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Cultivated oat ( Avena sativa L.) is an allohexaploid (AACCDD, 2 n  = 6 x  = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia 1 , 2 . Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A . sativa and close relatives of its diploid ( Avena longiglumis , AA, 2 n  = 14) and tetraploid ( Avena insularis , CCDD, 2 n  = 4 x  = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies. Assembly of the hexaploid oat genome and its diploid and tetraploid relatives clarifies the evolutionary history of oat and allows mapping of genes for agronomic traits.

Aquaglyceroporin 7 (AQP7) facilitates glycerol flux across the plasma membrane with a critical physiological role linked to metabolism, obesity, and associated diseases. Here, we present the single-particle cryo-EM structure of AQP7 determined at 2.55 Å resolution adopting two adhering tetramers, stabilized by extracellularly exposed loops, in a configuration like that of the well-characterized interaction of AQP0 tetramers. The central pore, in-between the four monomers, displays well-defined densities restricted by two leucine filters. Gas chromatography mass spectrometry (GC/MS) results show that the AQP7 sample contains glycerol 3-phosphate (Gro3P), which is compatible with the identified features in the central pore. AQP7 is shown to be highly expressed in human pancreatic α- and β- cells suggesting that the identified AQP7 octamer assembly, in addition to its function as glycerol channel, may serve as junction proteins within the endocrine pancreas. Glycerol flux across the plasma membrane is critical to metabolism and linked to disease. Here, authors present the cryo-EM structure of the glycerol channel AQP7 composed of two adhering tetramers and displaying well-defined densities in the central pore.

To evade immunosurveillance many cancers convert tryptophan (Trp) into kynurenine (Kyn), which induces immunotolerance and suppresses immune responses. Elevated Kyn amounts have been found in blood from patients with cutaneous melanoma. This study aimed to investigate whether higher Kyn abundance and lower Trp abundance can be detected on the surface of cutaneous melanoma lesions compared with adjacent non-lesional skin. Sixteen patients with suspected melanomas were enrolled in this study. All lesions were excised and histopathologically diagnosed: 7 lesions were diagnosed as invasive malignant melanomas (MM), 6 as melanomas in situ (MIS), and 3 as benign lesions (BL). Non-invasive metabolite sampling was performed by tape stripping of suspected skin lesions and adjacent healthy non-lesional (NL) skin. Trp, Kyn, tyrosine (Tyr), and phenylalanine (Phe) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Electrical impedance spectroscopy (EIS) measurements were conducted to assess skin barrier integrity. Levels of all metabolites, Tyr (x6), Phe (x6), Trp (x5), and Kyn (x3), were significantly higher in MM lesions compared with adjacent NL skin, resulting in an elevated Trp/Kyn ratio. Trp levels increased less than Phe and Tyr levels, suggesting a potential increase in Trp depletion. Skin resistance in MM lesions was half that of NL skin. No differences were observed between MIS or BL and NL skin. Non-invasive skin sampling revealed elevated Tyr, Phe, Trp and Kyn levels in MM skin, which is likely the result of compromised skin barrier at this stage of cutaneous melanoma.

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo . The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo . Mild methods involving pressurized CO 2 -ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO 2 -EtOH-H 2 O fluids (average molar composition, Χ CO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO 2 -limonene fluids ( Χ CO2 0.88) and neat supercritical CO 2 (scCO 2 ) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO 2 -limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO 2 -ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO 2 -limonene fluids facilitate subsequent enzymatic removal of DNA.

Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3 -targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r 2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3 , which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.

Leif Groop and colleagues report that, in prospective studies, a variant in the gene encoding melantonin receptor 1B influences future risk of type 2 diabetes. Insulin release from pancreatic beta cells in response to glucose was inhibited by melatonin. Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in β cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal β cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on β cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets. Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets. Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells. Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Background Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting d -xylose sugar to ethanol compared to the preferred sugar d -glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of d -xylose is similar to the response seen on low concentrations of d -glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene ( PGI1 ) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1 -wildtype strains in the presence of various types and combinations of sugars. Results Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1 , with the expected accumulation of intermediates upstream of the Pgi1p reaction on d -glucose and downstream intermediates on d -xylose. Moreover, the analysis revealed a preferential formation of d -fructose-6-phosphate from d -xylose, as opposed to the accumulation of d -fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on d -fructose. This may indicate a role of PFK27 in d -xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p ( d -glucose and d -galactose) were more affected than the response to those entering downstream of the reaction ( d -fructose and d -xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p ( d -glucose with d -fructose, or d -glucose with d -xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. Conclusions Overall, the sensing assays indicated that the previously observed d -xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of d -fructose-6-phosphate could represent attractive engineering targets for improved d -xylose utilization.

Mutations in the gene encoding glucokinase (GCK) cause a mild hereditary form of diabetes termed maturity-onset diabetes of the young (MODY)2 or GCK-MODY. The disease does not progress over time, and diabetes complications rarely develop. It has therefore been suggested that GCK-MODY represents a metabolically compensated condition, but experimental support for this notion is lacking. Here, we profiled metabolites in serum from patients with MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), and type 2 diabetes and from healthy individuals to characterize metabolic perturbations caused by specific mutations. Analysis of four GCK-MODY patients revealed a metabolite pattern similar to that of healthy individuals, while other forms of diabetes differed markedly in their metabolite profiles. Furthermore, despite elevated glucose concentrations, carriers of GCK mutations showed lower levels of free fatty acids and triglycerides than healthy control subjects. The metabolite profiling was confirmed by enzymatic assays and replicated in a cohort of 11 GCK-MODY patients. Elevated levels of fatty acids are known to associate with β-cell dysfunction, insulin resistance, and increased incidence of late complications. Our results show that GCK-MODY represents a metabolically normal condition, which may contribute to the lack of late complications and the nonprogressive nature of the disease.

Kraft lignin is the main source of technically produced lignin. For the development of valuable products based on Kraft lignin, its molecular structure is important. However, the chemical composition of Kraft lignin is still not well known. So far, the analysis of Kraft lignin by mass spectrometry (MS) has been mainly focused on monomeric compounds. Previous MS studies on lignin oligomers (LOs) considered only synthesised LO standards and/or lignins produced by processes other than the Kraft process. Furthermore, published MS methods suffer from using high resolution only in the MS1 stage in multiple-stage tandem MS methods. A high resolution in all MSn stages would provide more detailed information about LO fragmentation pathways. Since lignin samples are complex mixtures of a large number of similar phenolic compounds, the selection of tentative LOs in the MS data is challenging. In this study, we present a method for non-targeted analysis of LOs in Kraft lignin using ultra-high-performance liquid chromatography/high-resolution multiple-stage tandem mass spectrometry (UHPLC/HRMSn). A pre-selection strategy for LOs has been established based on a data-dependent neutral loss MS3 method in combination with a principal component analysis-quadratic discriminant analysis classification model (PCA-QDA). The method was optimised using a design of experiments (DOE) approach. The developed approach improved the pre-selection of tentative LOs in complex mixtures. From 587 detected peaks, 36 peaks were identified as LOs.