Non-transgenic Control Research Articles

IgG Immunocomplexes Drive the Differentiation of a Novel Subset of Osteoclasts Independent of RANKL and Inflammatory Cytokines.

Potentiation of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by IgG immunocomplexes (ICs) is generally considered an important pathway leading to cartilage and bone destruction in rheumatoid arthritis (RA). However, whether IgG ICs possess pro-osteoclastogenic potential independent of RANKL and inflammatory cytokines is unclear. Here we demonstrate that by fully cross-linking human FcγRIIa (hFcγRIIa) or co-ligating hFcγRIIa and TLR4, IgG ICs alone could drive the differentiation of human blood monocytes into nuclear factor of activated T cells cytoplasmic 1 (NFATc1-negative nonclassical osteoclasts (NOCs). Surprisingly, IgG ICs could also overrule RANKL-induced classical osteoclast (COC) differentiation in vitro. In mouse model of collagen-induced arthritis, hFcγRIIa-transgenic, but not nontransgenic control, mice suffered from cartilage/bone destruction accompanied by the presence of NFATc1- NOCs lining the eroded cartilage surface in affected joints. Our results not only identify a novel subset of IC-induced NOCs but also provide a possible explanation for the uncoupling of FcγR-mediated cartilage destruction from RANKL-related bone erosion in autoinflammatory arthritis. © 2021 American Society for Bone and Mineral Research (ASBMR)..

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30–40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

Activation tagging identifies Arabidopsis transcription factor AtMYB68 for heat and drought tolerance at yield determining reproductive stages.

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

Overexpression of a Sucrose Synthase Gene Indirectly Improves Cotton Fiber Quality Through Sucrose Cleavage.

The study aims to improve fiber traits of local cotton cultivar through genetic transformation of sucrose synthase (SuS) gene in cotton. Sucrose synthase (SuS) is an important factor that is involved in the conversion of sucrose to fructose and UDP-glucose, which are essential for the synthesis of cell wall cellulose. In the current study, we expressed a synthetic SuS gene in cotton plants under the control of a CaMV35S promoter. Amplification of an 813-bp fragment using gene-specific primers confirmed the successful introduction of SuS gene into the genome of cotton variety CEMB-00. High SuS mRNA expression was observed in two transgenic cotton plants, MA0023 and MA0034, when compared to the expression in two other transgenic cotton plants, MA0035 and MA0038. Experiments showed that SuS mRNA expression was positively correlated with SuS activity at the vegetative (54%) and reproductive stages (40%). Furthermore, location of transgene was found to be at chromosome no. 9 in the form of single insertion, while no signal was evident in non-transgenic control cotton plant when evaluated through fluorescent in situ hybridization and karyotyping analysis. Fiber analyses of the transgenic cotton plants showed increases of 11.7% fiber length, 18.65% fiber strength, and up to 5% cellulose contents. An improvement in the micronaire value of 4.21 was also observed in the MA0038 transgenic cotton line. Scanning electron microscopy (SEM) revealed that the fibers of the SuS transgenic cotton plants were highly spiral with a greater number of twists per unit length than the fibers of the non-transgenic control plants. These results determined that SuS gene expression influenced cotton fiber structure and quality, suggesting that SuS gene has great potential for cotton fiber quality improvement.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5′-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.

Comparative compositional analysis of cassava brown streak disease resistant 4046 cassava and its non-transgenic parental cultivar

ABSTRACT Compositional analysis is an important component of an integrated comparative approach to assessing the food and feed safety of new crops developed using biotechnology. As part of the safety assessment of cassava brown streak disease resistant 4046 cassava, a comprehensive assessment of proximates, minerals, amino acids, fatty acids, vitamins, anti-nutrients, and secondary metabolites was performed on leaf and storage root samples of 4046 cassava and its non-transgenic parental control, TME 204, collected from confined field trials in Kenya and Uganda over two successive cropping cycles. Among the 100 compositional components that were assessed in samples of 4046 and control TME 204 cassava roots (47 components) and leaves (53 components), there were no nutritionally relevant differences noted. Although there were statistically significant differences between the transgenic and control samples for some parameters, in most cases the magnitudes of these differences were small (20%), and in every case where comparative literature data were available, the mean values for 4046 and control cassava samples were within the range of normal variation reported for the compositional component in question. Overall, no consistent patterns emerged to suggest that biologically meaningful adverse changes in the composition or nutritive value of the leaves or storage roots occurred as an unintended or unexpected consequence of the genetic modification resulting in 4046 cassava. The data presented here provide convincing evidence of the safety of 4046 cassava with respect to its biochemical composition for food and feed, and it could be considered as safe as its non-transgenic control.

