Fold Increase In Expression Research Articles

Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Genome mining of Bacillus licheniformis MCC2514 for the identification of lasso peptide biosynthetic gene cluster and its characterization.

The probiotic strain Bacillus licheniformis MCC2514 has been shown to produce a strong antibacterial peptide and the whole genome sequence of this strain is also reported in our previous study. The present study is focused on the genome level investigation of this peptide antibiotic and its characterization. Genome mining of the culture revealed the presence of three putative bacteriocin clusters, viz. lichenicidin, sonorensin and lasso peptide. Hence, the mode of action of the peptide was investigated by reporter assay, scanning electron microscopy, and Fourier Transform Infrared spectroscopy. Additionally, the peptide treated groups of Kocuria rhizophila showed a reduction in the fold expression for transcription-related genes. The gene expression studies, quantitative β-galactosidase induction assay using the RNA stress reporter strain, yvgS along with the homology studies concluded that lasso peptide is responsible for the antibacterial activity of the peptide which acts as an inhibitor of RNA biosynthesis. Gene expression analysis showed a considerable increase in fold expression of lasso peptide genes at various fermentation hours. Also, the peptide was isolated, and its time-kill kinetics and minimum inhibitory concentration against the indicator pathogen K. rhizophila were examined. The peptide was also purified and the molecular weight was determined to be ~ 2kDa. Our study suggests that this bacteriocin can function as an effective antibacterial agent in food products as well as in therapeutics as it contains lasso peptide, which inhibits the RNA biosynthesis.

Transcriptional analysis of oxidative and nitrosative stress on oral opportunistic Candida albicans.

The yeast Candida albicans is one of the most aggressive opportunistic pathogens in immunocompromised patients. The ability of the yeast to withstand stresses and radicals is of great concern. In the present study, four isolates of C. albicans were taken from patients with oral candidiasis and grown on RPMI for 24 hours at 37°C. Then, they were exposed to various concentrations of oxidative (H2O2) and nitrosative (HNO3) stress for two hours, and gene expression rates were measured through RT-PCR. After initial biofilm formation steps and growth validation, RNA extracted from the yeast and gene expression status were evaluated. Upon treatment with H2O2, the gene expression profile for ALS1, MLH1, and EXO1 showed approximately a fold increase in expression. While within HNO3 the yeast gene expression exhibited a dramatic increase in ALS1 up to 217 folds, while others such as MLH1, HWP1, and ERG11 showed a one-fold increase in the expression rate. The findings of this research indicate a considerable expression activity within the biofilm of Candida albicans, increased rate of DNA mismatch repair and break fixation may indicate the ability of the yeast to tolerate high concentrations of free radicals. It paves the way toward understanding the pathogenicity of the yeast and its survival capability inside macrophages. The study also revealed that the biofilm strategy of the yeast is more active within these stresses.

MBD2a-NuRD Binds to the γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Bcl11a Mediated Silencing of Hb F Expression

