Enzymes Of Fatty Acid Synthesis Research Articles

PO-229 The fatty acid desaturase SCD1 as crucial factor for tumour progression in colon cancer

IntroductionAberrant activation of lipogenesis is a hallmark of cancer metabolism. However, the functional contribution of lipids to tumorigenesis is still uncovered. We analysed the clinical implication of key enzymes of fatty acid synthesis and processing, including FASN (fatty acid synthase), fatty acid elongase 6 (ELOVL6), the master transcription factor of lipid synthesis, SREBP1 (sterol regulatory element binding protein 1) and steaoryl-CoA desaturase 1 (SCD1). SCD1 is essential for the production of mono-unsaturated fatty acids (MUFAs) and crucially involved in colon cancer, suggested by our previous expression profiling data. To investigate its functional role, we generated SCD1 deficient colon cancer cells.Material and methodsSCD1, SREBP1, FASN and ELOVL6 were quantified by qPCR in 78 patients with stage III colon cancer, and clinical and prognostic significance was assessed by Kaplan-Meier analysis. SCD1 and SREBP1 were further analysed on protein level. SCD1-deficiency was implemented in colon cancer cells HCT116 by the CRISPR-Cas9 system. Mass spectrometry (MS) analysis was used to analyse the activity of SCD1; cell proliferation and survival of the SCD1 deficient clones were compared to parental cells.Results and discussionsExpression of SCD1 and FASN was highly significantly increased in tumours, whereas SREBF1 and ELOVL6 were significantly reduced. However, SCD1 and SREBP1 were upregulated in tumour tissue on protein level and their mRNA was significantly co-expressed. SCD1 was significantly associated with tumour grading and worse post-operative survival, and elevated expression of FASN and ELOVL6 with increased metastatic relapse.SCD1 deficiency was verified by DNA sequencing and immunoblotting, and MS analysis confirmed the deprivation of MUFAs. KO of SCD1 led to significantly decreased cell proliferation and survival under normal growth conditions, which was even more pronounced under reduced serum. This was fully rescued by addition of the SCD1 product oleate, suggesting that SCD1 is necessary for growth and survival especially in the absence of exogenous lipids.ConclusionSCD1, FASN and ELOVL6 are negative prognostic markers in stage III colon cancer. Functionally, SCD1 is essential for cell survival under serum depletion. Together, these data suggest that the cell-autonomous synthesis of fatty acid species in cancer cells is due to reduced access to exogenous lipids. Enhanced lipogenesis rises the requirement for the production of MUFAs by SCD1, e.g., to maintain survival and circumvent lipotoxicity.

Elevated O‐GlcNAc Exacerbates Pro‐Inflammatory Cytokine Secretion from CD4+ T cells