Overexpression of ICE1 gene in mungbean (Vigna radiata L.) for cold tolerance

The putative transgenic mungbean (Vigna radiata Var. OBGG-52) plants were regenerated on Murashige and Skoog (1962) medium supplemented with 1.0 mg/L 6-benzylaminopurine (BA). The transgenic status of transformants (T2 generation) was determined through PCR analysis using nptII and ICE1 gene-specific primers as well as uidA reporter gene expression. About 75% of the transformed plantlets were successfully established in the greenhouse. Overexpression of ICE1 gene in transgenic mungbean plants resulted in cold-tolerance at the seedling stage when compared to non-transformed plants. Seeds from T2 generation were further studied for cold tolerance through nutrient culture. Transgenic seeds had significantly increased germination and growth on a nutrient medium at 10–14 °C in growth chamber compared to the non-transgenic control. Moreover, the root and shoot length of transformed plants were significantly increased at 10–14 °C as compared with the non-transformed plants. Under cold stress, it was also observed that the transformed plantlets have significantly higher chlorophyll, proline, and lipid content as well as anti oxidative enzyme activity as compared to non-transformed plants. This study will help in mungbean improvement program for abiotic stress tolerance. Cold temperature damaged the crop productivity of mungbean (Vigna radiata L. Wilczek), a high protein rich legume crop. The overexpression of cold responsive transcription factor, INDUCER OF CBF EXPRESSION 1 (ICE1) in mungbean enhanced cold tolerance.

Overexpression of <italic>TaJRL53 </italic>enhances the <italic>Fusarium </italic>head blight resistance in wheat

<p indent=0mm>Jacalin-related lectins (JRLs) are a group of plant lectins with jacalin protein domain, which play important roles during plant response to biotic and abiotic stress. According their carbohydrate binding specificities, the JRLs were described as two types: galactose-binding acalins (gJRLs) and mannose-binding jacalins (mJRLs). Previous studies have shown that <italic>TaJRL53</italic> encodes a protein with Jacalin and Dirigent domains and can be up-regulated by <italic>Fusarium graminearum</italic> infection. Wheat FHB induced by this pathogen is a disastrous disease, which not only reduces the grain yield, but also contains deoxynivalenol and other toxins, which seriously affect the health of human and livestock. To dissect the gene’s function in FHB resistance, we silenced and overexpressed the <italic>TaJRL53</italic> in wheat respectively. As a result, attenuating <italic>TaJRL53</italic> increased susceptibility to <italic>Fusarium graminearum</italic>. And susceptible wheat transformed with <italic>TaJRL53</italic> over-expression showed higher <italic>Fusarium</italic> head blight resistance, the number of diseased spikelets (NDS) and length of diseased spike rachis (LDR) of transgenics wheat with <italic>TaJRL53</italic> overexpression were significantly reduced than those of wild type. After <italic>Fusarium graminearum</italic> infection, the expression of ROS synthesis pathway, JA synthesis and signaling pathways marker genes, and disease related genes in <italic>TaJRL53</italic> over-expression transgenic plants were higher than those in non-transgenic control. These results suggest that <italic>TaJRL53</italic> enhanced the FHB resistance in wheat through regulating ROS synthesis pathway and JA signal transduction pathways. This study enhanced the understanding of wheat JRLs family genes’ function and laid a foundation for the mechanism dissection of <italic>TaJRL53</italic> resistant to FHB in wheat.

Barley Plants Overexpressing Ferrochelatases (HvFC1 and HvFC2) Show Improved Photosynthetic Rates and Have Reduced Photo-Oxidative Damage under Drought Stress than Non-Transgenic Controls

We investigated the roles of two Ferrochelatases (FCs), which encode the terminal enzyme for heme biosynthesis, in drought and oxidative stress tolerance in model cereal plant barley (Hordeum vulgare). Three independent transgenic lines ectopically overexpressing either barley FC1 or FC2 were selected and evaluated under well-watered, drought, and oxidative stress conditions. Both HvFC1 and HvFC2 overexpressing transgenics showed delayed wilting and maintained higher photosynthetic performance relative to controls, after exposure to soil dehydration. In each case, HvFC overexpression significantly upregulated the nuclear genes associated with detoxification of reactive oxygen species (ROS) upon drought stress. Overexpression of HvFCs, also suppressed photo-oxidative damage induced by the deregulated tetrapyrrole biosynthesis mutant tigrinad12. Previous studies suggest that only FC1 is implicated in stress defense responses, however, our study demonstrated that both FC1 and FC2 affect drought stress tolerance. As FC-derived free heme was proposed as a chloroplast-to-nuclear signal, heme could act as an important signal, stimulating drought responsive nuclear gene expression. This study also highlighted tetrapyrrole biosynthetic enzymes as potential targets for engineering improved crop performance, both in well-watered and water-limited environments.