A mechanistic understanding of γ-globin silencing is of clinical importance because reversal of silencing in adult erythroid cells results in high level expression Hb F which is therapeutic in sickle cell anemia and beta thalassemia. We describe here studies to elucidate the mechanism by which MBD2a-NuRD is an obligate partner of Bcl11a in silencing of HbF. We have previously shown that depletion of MBD2 or disruption of the MBD2-NuRD chromatin remodeling complex, but not MBD3-NuRD complex results in high levels of Hb F despite a concomitant 2-3 fold increase in expression of Bcl11a, a major silencing transcription factor (Yu etal. Haematologica, 2019). At the same time, Bcl11a can interact with both the MBD3 and MBD2 NuRD complexes. To elucidate the specific role of MBD2-NuRD, Chromatin Immunoprecipitation-qPCR(ChIP-qPCR) assays carried out in both adult erythroid phenotype HUDEP-2 and primary progenitor derived adult erythroid cells showed binding at the γ-globin gene promoter. To clarify the role of MBD2-NuRD in establishing closed chromatin configuration at the γ-globin gene loci, we carried out ATAC seq assays which showed that MBD2 knockout resulted in the same pattern of chromatin opening as reported for Bcl11a knockout in HUDEP-2 cells, consistent with our previous data showing ~40-50% Hb F in MBD2 knockout cells. In order to examine the effect of MBD2-NuRD on nucleosome positioning over the γ-globin gene promoter, NOME-SEQ assays were performed in HUDEP 2 cells which showed a nucleosome positioned at -110 to + 36 relative to the transcription start site (TSS) in MBD +/+ cells but not MBD2 KO HUDEP-2 cells, such that the NFY-A transcriptional activator binding site but not the Bcl11a repressor binding site is occluded. MBD2 exists in two isoforms in erythroid cells, resulting from alternative translation initiation sites. MBD2a contains an arginine rich (GR) domain which is absent in MBD2b and MBD3. Addback of MBD2a but not MBD2b in MBD2 KO HUDEP-2 cells resulted in silencing of γ-globin gene expression. Editing of the endogenous MBD2 gene such that predominantly MBD2b was expressed relieved silencing. To understand how the GR domain contributes to silencing we carried out unbiased proteomic analysis of MBD2a vs MBD2b NuRD complexes, which identified only 3 proteins uniquely associated with MBD2a. One of these, PRMT5, has been implicated in silencing of HbF. Knockdown of PRMT5 resulted in a`from <1% to ~15% γ-globin RNA in parental HUDEP-2 cells and a 4-5 fold increase in primary human progenitor derived erythroid cells, but caused no significant further increase in MBD2 KO HUDEP-2 cells. ChIP qPCR assays showed that PRMT5 and its obligate binding partner MEP50 occupy the γ- promoter in parental HUDEP-2 cells and primary erythroid cells but not in MBD2 KO HUDEP-2 cells. Furthermore, knockdown of PRMT5 resulted in frequent displacement of the nucleosome positioned at -110 to + 36 relative to the TSS. In order to determine the role that the GR domain might have in occupancy of MBD2a-NuRD at the γ-promoter, in vitro binding studies where done, which showed a requirement for both CpG methylation and the GR domain for high affinity binding of the methyl cytosine binding domain of MBD2 to the proximal γ-globin gene promoter sequences. We conclude that MBD2a-NuRD is required for silencing of Hb F due to its ability to localize at the methylated γ-globin gene promoter in conjunction with Bcl11a and recruit PRMT5, thereby stably positioning a nucleosome that blocks binding of the transcriptional activator NFY-A. These results reinforce the concept that targeted disruption of this specific complex has therapeutic potential to increase HbF in sickle cell anemia and β thalassemia.

Dipteran endoparasitoid Exorista bombycis utilizes antihemocyte components against host defense of silkworm Bombyx mori.

Dipteran endoparasitoids avoid host immune response; however, antidefense components from the Dipteransare unknown. Infestation of commercial silkworm Bombyx mori Linnaeus (Lepidoptera: Bombycidae) by endoparasitoid Exorista bombycis Louis (Diptera: Tachinidae) induced immune reactions, cytotoxicity, granulation, degranulation, and augmented release of cytotoxic marker enzymelactate dehydrogenase(LDH), and degranulation-mediator enzyme β-hexosaminidase in hemocytes. In this study, by reverse phase high-performance liquid chromatography, fractions of E. bombycis larval tissue protein with antihemocytic activity are separated. From the fraction, peptides of hemocyte aggregation inhibitor protein (HAIP) and pyridoxamine phosphate oxidase (PNPO) are identified by mass spectrometry. Interacting partners of HAIP and PNPO are retrieved that further enhance the virulence of the parasitoid. PNPO and HAIP genes showed afour- to seven fold increase in expression in the integument of the parasitoid larva. Together, the dipteran endoparasitoid E. bombycis exploit antihemocyte activity to inhibit host defense reactions in addition to defense evasion contemplated.

Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System.

Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.

Melatonin enhances stress tolerance in pigeon pea by promoting flavonoid enrichment, particularly luteolin in response to salt stress.

