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DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

The metabolic syndrome (MetS), defined as the co-occurrence of disorders including obesity, dyslipidemia, insulin resistance, and hepatic steatosis, has become increasingly prevalent in the world over recent decades. Dietary and other environmental factors interacting with genetic predisposition are likely contributors to this epidemic. Among the involved dietary factors, excessive fructose consumption may be a key contributor. When fructose is consumed in large amounts, it can quickly produce many of the features of MetS both in humans and mice. The mechanisms by which fructose contributes to metabolic disease and its potential interactions with genetic factors in these processes remain uncertain. Here, we generated a small F2 genetic cohort of male mice derived from crossing fructose-sensitive and -resistant mouse strains to investigate the interrelationships between fructose-induced metabolic phenotypes and to identify hepatic transcriptional pathways that associate with these phenotypes. Our analysis indicates that the hepatic transcriptional pathways associated with fructose-induced hypertriglyceridemia and hyperinsulinemia are distinct from those that associate with fructose-mediated changes in body weight and liver triglyceride. These results suggest that multiple independent mechanisms and pathways may contribute to different aspects of fructose-induced metabolic disease.

Hunt et al. show that mitochondrial stress in Drosophila neurons activates ATF4 expression, resulting in increased L-2-hydroxyglutarate (L-2-HG) levels. Reducing L-2-HG levels improves neuronal activity, demonstrating that increased L-2-HG contributes to neuronal dysfunction., Mitochondrial stress contributes to a range of neurological diseases. Mitonuclear signaling pathways triggered by mitochondrial stress remodel cellular physiology and metabolism. How these signaling mechanisms contribute to neuronal dysfunction and disease is poorly understood. We find that mitochondrial stress in neurons activates the transcription factor ATF4 as part of the endoplasmic reticulum unfolded protein response (UPR) in Drosophila. We show that ATF4 activation reprograms nuclear gene expression and contributes to neuronal dysfunction. Mitochondrial stress causes an ATF4-dependent increase in the level of the metabolite L-2-hydroxyglutarate (L-2-HG) in the Drosophila brain. Reducing L-2-HG levels directly, by overexpressing L-2-HG dehydrogenase, improves neurological function. Modulation of L-2-HG levels by mitochondrial stress signaling therefore regulates neuronal function.

Genome-wide association on winter survival was conducted using data from image-based phenotyping method. Nine QTL were observed and three of them with candidate gene identified. Winter survival is an essential trait of winter wheat (Triticum aestivum L.) grown in regions with high risk of winterkill. We characterized a diversity panel of 450 Canadian wheat varieties that included mostly winter-growth habit wheats to identify key genetic factors that contribute to higher winter survival under field conditions. To more accurately quantify winter survival differences among varieties, image-based phenotyping methods, captured by unmanned aerial vehicle (UAV) and on ground level, were used to estimate the winter survival of each varieties. Winter survival index was developed to correct for emergence when evaluating winter survival. Winter survival measurement estimated by visual estimation, UAV imagery and ground imagery showed strong correlation with each other and had comparable broad-sense heritability. Genome-wide association studies resulted in the identification of seven quantitative trait loci (QTL) for winter survival including Vrn-A1. By using the recently released annotated sequence of the wheat genome and the available RNA-Seq data, two putative candidate genes underlying the QTL for winter survival were identified. However, our study showed that certain QTL was unique to specific winter survival measurement. Collectively, our study demonstrated the feasibility of using UAV-based imagery for the identification of loci associated with winter survival in wheat. The complexity of in-field condition make our result a valuable complement to indoor frost-tolerance studies in the identification of genetic factors not directly linked to freezing tolerance.

Mitochondrial complex I inhibition in the presence of a mito-antioxidant induces reductive stress, ISR, and cancer cell death., Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.

There are numerous mechanisms by which mammals respond to hypoxia. These include acute changes in pulmonary arterial tone due to smooth muscle cell contraction, acute increases in respiration triggered by the carotid body chemosensory cells, and chronic changes such as induction of red blood cell proliferation and angiogenesis by hypoxia inducible factor targets erythropoietin and vascular endothelial growth factor, respectively. Mitochondria account for the majority of oxygen consumption in the cell and have recently been appreciated to serve as signaling organelles required for the initiation or propagation of numerous homeostatic mechanisms. Mitochondria can influence cell signaling by production of reactive oxygen species and metabolites. Here we review recent evidence that mitochondrial signals can imitate acute and chronic hypoxia responses.