DP-2Ø2216-6 maize does not adversely affect rats in a 90-day feeding study

Maize plants containing event DP-2Ø2216-6 (DP202216), which confers herbicide tolerance through expression of phosphinothricin acetyltransferase and enhanced grain yield potential via temporal modulation of the native ZMM28 protein, were developed for commercialization. To address current regulatory expectations, a mandatory 90-day rodent feeding study was conducted to support the safety assessment. Diets containing 50% by weight of ground maize grain from DP202216, non-transgenic control, and 3 non-transgenic reference varieties, were fully characterized, along with the grain, and diets were fed to Crl:CD®(SD) rats for at least 90 days. As anticipated, no biologically-relevant effects or toxicologically-significant differences were observed on survival, body weight/gain, food consumption/efficiency, clinical and neurobehavioral evaluations, ophthalmology, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), organ weights, or gross and microscopic pathology parameters in rats fed a diet containing up to 50% DP202216 maize grain when compared with rats fed diets containing control or reference maize grains. The results of this study support the conclusion that maize grain from plants containing event DP-2Ø2216-6 is as safe and nutritious as maize grain not containing the event and add to the significant existing database of rodent subchronic studies demonstrating the absence of hazards from consumption of edible fractions of genetically modified plants.

Overexpression of annexin gene in rice (Oryza sativa L.) for salinity and water stress

The aim of the study is to develop transgenic rice with annexin genes (AnnBj2) to determine its salinity and water stress tolerance through Agrobacterium-mediated genetic transformation. The transgenic somatic embryos were developed on Murashige and Skoog medium fortified with 0.5 mg L−1 kinetin and 3.0 mg L−1 2,4-D. About 80% of the transformed somatic embryos germinated and was successfully established in the transgenic greenhouse. The transformed plantlets (T0 and T1 generation) were identified through PCR using NPT-II and AnnBj2 gene–specific primers as well as positive uidA reporter gene expression. Compared with non-transformed plants, the transgenic rice plants overexpressing AnnBj2 gene exhibited salt tolerance at the seedling stage. Seeds generated at T0 and T1 generations were further studied to determine their salinity tolerance through nutrient culture and pot culture experiments for water stress. When compared with the non-transgenic control, transgenic seeds (T0 and T1) had increased germination on a nutrient medium having 200 mM NaCl. The data showed that the shoot and root lengths were longer at 200 mM NaCl when compared with those of the non-transformed plants. Further, the biochemical assessment (photosynthetic pigment analysis, protein, proline content, and oxidative enzyme activity) was performed between transformed (T0 and T1) and non-transformed plantlets under stress. It was noted that the transformed plantlets were higher in chlorophyll and proline content as well as oxidative enzyme activity as compared with the non-transformed plants.

Early Stage Development of a Newcastle Disease Vaccine Candidate in Corn.

Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production. In the current study, we attempted to express fusion (F) and hemagglutinin-neuraminidase (HN) proteins (the protective antigens against NDV) under constitutive 35S and seed-specific Zein promoters, respectively. Almost 2–7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8–28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds). Similarly, 1.66 μg/ml of F protein in corn leaves, i.e., 0.5% of total soluble protein, and 2.4 μg/ml of HN protein in corn seed, i.e., 0.8% of total seed protein, were found when calculated through ELISA. Similar levels of immunological response were generated in chicks immunized through injection of E. coli-produced pET F and pET HN protein as in chickens orally fed leaves and seeds of maize with expressed immunogenic protein. Moreover, the detection of anti-NDV antibodies in the sera of chickens that were fed maize with immunogenic protein, and the absence of these antibodies in chickens fed a normal diet, confirmed the specificity of the antibodies generated through feeding, and demonstrated the potential of utilizing plants for producing more vaccine doses, vaccine generation at higher levels and against other infectious diseases.