Melatonin improves plant resistance to multiple stresses by participating in the biosynthesis of metabolites. Flavonoids are an important family of plant secondary metabolites and are widely recognized to be involved in resistance; however, the crosstalk between melatonin and flavonoid is largely unknown. We found that the resistance of pigeon pea (Cajanus cajan) to salt, drought, and heat stresses were significantly enhanced by pre-treatment with melatonin. Combined transcriptome and LC-ESI-MS/MS metabolomics analyses showed that melatonin significantly induced the enrichment of flavonoids and mediated the reprogramming of biosynthetic pathway genes. The highest fold-increase in expression in response to melatonin treatment was observed for the CcF3´H family, which encodes an enzyme that catalyses the biosynthesis of luteolin, and the transcription factor CcPCL1 directly bonded to the CcF3´H-5 promoter to enhance its expression. In addition, salt stress also induced the expression of CcPCL1 and CcF3´H-5, and their overexpression in transgenic plants greatly enhanced salt tolerance by promoting the biosynthesis of luteolin. Overall, our results indicated that pre-treatment of pigeon pea with melatonin promoted luteolin biosynthesis through the CcPCL1 and CcF3´H-5 pathways, resulting in salt tolerance. Our study shows that melatonin enhances plant tolerance to multiple stresses by mediating flavonoid biosynthesis, providing new avenues for studying the crosstalk between melatonin and flavonoids.

Symbiotic interplay of Piriformospora indica and Azotobacter chroococcum augments crop productivity and biofortification of Zinc and Iron

In the present study Piriformospora indica (Pi) a phyto-promotional fungus and Azotobacter chroococcumWR5 (AzWR5) a rhizobacterium, were symbiotically evaluated for their role in improving the nutritional quality of wheat (Triticum aestivum L.). Co-inoculation of Pi+AzWR5 modified root system architecture of host and along with increasing the proportion of finer roots by 88% and 92% in C306 and Hd2967 respectively. Furthermore, the synergistic impact of Pi+AzWR5 interplayed for enhanced accumulation of Zn and Fe in different plant parts including grains (3.12 and 1.33 fold respectively). Pi+AzWR5 increased the transfer factor of Zn (62%, 94%, 91% and 213%) and Fe (31%, 54%, 68% and 32%) in root, stem, leaves and grains, respectively, and translocation factor of Zn (20%, 18% and 63%) and Fe (18%, 29% and 29%) for root-stem, root-leaves and root-grains, respectively. In addition to these co-inoculation of endophytes led to several fold increase in expression of four ZIP transporter genes in roots and shoot. In addition to these symbiotic association of endophytes with host led to 3 fold increase in grain yield. We thereby conclude that co-inoculation of Pi+AzWR5 substantially improves mobilization of Zn and Fe from soil and increase its concentration in grains as well as improves crop yield.

Germin-like Protein 9-3, a Potential Allergen from a Rice Variety derived from Radiation-Mutation Breeding: An In Silico Study

New varieties of crops with a history of safe and widespread use derived from radiation-mutation breeding are generally regarded to be unlikely to produce food safety concerns compared with genetically modified (GM) crops. However, more recent molecular analysis revealed that there might be a greater likelihood that the former may bring about more unintended effects than genetic modification by foreign gene introduction. Thus, in this paper, an allergenicity prediction analysis was carried out on the microarray data of a stable rice mutant derived from γ irradiation-mutation breeding. For the first time, it was revealed that germin-like protein (GLP) 9-3, the transcript with the most significant fold increase in expression in a transcriptome data analyzed, may be a potential allergen in radiation-mutant rice. An immunoinformatic analysis of GLP 9-3 disclosed several linear and conformational (discontinuous) B-cell epitopes and MHCII-binders. These epitopes were located primarily on the random coil and surface-exposed regions based on inspection of the protein’s secondary structures. Additionally, the GLP 9-3 epitopes were predicted to be reactive in more than 50% of the population in top rice-producing and -consuming countries based on human leukocyte antigen (HLA) haplotypes. Since this is the first report on the up-regulation of GLP 9-3 allergen in mutant rice, it is recommended to investigate this molecule for further biochemical and clinical analyses.