Chronic inflammation is a feature of obesity and enhances the risk of atherosclerosis, cancer, diabetes, and autoimmunity. Specifically, pro-inflammatory TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell function. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a sensor of a cell's nutritional status. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells from mice fed a high fat and cholesterol “Western” diet. Naïve CD4+ T cells from obese mice have elevated O-GlcNAc levels compared to cells from mice fed standard chow. When polarized to a TH17 lineage, cells from obese mice secrete more IL-17A, the eponymous TH17 cytokine. Importantly, when naïve CD4+ T cells from lean and obese mice are polarized to a TH17 lineage in the presence of Thiamet-G (TMG, an inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA)) the cells from lean mice secrete levels of IL-17 comparable to those from obese mice and IL-17 secretion was exacerbated in cells from obese mice. Transcript levels of IL-17 are similarly elevated in both lean and obese mice TH17 cells treated with TMG. RORγt (retinoic acid-related orphan receptor gamma) acts as the TH17 master transcription factor, orchestrating the TH17 differentiation program. Uniquely among the CD4+ effector T cell master transcription factors, RORγt is regulated by fatty acid ligands which promote or repress its activity. Acetyl CoA carboxylase 1 (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt. We discovered that ACC1 is O-GlcNAcylated. Furthermore, lipidomics analysis identified clear differences in the lipid environment of T cells isolated from lean and obese mice −/+ TMG. Thus, abnormally elevated O-GlcNAc levels may favor the generation of ligands that enhance RORγt activity at the IL-17 promoter. More directly implicating O-GlcNAc's regulation of RORγt, chromatin immunoprecipation of RORγt and O-GlcNAc transferase (OGT, the enzyme that modifies proteins with O-GlcNAc) at the IL-17 promoter and an enhancer, CNS2 (conserved noncoding sequence 2), is increased with TMG treatment. Collectively, our data suggest that elevated O-GlcNAc levels increase pro-inflammatory IL-17 secretion from TH17 cells through alteration of the lipidome and direct effects on the IL-17 promoter complex. Further study into the molecular mechanism of how O-GlcNAcylation promotes pro-inflammatory TH17 T cell function will provide insight into how nutritional excess worsens inflammation. Support or Funding Information This research was supported by NIH grant R01DK100595-01, NIH grant R01DK091277-03, COBRE P30GM122731, and a KUMC Institute of Reproductive Health and Regenerative Medicine pilot grant. MM is supported by a KUMC Biomedical Research Training Program grant. Elevated O-GlcNAc increases pro-inflammatory IL-17 cytokine secretion from TH17 cells This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced skin lipid content in obese Japanese women mediated by decreased expression of rate-limiting lipogenic enzymes.

Skin barrier function is often deficient in obese individuals, but the underlying molecular mechanisms remain unclear. This study investigated how skin structure and lipid metabolism, factors strongly associated with barrier function, differed among 50 Japanese women of greatly varying body mass index (BMI). Subjects receiving breast reconstruction surgery were chosen for analysis to obtain skin samples from the same site. The subjects were classified into two groups, control (BMI < 25 kg/m2) and obese (25 kg/m2 ≤ BMI < 35 kg/m2), according to standards in Japan. Hematoxylin and eosin staining was used to assess skin thickness, Ki-67 immunostaining to examine keratinocyte proliferation, and real-time polymerase chain reaction to measure skin expression levels of genes associated with lipid metabolism. Total lipids, cholesterol, and fatty acids were also measured from these same skin samples. In the obese group, structural changes included epidermal thickening and an increase in the number of Ki-67-positive (proliferating) cells. Both skin cholesterol and fatty acid levels exhibited an “inverted-U” relationship with BMI, suggesting that there is an optimal BMI for peak lipid content and barrier function. Decreased lipid levels at higher BMI were accompanied by downregulated expression of PPARδ and other genes related to lipid metabolism, including those encoding acetyl-CoA carboxylase and HMG-CoA reductase, the rate-limiting enzymes for fatty acid and cholesterol synthesis, respectively. Thus, elevated BMI may lead to deficient skin barrier function by suppressing local lipid synthesis.

Measurement of AMPK-Induced Inhibition of Lipid Synthesis Flux in Cultured Cells.

AMP-activated protein kinase (AMPK) is a master regulator of multiple cellular metabolic pathways, including lipid metabolism. Some of the well-known substrates of AMPK are acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, regulatory enzymes of fatty acid and cholesterol synthesis, respectively. The discovery that both of them are inactivated by AMPK suggested the therapeutic potential of AMPK activation in the treatment of metabolic diseases associated with lipid disorders, such as nonalcoholic fatty liver disease (NAFLD). Here we describe a method to measure lipid synthesis flux in intact cells from the saponifiable (including fatty acids) and non-saponifiable (including sterols) fractions of lipid extracts.

Co-conspirators in a new mechanism for the degradation of Δ9-desaturase

Δ9-Desaturases are central enzymes in unsaturated fatty acid synthesis regulated at the transcriptional and mRNA levels and by proteasomal degradation. A new study by Murakami et al. uncovers a novel regulatory pathway in which an N-terminal di-proline motif in the Drosophila Δ9-desaturase mediates protein degradation by a calcium-dependent cysteine protease in response to unsaturated fatty acids. This study provides new details of desaturase regulation with therapeutic implications for the treatment of metabolic syndrome.

