Development and Validation of a Multiclass Model Defining Molecular Archetypes of Kidney Transplant Rejection: A Large Cohort Study of the Banff Human Organ Transplant Gene Expression Panel.

Gene expression profiling from formalin-fixed paraffin-embedded (FFPE) renal allograft biopsies is a promising approach for feasibly providing a molecular diagnosis of rejection. However, large-scale studies evaluating the performance of models using NanoString platform data to define molecular archetypes of rejection are lacking. We tested a diverse retrospective cohort of over 1400 FFPE biopsy specimens, rescored according to Banff 2019 criteria and representing 10 of 11 United Network of Organ Sharing regions, using the Banff Human Organ Transplant panel from NanoString and developed a multiclass model from the gene expression data to assign relative probabilities of 4 molecular archetypes: No Rejection, Antibody-Mediated Rejection, T Cell-Mediated Rejection, and Mixed Rejection. Using Least Absolute Shrinkage and Selection Operator regularized regression with 10-fold cross-validation fitted to 1050 biopsies in the discovery cohort and technically validated on an additional 345 biopsies, our model achieved overall accuracy of 85% in the discovery cohort and 80% in the validation cohort, with ≥75% positive predictive value for each class, except for the Mixed Rejection class in the validation cohort (positive predictive value, 53%). This study represents the technical validation of the first model built from a large and diverse sample of diagnostic FFPE biopsy specimens to define and classify molecular archetypes of histologically defined diagnoses as derived from Banff Human Organ Transplant panel gene expression profiling data.

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

A point mutation in the kinase domain of CRK10 leads to xylem vessel collapse and activation of defence responses in Arabidopsis.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.

Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum.

The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, particularly in galactolipids such as mono- and di-galactosyldiacylglycerol. LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analyzed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of triacylglycerol. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.

Using field evaluation and systematic iteration to rationalize the accumulation of omega-3 long-chain polyunsaturated fatty acids in transgenic Camelina sativa.

The Brassicaceae Camelina sativa (gold of pleasure) is now an established niche crop and being used as a transgenic host for a range of novel seed traits. Most notable of these is the accumulation of omega-3 long-chain polyunsaturates such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), fatty acids normally only found in marine organisms. As part of continued efforts to optimize the accumulation of these non-native fatty acids via seed-specific expression of algal genes, a new series of iterative constructs was built and introduced into Camelina. Seed fatty acid composition was determined, and the presence of EPA and DHA was confirmed. To provide an additional level of evaluation, full environmental release was carried out on selected events, providing a real-world gauntlet against which to assess the performance of these novel lines. Composition of the seed oil triacylglycerol was determined by mass spectrometry, allowing for conclusions as to the contribution of different activities to the final accumulation of EPA and DHA. Since these data were derived from field-grown material, they also represent a robust demonstration of the stability of the omega-3 LC-PUFA trait in Camelina. We propose that field trialling should be routinely incorporated in the plant synthetic biology 'design-build-test-learn' cycle.

Impairment of DHA synthesis alters the expression of neuronal plasticity markers and the brain inflammatory status in mice.

Docosahexaenoic acid (DHA) is a ω-3 fatty acid typically obtained from the diet or endogenously synthesized through the action of elongases (ELOVLs) and desaturases. DHA is a key central nervous system constituent and the precursor of several molecules that regulate the resolution of inflammation. In the present study, we questioned whether the impaired synthesis of DHA affected neural plasticity and inflammatory status in the adult brain. To address this question, we investigated neural and inflammatory markers from mice deficient for ELOVL2 (Elovl2-/- ), the key enzyme in DHA synthesis. From our findings, Elovl2-/- mice showed an altered expression of markers involved in synaptic plasticity, learning, and memory formation such as Egr-1, Arc1, and BDNF specifically in the cerebral cortex, impacting behavioral functions only marginally. In parallel, we also found that DHA-deficient mice were characterized by an increased expression of pro-inflammatory molecules, namely TNF, IL-1ß, iNOS, caspase-1 as well as the activation and morphologic changes of microglia in the absence of any brain injury or disease. Reintroducing DHA in the diet of Elovl2-/- mice reversed such alterations in brain plasticity and inflammation. Hence, impairment of systemic DHA synthesis can modify the brain inflammatory and neural plasticity status, supporting the view that DHA is an essential fatty acid with an important role in keeping inflammation within its physiologic boundary and in shaping neuronal functions in the central nervous system.