Evaluation of broiler performance and carcass yields when fed diets containing maize grain from transgenic product DP-2Ø2216-6

Summary Nutritional equivalency feeding studies with diets containing genetically modified grain continue to be required by some regulatory agencies as part of the safety assessment of new events. These studies have long been conducted using broiler chickens, as they are very sensitive to nutritional deficiencies and antinutrients in the diet owing to their very rapid growth rate. A new transgenic maize product containing event DP-2O2216-6 (DP202216) with enhanced grain yield potential and an herbicide-tolerance trait has been developed. The nutritional equivalency of grain produced from maize containing this event with conventional grain was evaluated in a 42-D feeding trial with male and female Ross 708 broiler chickens. Broilers consuming diets produced with grain from DP202216 maize plants performed as well and produced similar organ, carcass, and parts yields as broilers consuming diets produced with nontransgenic near-isogenic control grain. In addition, the ranges of observed performance and carcass and organ yield trait values from DP202216 and control groups were similar to the respective ranges observed for broilers fed diets containing nontransgenic commercially available maize grains. Based on these results, it is concluded that grain from DP202216 maize is nutritionally equivalent to grain from nontransgenic near-isogenic maize, consistent with previously published reports on whole-food (where the edible crop tissue is incorporated in the feed) animal feeding studies for other genetically modified events.

Abstract 3740: Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma

Abstract Introduction: This study aimed to clarify the role of phosphatidylcholine (PC) and choline metabolites in β-catenin-driven hepatocellular carcinoma (HCC) using human HCC cell lines and transgenic zebrafish expressing hepatocyte-specific activated β-catenin. Background: β-catenin is widely believed to be a major oncogene in HCC. PC is one of the major constituents of biological membranes and is synthesized via the phosphatidylethanolamine N-methyltransferase (PEMT) and cytidine diphosphate (CDP)-choline pathways in the liver. Several studies have reported that changes in gene expression and enzyme activity lead to altered PC synthesis in cancer, which contributes to malignant growth. However, the role of altered PC synthesis in HCC and the contribution of activated β-catenin to changes in PC metabolism have not been fully explored. Methods and Results: Transgenic zebrafish expressing hepatocyte-specific activated β-catenin develop HCC as adults and show significant liver enlargement during larval development due to increased cell proliferation. We measured PC levels in transgenic zebrafish HCC and non-transgenic control livers using liquid chromatography-mass spectrometry (LC-MS). PC levels in transgenic zebrafish HCC were significantly higher than in controls. Using a colorimetric assay kit, we found that PC levels were significantly higher in human HCC cell lines (SNU398, C3A and PLC/PRF/5) than in immortalized human hepatocytes (THLE2). To clarify the role of PC and choline metabolites in β-catenin-driven liver overgrowth, we treated larval zebrafish with bezafibrate (PEMT inhibitor) or meclofenoxate (CPT/CEPT inhibitor). We found that both of these inhibitors of PC lipid synthesis reduced larval liver size and decreased the number of EdU-labeled proliferative cells in transgenic zebrafish. Histologically, nonalcoholic steatohepatitis (NASH)-like changes, such as steatosis, ballooning, inflammation, and hepatocellular necrosis, were detected in the livers of transgenic larvae treated with bezafibrate or meclofenoxate. To characterize the role of PC in human HCC cell lines, we treated SNU398, C3A and PLC/PRF/5 cells with bezafibrate or meclofenoxate and measured PC levels by colorimetric assay and viability by CellTiter-Glo Assay. We found that both drugs decreased PC levels and viability in these HCC cell lines. Furthermore, SNU398 cells overexpressing activated β-catenin using a Tet-on system demonstrated higher PC levels and greater viability than SNU398 control cells. Conclusion: Our results suggest that increased PC synthesis is associated with cell proliferation, zebrafish larval liver overgrowth, and hepatocarcinogenesis. These results also suggest that activated β-catenin may regulate choline metabolites and contribute to an increase in PC production. Citation Format: Junko Kuramoto, Richard Smith, Sharanya M. Kalasekar, Alexis Fulbright, Gregory S. Ducker, Kimberley J. Evason. Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3740.