Mitochondrial complex I regenerates NAD+ and proton pumps for TCA cycle function and ATP production, respectively. Mitochondrial complex I dysfunction has been implicated in many brain pathologies including Leigh syndrome and Parkinson's disease. We sought to determine whether NAD+ regeneration or proton pumping, i.e., bioenergetics, is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NADH dehydrogenase (NDI1), a single enzyme that can replace the NAD+ regeneration capability of the 45-subunit mammalian mitochondrial complex I without proton pumping. NDI1 expression was sufficient to dramatically prolong lifespan without significantly improving motor function in a mouse model of Leigh syndrome driven by the loss of NDUFS4, a subunit of mitochondrial complex I. Therefore, mitochondrial complex I activity in the brain supports organismal survival through its NAD+ regeneration capacity, while optimal motor control requires the bioenergetic function of mitochondrial complex I.

Thiocyanate (SCN) is used by the innate immune system, but less is known about its impact on inflammation and oxidative stress. Granulocytes oxidize SCN to evolve the bactericidal hypothiocyanous acid, which we previously demonstrated is metabolized by mammalian, but not bacterial, thioredoxin reductase (TrxR). There is also evidence that SCN is dysregulated in cystic fibrosis (CF), a disease marked by chronic infection and airway inflammation. To investigate antiinflammatory effects of SCN, we administered nebulized SCN or saline to β epithelial sodium channel (βENaC) mice, a phenotypic CF model. SCN significantly decreased airway neutrophil infiltrate and restored the redox ratio of glutathione in lung tissue and airway epithelial lining fluid to levels comparable to wild type. Furthermore, in Pseudomonas aeruginosa–infected βENaC and wild-type mice, SCN decreased inflammation, proinflammatory cytokines, and bacterial load. SCN also decreased airway neutrophil chemokine keratinocyte chemoattractant (also known as C-X-C motif chemokine ligand 1) and glutathione sulfonamide, a biomarker of granulocyte oxidative activity, in uninfected βENaC mice. Lung tissue TrxR activity and expression increased in inflamed lung tissue, providing in vivo evidence for the link between hypothiocyanous acid metabolism by TrxR and the promotion of selective biocide of pathogens. SCN treatment both suppressed inflammation and improved host defense, suggesting that nebulized SCN may have important therapeutic utility in diseases of both chronic airway inflammation and persistent bacterial infection, such as CF.

Carotenoids serve antioxidant functions in plant photosynthetic processes, as well as in actions of disease reduction in mammalian systems. Lutein and zeaxanthin are important dietary carotenoids in suppressing aging eye diseases. Age-related macular degeneration now affects more than 1.75 million individuals in the US. Sweet corn (Zea mays var. rugosa) is one of only a few vegetable sources high in zeaxanthin. Mesotrione herbicide is currently labeled for selective pre- and post-emergence weed control in sweet corn production. Mesotrione competitively inhibits phytoene desaturase, a critical enzyme in carotenoid biosynthesis, which results in bleaching of leaf tissues in susceptible species and eventual plant death. Sweet corn is tolerant to mesotrione applications; however, differing sensitivity exists among genotypes. What remains unclear is the impact of mesotrione on carotenoid concentrations in mature sweet corn kernels following post-emergent applications to young corn plants. Our research objective was to measure mature kernel carotenoid concentrations in response to post-emergence applications of mesotrione to genotypes of different sensitivities. Post-emergence treatments included mesotrione applied alone, or in mixtures with the photosystem II inhibitor atrazine applied to corn plants of either 5-10 or 15-20 cm in height. Kernels were harvested from mature plants and measured for carotenoid concentrations. Mesotrione applied alone, or in mixtures with atrazine acted to increase concentrations of kernel antheraxanthin, lutein, and zeaxanthin in several sweet corn genotypes. Mesotrione applications resulted in greater pools of kernel carotenoids once the sweet corn genotypes overcame initial herbicidal photo-oxidative stress. This is the first report of herbicides directly up regulating a key biochemical pathway linked to the nutritional quality of a vegetable crop.

Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings.

