying spatial organization of PC metabolism as part of the ER-chloroplast metabolic interactions., (© 2019 American Society of Plant Biologists. All rights reserved.)

Main Conclusion: In vivo and in vitro analyses of Euphorbiaceae species' triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils. Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.

Current estimates of global flood exposure are made using datasets that distribute population counts homogenously across large lowland floodplain areas. When intersected with simulated water depths, this results in a significant mis-estimation. Here, we use new highly resolved population information to show that, in reality, humans make more rational decisions about flood risk than current demographic data suggest. In the new data, populations are correctly represented as risk-averse, largely avoiding obvious flood zones. The results also show that existing demographic datasets struggle to represent concentrations of exposure, with the total exposed population being spread over larger areas. In this analysis we use flood hazard data from a ~90 m resolution hydrodynamic inundation model to demonstrate the impact of different population distributions on flood exposure calculations for 18 developing countries spread across Africa, Asia and Latin America. The results suggest that many published large-scale flood exposure estimates may require significant revision.

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with β-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper, we report a method for the separation of hydroxy fatty acid and non-hydroxy fatty acid containing neutral lipid classes via normal phase HPLC with UV detection on a PVA-Sil column. The hexane/isopropanol/methanol/water based method separates all the neutral lipids in 21 min, and subsequently flushes through the polar lipids by 27 min such that prefractionation of neutral and polar lipids are not required, and the column is re-equilibrated for the next run in 15 min, for a total run time of 45 min per sample. The separation was demonstrated at both 1.0 mL/min and 1.5 mL/min for added applicability for fraction collection or inline analysis. Separation of various hydroxy fatty acid containing lipids was demonstrated from three different plant species Ricinus communis, Physaria fendleri, and engineered Arabidopsis thaliana. Additionally, we have combined this method with an in-line liquid scintillation counter for the separation and quantification of 14 C labeled lipids obtained from in vivo metabolic flux experiments conducted in the developing seeds of Arabidopsis thaliana., (Copyright © 2018 Elsevier B.V. All rights reserved.)