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while ß-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

High level accumulation of EPA and DHA in field-grown transgenic Camelina - a multi-territory evaluation of TAG accumulation and heterogeneity.

The transgene-directed accumulation of non-native omega-3 long chain polyunsaturated fatty acids in the seed oil of Camelina sativa (Camelina) was evaluated in the field, in distinct geographical and regulatory locations. A construct, DHA2015.1, containing an optimal combination of biosynthetic genes, was selected for experimental field release in the UK, USA and Canada, and the accumulation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) determined. The occurrence of these fatty acids in different triacylglycerol species was monitored and found to follow a broad trend irrespective of the agricultural environment. This is a clear demonstration of the stability and robust nature of the transgenic trait for omega-3 long chain polyunsaturated fatty acids in Camelina. Examination of non-seed tissues for the unintended accumulation of EPA and DHA failed to identify their presence in leaf, stem, flower, anther or capsule shell material, confirming the seed-specific accumulation of these novel fatty acids. Collectively, these data confirm the promise of GM plant-based sources of so-called omega-3 fish oils as a sustainable replacement for oceanically derived oils.

Sphingolipids: towards an integrated view of metabolism during the plant stress response.

Plants exist in an environment of changing abiotic and biotic stresses. They have developed a complex set of strategies to respond to these stresses and over recent years it has become clear that sphingolipids are a key player in these responses. Sphingolipids are not universally present in all three domains of life. Many bacteria and archaea do not produce sphingolipids but they are ubiquitous in eukaryotes and have been intensively studied in yeast and mammals. During the last decade there has been a steadily increasing interest in plant sphingolipids. Plant sphingolipids exhibit structural differences when compared with their mammalian counterparts and it is now clear that they perform some unique functions. Sphingolipids are recognised as critical components of the plant plasma membrane and endomembrane system. Besides being important structural elements of plant membranes, their particular structure contributes to the fluidity and biophysical order. Sphingolipids are also involved in multiple cellular and regulatory processes including vesicle trafficking, plant development and defence. This review will focus on our current knowledge as to the function of sphingolipids during plant stress responses, not only as structural components of biological membranes, but also as signalling mediators.

Dietary supplementation with seed oil from transgenic Camelina sativa induces similar increments in plasma and erythrocyte DHA and EPA to fish oil in healthy humans.

EPA and DHA are required for normal cell function and can also induce health benefits. Oily fish are the main source of EPA and DHA for human consumption. However, food choices and concerns about the sustainability of marine fish stocks limit the effectiveness of dietary recommendations for EPA + DHA intakes. Seed oils from transgenic plants that contain EPA + DHA are a potential alternative source of EPA and DHA. The present study investigated whether dietary supplementation with transgenic Camelina sativa seed oil (CSO) that contained EPA and DHA was as effective as fish oil (FO) in increasing EPA and DHA concentrations when consumed as a dietary supplement in a blinded crossover study. Healthy men and women (n 31; age 53 (range 20-74) years) were randomised to consume 450 mg/d EPA + DHA provided either as either CSO or FO for 8 weeks, followed by 6 weeks washout and then switched to consuming the other test oil. Fasting venous blood samples were collected at the start and end of each supplementation period. Consuming the test oils significantly (P < 0·05) increased EPA and DHA concentrations in plasma TAG, phosphatidylcholine and cholesteryl esters. There were no significant differences between test oils in the increments of EPA and DHA. There was no significant difference between test oils in the increase in the proportion of erythrocyte EPA + DHA (CSO, 12 %; P < 0·0001 and FO, 8 %; P = 0·02). Together, these findings show that consuming CSO is as effective as FO for increasing EPA and DHA concentrations in humans.

Postprandial incorporation of EPA and DHA from transgenic Camelina sativa oil into blood lipids is equivalent to that from fish oil in healthy humans.