Abstract LB236: Utilizing endometrial tumor organoids to model cancer immunomodulation

Abstract Endometrial cancer is the most common gynecologic malignancy in the United States. Most women present with early stage disease and have a favorable prognosis. However, the treatment options for recurrent endometrial cancer is limited. Immunotherapy has become increasingly popular in the treatment of recurrent endometrial cancer. A hallmark of malignant cells is immune-evasion at both the genetic level and in the tumor microenvironment. We hypothesized that an endometrial cancer organoid model could be utilized to study the tumor immune microenvironment. Major Histocompatibility Complex (MHC) is a protein receptor recognized with its antigen by immune cells to screen either non-self or tumorous cells. It has a crucial role in eliminating non-self cells, such as cancer cells. We first assessed the genes involved in the regulation of MHC I and II in cultured normal and tumor organoids. RNA-Sequencing of fifty-six patients from our living organoid biobank were analyzed and we found an immunologic cluster of high-grade endometrial cancers that display significant downregulation in both MHC I and II. This was confirmed with real time-PCR which showed that high-grade cancers organoids had downregulated MHC I and II compare to organoids derived from low-grade cancer tissues and benign tissues. We next sought to demonstrate that both normal and endometrial cancer organoids retain the ability to present external antigen by using DQ-Ovalbumin. We demonstrated that endometrial organoids retain the ability to present antigen, but cancer organoids display reduced efficiency in the antigen processing. Next, we stimulated endometrial cancer organoids with interferon-gamma (IFNg), a well known cytokine for enhancing immunological response, to increase the expression of MHC I and II in cancer organoids. IFNg led to a 2-6 fold increase in MHC expression in low-grade cancer organoids, but a 5-40 fold increase in expression in high-grade cancer organoids utilizing real time-PCR. Finally, we implement the organoid-immune cell co-culture system with patient-derived organoids and CD8+ cytotoxic T-Cells in the allogeneic and syngeneic settings. Endometrial cancer organoids and matched patient-derived cytotoxic T-Cells were co-cultured for 48 hours and imaged for 16 hours. Both migratory effect and tumor killing effect were compared. As expected, in the allogeneic setting CD8+ T-Cells efficiently colocalized and killed cancer cells in the vehicle and IFNg stimulated groups. In the syngeneic setting, CD8+ T-Cells did not colocalize or kill cancer cells in the vehicle, whereas they significantly colocalized and killed cancer cells when stimulated with IFNg. Our data demonstrate that high-grade endometrial cancer cells downregulate MHC I and II, suggesting that they can evade the host immune system. We also showed that this effect is patient-specific and can be overcome by stimulating with interferon-gamma. Furthermore, we demonstrated that increasing MHC I and II among cancer organoids enhances the target cell recognition and apoptosis induction by CD8+ T-cells. Collectively, our finding offers an approach to study the tumor microenvironment that can be individualized for each patient through the use of patient-derived cancer organoids. This technology can be further developed to study targeted therapies and provides a much needed pre-clinical model for the study of endometrial cancer. Citation Format: Aaron Nizam, Charlie Chung, Gary L. Goldberg, Semir Beyaz. Utilizing endometrial tumor organoids to model cancer immunomodulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB236.

Root colonizing Burkholderia sp. AQ12 enhanced rice growth and upregulated tillering-responsive genes in rice

Association of 1-aminocyclo propane-1-carboxylate (ACC) deaminase producing bacteria with plants under abiotic stress conditions leads to stress alleviation not only by the regulation of ethylene concentration but also by the activation of numerous physiological and genetic mechanisms in plants. The objective of the current study was to evaluate the inoculation response of a plant-beneficial bacterial strain AQ12 on growth, physiological parameters, and expression of tiller responsive genes of rice plants grown under salt stress. The strain was identified as Burkholderia sp. based on 16S rRNA gene sequence analysis and it demonstrated promising ACC deaminase, indole-3-acetic acid (IAA) production, and phosphate solubilization traits in the presence of varying salt levels. The molecular confirmation of ACC deaminase activity was made by amplification and sequencing of the acdS gene. Inoculation of rice plants with the rifampicin-resistant derivative of Burkholderia sp. AQ12 resulted in a significant increase in length, dry matter, and cellular antioxidant enzyme levels together with the reduced levels of reactive oxygen species up to 50 mM salt stress. Furthermore, the inoculated plants revealed more than 2.0 fold increase in expression of the MOC1, OSH1, and OsTB1 genes up to 50 mM salt stress. It was concluded that inoculation of Burkholderia sp. AQ12 helped rice plants alleviate harmful salt stress effects by improving the growth, boosting the antioxidative defense system, and triggering the expression of tillering-responsive genes.