In vivo evidence on the functional variation within fatty acid synthase gene associated with lipid metabolism in bovine longissimus dorsi muscle tissue.

In Korean cattle, intramuscular fat (IMF), or marbling, of the longissimus dorsi muscle (LM) cross section is one of the most important indicators of beef quality and are influenced by environmental and genetic factors. This study was to evaluate the effect of SNPs on the beef quality in Korean cattle for functional studies, such as site-directed mutagenesis based on bovine adipocytes. The fatty acid synthase (FASN) gene plays an important role in lipogenesis. FASN is an essential metabolic and multifunctional enzyme in fatty acid synthesis. Several studies have reported that SNPs g.841G, g.16024A, g.16039T, and g.17924G have a significant impact on marbling scores in Korean cattle and Japanese Black cattle population. These SNPs are located in transcription factor binding sites, the beta-ketoacyl reductase, and thioesterase domains. Our results revealed that the g.17924 A>G SNP is located in the thioesterase domain of the FASN protein, and changes from polar, neutral, and hydrophilic to nonpolar, aliphatic, and hydrophobic, respectively. In in vivo LM tissue of Korean cattle, the g.17924A>G SNP has an effect on increasing fat deposition. Therefore, g.17924A>G SNP could be a causal mutation for increasing fat deposition in Korean cattle LM tissue.

Expression of AMPK, SIRT1, and ACC Differs between Winter- and Summer-Acclimatized Djungarian Hamsters.

The wintering strategy of the Djungarian hamster (Phodopus sungorus) includes a naturally occurring decrease in food intake and body mass. Our aim was to investigate the conceivable role of the metabolic regulators, AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), in the seasonal adaptation of the Djungarian hamster. In addition, a rate-limiting enzyme in fatty acid synthesis and oxidation, acetyl CoA carboxylase (ACC), was studied. Relative protein expressions and phosphorylated forms (pAMPK and pACC) were determined by Western blot from subcutaneous white adipose tissues (sWAT), abdominal white adipose tissues (aWAT), interscapular brown adipose tissues (iBAT), skeletal muscle, and hypothalamus of winter- and summer-acclimatized hamsters. The winter group had higher AMPK expression in sWAT, aWAT, and iBAT, but the relative amount of phosphorylated protein (pAMPK/AMPK ratio) was lower in these tissues. Furthermore, ACC expression was higher in sWAT and iBAT of the winter animals. pACC (inactive form) levels were higher in all adipose tissues, yet a lower pACC/ACC ratio was detected in iBAT of the winter hamsters. Muscle AMPK expression was lower but pAMPK/AMPK ratio higher in the winter group. SIRT1 expression was higher in muscle and all adipose tissues of the winter hamsters. Hypothalamic protein expressions did not differ between the groups. Higher expressions of AMPK, ACC, and SIRT1 in WAT and iBAT of the winter hamsters suggest a role in the regulation of lipid reserves and increased thermogenic capacity characteristic to the winter-adapted Djungarian hamsters.

Anti-atherosclerotic effect of hesperidin in LDLr−/− mice and its possible mechanism