EPA and DHA are important components of cell membranes. Since humans have limited ability for EPA and DHA synthesis, these must be obtained from the diet, primarily from oily fish. Dietary EPA and DHA intakes are constrained by the size of fish stocks and by food choice. Seed oil from transgenic plants that synthesise EPA and DHA represents a potential alternative source of these fatty acids, but this has not been tested in humans. We hypothesised that incorporation of EPA and DHA into blood lipids from transgenic Camelina sativa seed oil (CSO) is equivalent to that from fish oil. Healthy men and women (18-30 years or 50-65 years) consumed 450 mg EPA + DHA from either CSO or commercial blended fish oil (BFO) in test meals in a double-blind, postprandial cross-over trial. There were no significant differences between test oils or sexes in EPA and DHA incorporation into plasma TAG, phosphatidylcholine or NEFA over 8 h. There were no significant differences between test oils, age groups or sexes in postprandial VLDL, LDL or HDL sizes or concentrations. There were no significant differences between test oils in postprandial plasma TNFα, IL 6 or 10, or soluble intercellular cell adhesion molecule-1 concentrations in younger participants. These findings show that incorporation into blood lipids of EPA and DHA consumed as CSO was equivalent to BFO and that such transgenic plant oils are a suitable dietary source of EPA and DHA in humans.

Pan genome of the phytoplankton Emiliania underpins its global distribution.

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

Chlamydomonas carries out fatty acid ß-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase.

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2 O2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) ß-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA ß-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of ß-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA ß-oxidation involving H2 O2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.

Oil from transgenic Camelina sativa containing over 25 % n-3 long-chain PUFA as the major lipid source in feed for Atlantic salmon (Salmo salar).

Facing a bottleneck in the growth of aquaculture, and a gap in the supply and demand of the highly beneficial n-3 long-chain PUFA (LC-PUFA), sustainable alternatives to traditional marine-based feeds are required. Therefore, in the present trial, a novel oil obtained from a genetically engineered oilseed crop, Camelina sativa, that supplied over 25 % n-3 LC-PUFA was tested as a sole dietary-added lipid source in Atlantic salmon (Salmo salar) feed. Three groups of fish were fed three experimental diets for 12 weeks with the same basal composition and containing 20 % added oil supplied by either a blend of fish oil and rapeseed oil (1:3) (COM) reflecting current commercial formulations, wild-type Camelina oil (WCO) or the novel transgenic Camelina oil (TCO). There were no negative effects on the growth, survival rate or health of the fish. The whole fish and flesh n-3 LC-PUFA levels were highest in fish fed TCO, with levels more than 2-fold higher compared with those of fish fed the COM and WCO diets, respectively. Diet TCO had no negative impacts on the evaluated immune and physiological parameters of head kidney monocytes. The transcriptomic responses of liver and mid-intestine showed only mild effects on metabolism genes. Overall, the results clearly indicated that the oil from transgenic Camelina was highly efficient in supplying n-3 LC-PUFA providing levels double that obtained with a current commercial standard, and similar to those a decade ago before substantial dietary fishmeal and oil replacement.

Plant sphingolipids: Their importance in cellular organization and adaption.

Sphingolipids and their phosphorylated derivatives are ubiquitous bio-active components of cells. They are structural elements in the lipid bilayer and contribute to the dynamic nature of the membrane. They have been implicated in many cellular processes in yeast and animal cells, including aspects of signaling, apoptosis, and senescence. Although sphingolipids have a better defined role in animal systems, they have been shown to be central to many essential processes in plants including but not limited to, pollen development, signal transduction and in the response to biotic and abiotic stress. A fuller understanding of the roles of sphingolipids within plants has been facilitated by classical biochemical studies and the identification of mutants of model species. Recently the development of powerful mass spectrometry techniques hailed the advent of the emerging field of lipidomics enabling more accurate sphingolipid detection and quantitation. This review will consider plant sphingolipid biosynthesis and function in the context of these new developments. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Synthetic redesign of plant lipid metabolism.

Plant seed lipid metabolism is an area of intensive research, including many examples of transgenic events in which oil composition has been modified. In the selected examples described in this review, progress towards the predictive manipulation of metabolism and the reconstitution of desired traits in a non-native host is considered. The advantages of a particular oilseed crop, Camelina sativa, as a flexible and utilitarian chassis for advanced metabolic engineering and applied synthetic biology are considered, as are the issues that still represent gaps in our ability to predictably alter plant lipid biosynthesis. Opportunities to deliver useful bio-based products via transgenic plants are described, some of which represent the most complex genetic engineering in plants to date. Future prospects are considered, with a focus on the desire to transition to more (computationally) directed manipulations of metabolism.

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl-CoA substrate pool, through the co-expression of acyl-ACP thioesterases, to direct the accumulation of medium-chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl-CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non-native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl-ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl-CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co-expression of acyl-ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co-expression of the same acyl-ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.