Pilea umbrosa ameliorate CCl4 induced hepatic injuries by regulating endoplasmic reticulum stress, pro-inflammatory and fibrosis genes in rat

BackgroundPilea umbrosa (Urticaceae) is used by local communities (district Abbotabad) for liver disorders, as anticancer, in rheumatism and in skin disorders.MethodsMethanol extract of P. umbrosa (PUM) was investigated for the presence of polyphenolic constituents by HPLC-DAD analysis. PUM (150 mg/kg and 300 mg/kg) was administered on alternate days for eight weeks in rats exposed with carbon tetrachloride (CCl4). Serum analysis was performed for liver function tests while in liver tissues level of antioxidant enzymes and biochemical markers were also studied. In addition, semi quantitative estimation of antioxidant genes, endoplasmic reticulum (ER) induced stress markers, pro-inflammatory cytokines and fibrosis related genes were carried out on liver tissues by RT-PCR analysis. Liver tissues were also studied for histopathological injuries.ResultsLevel of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione (GSH) decreased (p < 0.05) whereas level of thiobarbituric acid reactive substance (TBARS), H2O2 and nitrite increased in liver tissues of CCl4 treated rat. Likewise increase in the level of serum markers; alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin was observed. Moreover, CCl4 caused many fold increase in expression of ER stress markers; glucose regulated protein (GRP-78), x-box binding protein1-total (XBP-1 t), x-box binding protein1-unspliced (XBP-1 u) and x-box binding protein1-spliced (XBP-1 s). The level of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) was aggregated whereas suppressed the level of antioxidant enzymes; γ-glutamylcysteine ligase (GCLC), protein disulfide isomerase (PDI) and nuclear erythroid 2 p45-related factor 2 (Nrf-2). Additionally, level of fibrosis markers; transforming growth factor-β (TGF-β), Smad-3 and collagen type 1 (Col1-α) increased with CCl4 induced liver toxicity. Histopathological scrutiny depicted damaged liver cells, neutrophils infiltration and dilated sinusoids in CCl4 intoxicated rats. PUM was enriched with rutin, catechin, caffeic acid and apigenin as evidenced by HPLC analysis. Simultaneous administration of PUM and CCl4 in rats retrieved the normal expression of these markers and prevented hepatic injuries.ConclusionCollectively these results suggest that PUM constituted of strong antioxidant chemicals and could be a potential therapeutic agent for stress related liver disorders.