The circadian nuclear receptor RORα negatively regulates cerebral ischemia–reperfusion injury and mediates the neuroprotective effects of melatonin

Disruptions of the circadian rhythm and reduced circulating levels of the circadian hormone melatonin predispose to ischemic stroke. Although the nuclear receptor RORα is considered as a circadian rhythm regulator and a mediator of certain melatonin effects, its potential role in cerebral ischemia-reperfusion (CI/R) injury and in the neuroprotective effects of melatonin remain undefined. Here, we observed that CI/R injury in RORα-deficient mice was associated with greater cerebral infarct size, brain edema, and cerebral apoptosis compared with wild-type model. In contrast, transgenic mice with brain-specific overexpression of RORα versus non-transgenic controls exerted significantly reduced infarct volume, brain edema and apoptotic response induced by CI/R. Mechanistically, RORα deficiency was found to exacerbate apoptosis pathways mediated by endoplasmic-reticulum stress and mitochondria and aggravate oxidative/nitrative stress after CI/R. Further studies revealed that RORα deficiency intensified the activation of nuclear factor-κB signaling induced by CI/R. Given the emerging evidence of RORα as an essential melatonin activity mediator, we further investigated the RORα roles in melatonin-exerted neuroprotection against acute ischemic stroke. Melatonin treatment significantly decreased infarct volume and cerebral apoptosis; mitigated endoplasmic reticulum stress and mitochondrial dysfunction; and inhibited CI/R injury-induced oxidative/nitrative stress and nuclear factor-κB activation, which was eradicated in RORα-deficient mice. Collectively, current findings suggest that RORα is a novel endogenous neuroprotective receptor, and a pivotal mediator of melatonin's suppressive effects against CI/R injury.

Silencing Folylpolyglutamate Synthetase1 (FPGS1) in Switchgrass (Panicum virgatum L.) Improves Lignocellulosic Biofuel Production.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

Alcohol Enhances Responses to High Frequency Stimulation in Hippocampus from Transgenic Mice with Increased Astrocyte Expression of IL-6.

Recent studies show that alcohol exposure can induce glial production of neuroimmune factors in the CNS. Of these, IL-6 has gained attention because it is involved in a number of important physiological and pathophysiological processes that could be affected by alcohol-induced CNS production of IL-6, particularly under conditions of excessive alcohol use. For example, IL-6 has been shown to play a role in hippocampal behaviors and synaptic plasticity (long-term potentiation; LTP) associated with memory and learning. Surprisingly, in our in vitro studies of LTP at the Schaffer collateral to CA1 pyramidal neuron synapse in hippocampus from transgenic mice that express elevated levels of astrocyte produced IL-6 (TG), LTP was not altered by the increased levels of IL-6. However, exposure to acute alcohol revealed neuroadaptive changes that served to protect LTP against the alcohol-induced reduction of LTP observed in hippocampus from non-transgenic control mice (WT). Here we examined the induction phase of LTP to assess if presynaptic neuroadaptive changes occurred in the hippocampus of TG mice that contributed to the resistance of LTP to alcohol. Results are consistent with a role for IL-6-induced neuroadaptive effects on presynaptic mechanisms involved in transmitter release in the resistance of LTP to alcohol in hippocampus from the TG mice. These actions are important with respect to a role for IL-6 in physiological and pathophysiological processes in the CNS and in CNS actions of alcohol, especially when excessive alcohol used is comorbid with conditions associated with elevated levels of IL-6 in the CNS.

The potential and efficacy of Allium sativum leaf lectin (ASAL) against sap-sucking insect pests of transgenic maize

The gene sequence encoding the mannose-binding homodimeric protein Allium sativum leaf agglutinin (ASAL) was introduced into maize inbred lines to achieve resistance against sap-sucking corn leaf aphids in transgenic lines. Maize transformants were generated after the co-cultivation of immature maize embryos with the Agrobacterium strain LB4404 harbouring a recombinant Ti-binary ASAL construct driven by the polyubiquitin promoter. The preliminary screening of maize transformants was performed via GUS histochemical analysis, and PCR and Southern blotting confirmed the insertion of the transgene and its stable integration in five transgenic maize lines. Transcript abundance was quantified by a quantitative real-time PCR assay, which revealed variable expression of the ASAL transgene among five transgenic maize lines. The highest mRNA expression of the ASAL gene was found in the A23 transgenic maize line, while the lowest expression was found in the AU1 transgenic maize line. In planta bioassays in the T1 progeny of the transgenic maize lines revealed high resistance against corn leaf aphids compared to the control non-transgenic line. The mortality of the infesting aphids (Rhopalosiphum maidi) was found to vary from 40 to 71% compared to that of the non-transgenic control maize line.