Papadopoulos, Y. A., McElroy, M. S., Fillmore, S. A. E., McRae, K. B., Duyinsveld, J. L. and Fredeen, A. H. 2012. Sward complexity and grass species composition affects the performance of grass-white clover pasture mixtures. Can. J. Plant Sci. 92: 1199–1205. The productivity of managed permanent pastures is closely associated with the species composition of seeded mixtures. Ecological theory suggests that increasing plant species' diversity will result in higher productivity, resilience, and resistance to invasive species. To better understand the relationship between sward species composition and pasture productivity, mixtures of four common pasture grass species, timothy (Phleum pratense L.), Kentucky bluegrass (Poa pratensis L.), reed canarygrass (Phalaris arundinacea L.), and meadow fescue (Festuca pratensis L.), were seeded in 2004 in binary (two-grass), tertiary (three-grass), and quaternary (four-grass) combinations with white clover (Trifolium repens L.). Plots were rotationally grazed for 5 yr, with yield determined in the first post-establishment year (2005) and in 3 subsequent production years (2007, 2008, and 2009). Mean dry matter yield (DMY) increased appreciably from post-establishment (3801 kg ha−1) to the production years (6613 kg ha−1). Contrasts revealed significantly higher DMY production in quaternary mixtures versus less complex plots in production years. Repeated measure analysis found significant quadratic trends in DMY for plots containing bluegrass and timothy, showing different patterns of growth between the respective mixtures. Principal component analysis (PCA) of averaged yields over the production years revealed a strong association between seeded grass growth and DMY. This relationship between DMY and seeded grass growth was strongest in swards containing a combination of timothy and bluegrass. In general, the growth of unseeded grasses and forbs increased in mixtures with large proportions of timothy and reed canarygrass. In spite of the fact that mixtures containing timothy and bluegrass were shown to produce high DMY, it appears the aggressiveness of bluegrass suppressed the yield potential of timothy. White clover yields and unseeded grasses/forbs were both significantly lower in mixtures containing bluegrass. Also, the PCA revealed a favourable compatibility between meadow fescue and white clover growth, while bluegrass appears to suppress meadow fescue growth. Results show that species complexity increases sward productivity over the long term, and that the presence of two grass species, in particular (timothy and bluegrass), has considerable influence on DMY.

Amicarbazone is a photosystem II (PSII)-inhibiting herbicide in the triazolinone family, which is similar in mode of action to the triazines. Annual bluegrass is a cool-season weed and has shown resistance to some PSII-inhibiting herbicides. The objective was to evaluate triazine-resistant and -susceptible annual bluegrass populations for potential cross-resistance to amicarbazone. Two triazine-resistant (MS-01, MS-02) and triazine-susceptible (AL-01, COM-01) annual bluegrass populations were treated with amicarbazone, atrazine, and simazine at 0.26, 1.7, and 1.7 kg ai ha−1, respectively. All herbicide treatments controlled the susceptible populations greater than 94% 2 wk after treatment (WAT). No visual injury of MS-01 and MS-02 was observed at any time following herbicide treatment. Quantum yield (ΦPSII) of annual bluegrass was measured 0 to 72 h after application (HAA) to determine the photochemical effects of amicarbazone compared to other PSII inhibitors. ΦPSIIof triazine-susceptible populations was reduced at all measurement times by all three herbicides. However, amicarbazone decreased ΦPSIIof susceptible populations faster and greater than atrazine and simazine at most measurement times. Amicarbazone did not reduce ΦPSIIof the MS-01 population. Amicarbazone significantly reduced ΦPSIIof the MS-02 population during several measurement timings; however, these reductions were short-lived compared to the susceptible populations and no trend in ΦPSIIreduction was observed. Sequencing of thepsbAgene revealed a Ser to Gly substitution at amino acid position 264 known to confer resistance to triazine herbicides. These data indicate amicarbazone efficiently inhibited PSII of susceptible annual bluegrass populations; however, triazine-resistant annual bluegrass populations with Ser264to Gly mutations are cross-resistant to amicarbazone.

Amicarbazone is a photosystem II (PSII) inhibiting herbicide of the triazolinone herbicide family. Greenhouse experiments were conducted to compare the effects of amicarbazone and atrazine on annual bluegrass control and quantum yield (ΦPSII) when applied at three treatment placements (soil-only, foliage-only, and foliage + soil). Herbicide rates for amicarbazone and atrazine were 0.53 and 2.25 kg ha−1, respectively. Amicarbazone applied soil-only and foliage + soil controlled annual bluegrass 57 and 59%, respectively, 1 wk after treatment (WAT). Atrazine applied to foliage + soil controlled annual bluegrass 48% at 1 WAT. All soil-only and foliage + soil treatments were similar 2 WAT. Foliage-only application of amicarbazone provided significantly less control than other amicarbazone treatments throughout the study. Amicarbazone applied soil-only and foliage + soil controlled annual bluegrass 100% at 3 WAT. Soil-only and foliage + soil applications of atrazine and amicarbazone had similar reductions in quantum yield (ΦPSII) at 1 to 3 WAT. Foliar-applied amicarbazone reduced ΦPSII 78, 84, and 86% at 1, 2, and 3 WAT, respectively. The rapid reduction in annual bluegrass ΦPSII and the increase in control resulting from soil contact of amicarbazone indicate root exposure of amicarbazone is beneficial for annual bluegrass control.