PALMATINE ACTIVATES INSULIN RELEASE VIA PI3K – GLUT4 INSULIN DEPENDENT PATHWAY EX‐VIVO

BackgroundPalmatine is a protoberberine alkaloid which identified to be the bioactive compound present in stem extract of Cosininum Fenestratum plant. Our previous research have reported the plasma glucose lowering activity of Palmatine and its effect on liver, kidney and antioxidant enzymes parameters in STZ induced diabetic rat model. We also reported that the molecular mechanism of action of palmatine insulin release is through activation insulin‐dependent pathway in vivo and in vitro.ObjectiveThe focus of this study was to determine the effect of Palmatine on insulin dependent signaling pathway in insulin resistance rat skeletal muscle ex‐vivo.MethodNormal Male SD rats (180–200g) were sacrificed and skeletal muscles were harvested. The smaller size skeletal muscles from rats were mounted in the Krebs‐Henseleit buffer (KHB) buffer to maintain the resting position. The tissues were then incubated at 37°C under 95% O2‐ 5%CO2 in Glucose free KHB containing 01% BSA and 1mM pyruvate for 1hr, and KHB in 500nmol/L insulin and 125nmol/L glucose for 24hrs. After incubation with insulin/glucose KHB, tissues were transferred to KHB buffer with 250mmol/L glucose for 5hrs, and then KHB with insulin for 30 mints. After induction tissue were replaced with fresh KHB drug was administered by pipetting drug into the organ bath and was allowed to incubate for 24hrs. RNA was extracted and converted to cDNA according manufacturer protocol and the expression of IRS‐1, PI3K, PKC, AKT2 and GLUT4 genes involved in insulin dependent signaling pathway were evaluated using qPCR.ResultPalmatine showed an increase in fold expression of PI3K, PKC, AKT2 and GLUT 4 compared to negative control. The result was similar to standard drug metformin. The increase in fold expression of the insulin dependent pathway genes shows they were revived as a result of Palmatine treatment.ConclusionPalmatine were able to rejuvenate PI3K, PKC, AKT2 and GLUT 4 in Palmatine treated group, which is usual impaired in insulin resistance skeletal muscle. The result is in consonance with the previous finding of in vitro and in vivo. Currently work is on‐going to investigate the effect of palmatine on the insulin‐independent pathway.Support or Funding InformationUCSI University

Bacillus subtilis CBR05 induces Vitamin B6 biosynthesis in tomato through the de novo pathway in contributing disease resistance against Xanthomonas campestris pv. vesicatoria

Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.

SUN-191 Stress Opens Tim23 Complex Resulting Faster Steroid Metabolism

The movement and distribution of mitochondria is essential for cellular function in response to physiological demand. Thus, coordinated mitochondrial action is necessary in cell development. The molecular basis of mitochondrial redistribution depends on the different steps of protein folding as the rapid burst of movements is interrupted by variables, including environmental and hormonal stressors, such as adrenocorticotropic hormone (ACTH). In adrenal and gonads, CYP11A1, catalyzes cholesterol to pregnenolone conversion. During stress, the ER and mitochondria comes close to each other, resulting in the formation of a complex consisting of the translocase of outer mitochondrial membrane 22, steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2) via its intermembrane space exposed charged unstructured loop region. An excess of ACTH inhibits organelle organization and fiber formation as visualized by transmission electron microscopy. We created stress in cells by depriving serum. Next isolation of mitochondrial complex with digitonin and analysis through a native-gradient PAGE formed a 400-kDa complex in the absence of serum. However an additional complex of 650 kDa was observed with serum. Western staining of the stressed cells with Tom22, 3βHSD2 and Tim23 antibodies independently showed several fold increase in expression of Tim23 and 3βHSD2 after 8 hour and a three fold increase Tom 22 after 2 hour. The expression of mitochondrial VDAC2 or cytoplasmic calnexin was unchanged. We conclude that an increase in Tim23 expression possibly opens the TIM23 complex resulting rapid interaction with the MAM and ER proteins for faster metabolic activity.

Magnaporthe grisea infection modifies expression of anti-oxidant genes in finger millet [Eleusine coracana (L.) Gaertn.

Magnaporthe grisea causes blast disease in finger millet, and has severe economic impact on the crop. Plant defense against fungal pathogens is a complex phenomenon, involving series of reorganizations at morphological and molecular levels. Among these, generation of reactive oxygen species (ROS) is one of the most significant events. The accumulation of ROS in plant cell is regulated by a set of well-known anti-oxidant enzymes. Differential expression of these enzymes at the transcriptional level determines the compatibility of a plant-pathogen interaction. We studied the differential expression of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), class III peroxidase (POX) and lipoxygenase (LOX) in a blast resistant and susceptible genotype at two time points after inoculation [48 h post inoculation (hpi) and 96 hpi] using quantitative PCR. There was an increase in fold expression of SOD between both time points in the resistant genotype and a uniform expression in the susceptible genotype. With regard to APX the resistant genotype showed an increase in expression with time while the susceptible genotype displayed a significant decline in fold expression. Expression of GPX, POX and LOX displayed a descending trend in the resistant genotype and ascending trend in the susceptible one from 48 hpi to 96 hpi.