2043-P: Hyaluronan Produced by Inflamed Islet Endothelial Cells Contributes to HIAPP Aggregation

In type 2 diabetes (T2D), aggregation of human islet amyloid polypeptide (hIAPP) occurs within the islet extracellular matrix (ECM) and is toxic to β cells. We recently showed that hIAPP aggregation also induces a pro-inflammatory response in islet endothelial cells (ECs). In most tissues, this inflammatory response in ECs is mediated by upregulating production of ECM components, such as hyaluronan (HA). Indeed, we find that HA accumulates in the islet ECM of hIAPP transgenic mice, where it was colocalized with amyloid deposits, while no HA accumulation occurred in non-transgenic littermate controls (which do not develop islet amyloid). Therefore, we hypothesized that hIAPP aggregation results in HA production from islet ECs, and that this HA production exacerbates hIAPP deposition/toxicity. To test this hypothesis, we utilized immortalized murine islet ECs (MS-1 cells), which under normal conditions express low levels of HA synthase (Has isoforms 2 and 3). Treatment of these cells for 48 h with hIAPP (20 µM) increased Has2 and Has3 mRNA levels (23±4 and 8±1 fold over vehicle; n=4, p&amp;lt;0.05 for both), which was abrogated upon co-incubation with Congo red, an inhibitor of hIAPP aggregation (1.4±0.3 and 1.4±0.2 for Has2 and Has3, respectively; n=4). Next, we used a thioflavin T assay, wherein fluorescence is proportional to fibril formation from amyloidogenic peptides, to determine the kinetics of hIAPP aggregation ± HA. We found co-incubation of HA with synthetic hIAPP peptide resulted in a marked acceleration (log time of 1 vs. 40 hours) and increase in maximal (greater than 2-fold) hIAPP accretion. In sum, exposure of islet ECs to aggregated hIAPP increases HA synthesis by these cells, which in turn further exacerbates aggregation of hIAPP to form amyloid. Thus, interventions targeting endothelial cell inflammation and/or HA production may be useful in blocking amyloid formation and its toxic effects in T2D. Disclosure J.J. Castillo: None. M.F. Hogan: None. A. Aplin: None. N. Esser: None. A.T. Templin: None. R.L. Hull: None. Funding National Institutes of Health (T32HL007028)

1884-P: Cardiomyocyte-Specific PKA Inhibition Ameliorates Cardiac Dysfunction Induced by Obesity

Obesity and type 2 diabetic (T2D) cause cardiac dysfunction, which could be related to chronically activated sympathoadrenergic system (SAS). SAS system activation leads to the activation of PKA in cardiomyocytes. In this study, we aimed to test the hypothesis that chronically activated PKA plays an important role in obese induced cardiac dysfunction. Methods and Results: 1. Cardiomyocyte specific PKA inhibition (cPKAi) transgenic (TG) mouse model (n=18) and nontransgenic littermate control (NLC, n=20) mice (3-month old) were fed with high fat food (D12451, Research Diet) or normal chow for 6 months.2. In 3-month old male TG mice, intraperitoneal glucose injection did not increase blood glucose as high as in 3-month old NLC mice. The area under the curve of cPKAi TG mice was significantly less than that of control mice. cPKAi transgenic mice had decreased cardiac function after overnight fasting while control mice did not have cardiac function decrement even though both types of mice had the same extend of glucose reduction after overnight fasting. 3. The BW increase was more in TG mice. 4. After HFD feeding, cPKAi TG group had better cardiac function: (1). The maximal systolic pressure (mmHg) of TG (107.8±4.49) was significantly higher than that of NLC (83.0±8.1) mice. (2). The end diastolic pressure (EDP, mmHg) of TG (2.7 ± 2.2) was significantly lower in TG mice (11.5 ± 3.3); (3). Maximum dp/dt was significantly greater in TG than in NLC mice; (4). The relaxation rate of the heart was significantly better in TG than in NLC mice; 5. The HW/BW (mg/g) was significantly less in TG mice (4.6 ± 0.2) than in control mice (5.2 ± 0.2). 6. HFD reduced myocytes contractions, Ca transients, and Ca currents in both groups but the reduction was less in TG myocytes. 7. TG hearts had less inflammatory cell infiltration and fibrosis than control hearts. Conclusion: cPKAi enhances cardiac glucose uptake and ameliorates cardiac remodeling and preserves cardiac function in HFD-induced obese. Disclosure X. Chen: None. Funding American Diabetes Association (1-19-IBS-210)