It is widely reported that Listeria monocytogenes can infect virtually all cell types, however, the degree to which this facultative intracellular pathogen can infect lymphocytes has not been well characterized. Previous studies have shown that a subset of lymphocytes, including activated T cells, are susceptible to apoptosis following exposure to L. monocytogenes, but the ability of the bacteria to replicate in the cytosol of lymphocytes prior to cell death was not examined. In this report, we demonstrate that intracellular L. monocytogenes can survive and multiply in vitro in a variety of transformed cell lines of lymphocytic origin. Intracellular L. monocytogenes were also recovered from splenic B cells, T cells, and NK cells following intravenous infection of mice. In fact, lymphocyte-associated L. monocytogenes comprised a substantial portion of the total bacterial burden in the spleen throughout the course of murine infection and B cell-deficient mice had significantly lower titers of bacteria present in the spleen following intravenous infection. These results suggest that lymphocytes can be a reservoir for L. monocytogenes growth in vivo.

Detection of cytotoxic activity by pathogen-specific T cells of unknown antigenic specificity is difficult due to the limitations of using infected cells, instead of peptide-pulsed cells, as targets. We report here that the recently described CD107 mobilization assay readily allowed for the ex vivo detection of cytotoxic T lymphocytes (CTL) with a novel MHC-Ib restriction that specifically recognized Listeria monocytogenes-infected macrophages. The CD107 mobilization assay is likely to be a useful tool for detection of CD8(+) T cells that recognize a wide variety of intracellular pathogens.

Carotenoids are important photoprotectant and light-harvesting pigments within the photosynthetic apparatus. Little information is available regarding carotenoid physiology in creeping bentgrass (Agrostis stolonifera L.). Research was conducted to investigate relative high and low irradiance adaptation of creeping bentgrass with respect to β-carotene and xanthophyll composition. 'Crenshaw' creeping bentgrass plants were acclimated for 7 d to relative high [47.9 mol m -2 d -1 photosynthetically active radation (PAR)] or low irradiance (4.7 mol m 2 d -1 PAR). After the acclimation period, plants were transferred from high to low (low irradiance treatment) and low to high (high irradiance treatment) irradiance. Clippings were harvested at 0, 24, 72, and 168 h after the acclimation period. Zeaxanthin and antheraxanthin decreased from 5.1 and 3.4 to 0.9 and 0.6 mg 100 g -1 fresh weight (FW), respectively, over 168 h in low irradiance. As the turf adapted to low irradiance, violaxanthin, lutein, and lutein-5,6-epoxide (epoxylutein) increased at 24 h, but levels decreased from 24 to 168 h. Zeaxanthin and antheraxanthin increased in high irradiance, while violaxanthin and β-carotene decreased. Lutein was the predominant carotenoid quantified regardless of irradiance treatment. Cumulative zeaxanthin, antheraxanthin, and violaxanthin increased as a percentage of the total carotenoids as the turfgrass adapted to high irradiance, but decreased in low irradiance. Conversely, neoxanthin and β-carotene decreased in high irradiance and increased in low irradiance. Creeping bentgrass produces carotenoid amounts comparable to other plant species potentially attributable to selection efforts of more stress-tolerant varieties.

A subset of CD8+T cells can rapidly secrete gamma interferon (IFN-γ) in an antigen-independent and interleukin-12 (IL-12)- and IL-18-dependent manner within 16 h of infection with the intracellular bacterial pathogenListeria monocytogenes. This rapid IFN-γ response is robust enough to be detected directly ex vivo and is not observed following infection with intracellular bacterial pathogens that remain sequestered within host cell vacuoles. We demonstrate here that three distinct pathways can lead to rapid secretion of IFN-γ by CD8+T cells duringL. monocytogenesinfection: (i) a direct cytokine-inducing activity encoded by the cholesterol-dependent cytolysin (CDC) listeriolysin O (LLO) acts within the infected cell, (ii) the pore-forming activity of LLO promotes cytosolic localization of bacterial products that trigger cytosol-specific signaling pathways, and (iii) the sustained presence of high concentrations of bacterial products can exogenously trigger cytokine production. Although it has been suggested that CDC protein toxins may act as Toll-like receptor 4 (TLR4) agonists to trigger proinflammatory cytokine secretion, we show in this report that TLR4 signaling is not required to induce a maximal rapid IFN-γ response by CD8+T cells. The results presented here indicate that multiple mechanisms contribute to the induction of rapid IFN-γ secretion by CD8+T cells duringListeriainfection and that care must be taken when interpreting the results of in vitro assays, since the contribution of each pathway can vary depending on how the assay is performed.