Hesperidin, a citrus bioflavonoid, exerts numerous pharmacological activities. However, its protective effect against atherosclerosis in vivo remains poorly understood. In the present study, we aimed to observe the effects of hesperidin on high fat diet (HFD)-induced atherosclerosis using LDL receptor deficient (LDLr-/-) mice. After 12 weeks of treatment, the animals were sacrificed. The blood samples were collected for further analysis. Mouse peritoneal macrophages were collected. Hepatic lipid content, quantification of atherosclerosis, assessment of oxidative stress and inflammation, gene expressions were performed on liver and aorta samples. The data showed that hesperidin ameliorated HFD-induced weight gain, improved insulin resistance and ameliorated hyperlipidemia. Hesperidin suppressed HFD-induced hepatic steatosis, atherosclerotic plaque area and macrophage foam cell formation. Further study showed that hesperidin down-regulated expressions of acetyl coenzyme A carboxylase alpha (ACCα) and fatty acid synthase (FAS) which are two key enzymes in fatty acid and triglyceride synthesis in liver; and upregulated expression of hepatic ATP-binding cassette transporters G8 (ABCG8), macrophage ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) which are transporters involved in the process of reverse cholesterol transport. Hesperidin also reduced oxidative stress by normalizing activities of antioxidant enzymes and inflammation in HFD-fed LDLr−/− mice. These findings suggest that hesperidin reduced atherosclerosis via its pleiotropic effects, including improvement of insulin resistance, amelioration of lipid profiles, inhibition of macrophage foam cell formation, anti-oxidative effect and anti-inflammatory action.

Regulation of malonyl-CoA-acyl carrier protein transacylase network in umbilical cord blood affected by intrauterine hyperglycemia.

BackgroundGestational diabetes mellitus (GDM) has been shown to be associated with high risk of diabetes in offspring. However, the mechanisms involved in the insulin resistance in offspring are still unclear. Mitochondrial dysfunction is related with insulin resistance. In mitochondria, malonyl-CoA-acyl carrier protein transacylase (MCAT) is the key enzyme of mitochondrial fatty acid synthesis and is estimated to contribute to insulin resistance. In this study, we aimed to examine the role of MCAT and its network in the umbilical cord blood in GDM-induced offspring insulin resistance.MethodsWe isolated lymphocytes from umbilical cord vein blood in 6 GDM patients and 6 controls and examined the differences of RNA by RNA sequencing. qRT-PCR and western blot were used to measure mRNA and protein changes. Bisulfite genomic sequencing PCR was applied to detect DNA methylation.ResultsWe found more than 400 genes were differentially regulated in the lymphocytes of umbilical cord blood from GDM patients and these genes were mainly enriched in immune system and endocrine system, which relate to mitochondrial dysfunction and insulin resistance. MCAT closely related with PTPN1 (Protein Tyrosine Phosphatase, Non-Receptor Type1) and STAT5A (Signal Transducer And Activator of Transcription 5A), which were all increased in umbilical cord blood from GDM patients. Increase in MCAT may be due to decreased MCAT DNA methylation.ConclusionMCAT and its network with PTPN1, STAT5A are regulated in umbilical cord blood affected by maternal intrauterine hyperglycemia.

Abstract P175: Chronic Exercise Attenuates Blood Pressure Elevation and Albuminuria With Improvement of Renal Lipid Metabolism in High Fructose-fed Rats

High fructose diet (HFr) can lead to metabolic disorder, hypertension, and renal disease. Although chronic exercise (Ex) provides various beneficial effects on hypertension and kidney disease, the precise mechanism is not fully clarified. Thus, present study examined the effects of Ex on the blood pressure, renal function and renal lipid metabolism in rats fed with HFr. Sprague-Dawley rats were allocated to 3 groups. The HFr and Ex groups were fed with HFr (60%, w/w), the control group was fed with the diet in which fructose was replaced by starch. The Ex group underwent treadmill exercise at aerobic intensity. After 12 weeks, renal triglyceride (TG) content were measured, and expression of enzymes and regulators of fatty acid metabolism were analyzed by Western blot. HFr increased systolic blood pressure (SBP) and albuminuria and Ex decreased the HFr-increased SBP and albuminuria (85±4 vs. 122±9 vs. 91±4 mmHg, P&lt;0.01, 326±67 vs. 534±79 vs. 176±54 mg/day, P&lt;0.01). HFr increased plasma TG and uric acid (UA) and Ex decreased the HFr-increased TG and UA (124±20 vs. 474±35 vs. 238±23 mg/dL, P&lt;0.01, 1.15±0.10 vs. 2.14±0.10 vs. 1.50±0.13 mg/dL, P&lt;0.01), whereas HFr or Ex did not affect plasma creatinine. HFr increased renal TG content and Ex decreased the HFr-increased TG content (12.2±0.5 vs. 14.1±0.5 vs. 10.3±1.1 mg/100mg tissue, P&lt;0.01). Among enzymes of fatty acid synthesis, HFr increased the renal expression of fatty acid synthase (FAS), and Ex decrease the expression of FAS (P&lt;0.01). Among enzymes and regulators of fatty acid oxidation, HFr decreased the renal expression of PPARα, carnitine palmitoyltransferase I (CPTI), medium-chain acyl-CoA dehydrogenase (MCAD) (P&lt;0.05), and Ex increased the expression of PPARα, CPTI, MCAD, and acyl-coenzyme A oxidase (ACOX). These results indicated that Ex attenuates blood pressure elevation, albuminuria with an improvement of renal lipid metabolism in the HFr-fed rats. These effects of Ex may relate to an improvement of the renal lipid metabolism.