Abstract 2595: Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer

Abstract The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer. Citation Format: Chike Osude, Leo Lin, Brad Foster, Neelu Puri. Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2595.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard

Sulfur mustard (2,2′-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2–15 fold increase in expression of the proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20–1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

Abstract 3531: YAP1-mediated circadian oscillation in sarcoma

Abstract Soft tissue sarcomas have presented a unique challenge to researchers due to their heterogeneity. One common mechanism underlying more than 25% of diagnosed sarcomas is the deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of a kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1 allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of muscle tissue from the LSL-KrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces cell proliferation and increases expression of circadian rhythm genes including PER1. UPS is a commonly diagnosed and aggressive type of muscle-derived sarcoma. The KP model recapitulates human UPS morphologically and histologically, as well as by gene expression profiling. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA level independent of p53. However, our findings represent a novel link between the circadian clock and the hippo pathway. Notably, deletion of YAP1 in KP tumors leads to a statistically significant 2.5 fold increase in expression of PER1. The study has two specific aims: 1) to identify the YAP1-dependent function of PER1 in sarcoma and 2) determine whether YAP1 directly or indirectly regulates PER1. We have validated the microarray results in KP tumor derived cell lines as well as in KP and KPY tumor tissue. I have also confirmed PER1 suppression in KP cells under YAP1 short hairpin RNA conditions. Our lab has previously observed that treatment with epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, significantly decreased sarcoma growth in vivo and in vitro when administered alone and had an additive effect when combined. These effects can be explained in part by the finding that treatment with these inhibitors significantly reduces YAP1 expression. Under these conditions, as well as YAP1 knockdown conditions, I demonstrated via qRT-PCR and western blot that PER1 is significantly increased at both the protein and transcriptional levels in KP cells. Additionally, preliminary evidence from an MTT proliferation assay showed loss of PER1 increased sarcoma cell proliferation. Further supporting the hypothesis that PER1 modulation impacts sarcoma proliferation, it has been reported that MyoD, the master regulator of muscle cell differentiation, is itself a clock-controlled gene. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic treatments can cause re-expression of PER1 which functions to inhibit cell proliferation and may promote differentiation. Citation Format: Gloria Marino, Shuai Ye, Koreana Pak, Jennifer Shah, Jason Godfrey, Susan Chor, Shaun Egolf, T.S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2017-3531

Immunotherapeutic target expression on breast tumors can be amplified by hormone receptor antagonism: a novel strategy for enhancing efficacy of targeted immunotherapy.

Immunotherapy has historically been successful in highly antigenic tumors but has shown limited therapeutic efficacy in non-antigenic tumors such as breast cancers. Our previous studies in autoimmunity have demonstrated that increased antigen load within a tissue enhances immune reactivity against it. We therefore hypothesized that enhancing expression of target proteins on breast tumors can increase efficacy of targeted immunotherapy. We hypothesized that antagonism of the estrogen receptor (ER) can increase expression of targets that are hormonally regulated and facilitate enhanced tumor recognition by targeted immunotherapy. We used a lactation protein α-Lactalbumin, a known immunotherapeutic target on breast tumors, as our model target antigen. Enhancement of target protein expression in human and murine breast tumors was tested in vitro and in vivo by ER antagonism using clinically established ER modulators, Tamoxifen and Fulvestrant. We show that antagonism of the ER can induce a 2–3 fold increase in expression of target proteins on tumors leaving the normal breast tissue unaffected. Tumor progression studies in 4T1 tumor-bearing mice show that efficacy of adoptively transferred cell based targeted immunotherapy was enhanced by target antigen amplification resulting in significantly higher tumor inhibition. However, in spite of increased target expression, anti-tumor efficacy of direct immunization was not enhanced probably due to other limiting factors involved in the immune priming process. Our study provides a novel combinatorial clinical strategy for enhancing efficacy of immunotherapy not only on breast tumors but potentially also for other hormonally driven tumors such as those of the prostate, testis and ovary.