Full-length sequencing and identification of novel polymorphisms in the ACACA gene of Valle del Belice sheep breed.

The essential role of the acetyl-CoA carboxylase (ACACA) enzyme in milk fatty acid (FA) synthesis suggests that it may be responsible for the phenotypic variability observed inmilk.Before attempting association analyses between this gene and/or enzyme and phenotypic traits, a study on the genetic variability within this locus is required. The aim of this work was to sequence the entire coding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the presence of 23 polymorphic sites. The most polymorphic was exon 53 which showed presence of 12 single-nucleotide polymorphisms (SNPs), ofwhich eightweremissensemutations, caused amino acid changes and therefore may affect protein function or stability causing variation in phenotype. The identified polymorphisms showed high variability of the ACACA gene. Sequences analysis allowed to find six new SNPs in exon 53 (6832C>T; 6835C>A; 6840G>A; 6847G>T; 6852C>T and 6860G>C). A total of 31 haplotypes were inferred. Although this study could not provide association study with production traits, it shows finding of novel SNPs that might be important in future studies and laid the basis for further association analyses needed to evaluate the potential use of these SNPs as genetic markers for fat content and FAs composition in milk of Valle del Belice sheep breed.

Expression of fatty acid synthase and E-cadherin markers in cancer of the prostate

Prostatic carcinomas are considered one of the most common malignancies in male population and have variable behavior and clinical course that affect the treatment options. Many molecular markers had been discovered to improve both early detection, prognosis, and risk prediction for patients with prostatic carcinoma. Fatty acid synthase (FASN) is a key enzyme for de-novo fatty acid synthesis, and E-cadherin is considered one of the vital glycoproteins in cell–cell adhesion in epithelial tissues. The aim of this work was to evaluate the expression of FASN and E-cadherin as prognostic markers in prostatic carcinoma. The immunohistochemical expression of FASN and E-cadherin was evaluated in 10 cases of high-grade prostatic intraepithelial neoplasia (HGPIN) and 45 cases of prostatic carcinoma. FASN was expressed in 80% of HGPIN and 76% of cases of prostatic carcinoma. A statistically significant relation between the Gleason score and FASN immunoreactivity was recorded (P=0.0012). E-cadherin showed preserved membranous staining in all cases of HGPIN (100%) and 62.3% of cases of prostatic carcinoma. We found a statistically significant association between the Gleason score and reduced E-cadherin expression (P=0.0021). Increased FASN immunohistochemical expression and reduced E-cadherin expression are associated with increased aggressiveness of prostatic carcinoma and bad prognosis. Combined detection of the expression of both markers may provide an essential predictor for prognosis of prostatic carcinoma.

O-GlcNAc Regulates CD4+ T Cell Differentiation

Abstract Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus.

The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.

An Induced Chromosomal Translocation in Soybean Disrupts a KASI Ortholog and Is Associated with a High-Sucrose and Low-Oil Seed Phenotype.

Mutagenesis is a useful tool in many crop species to induce heritable genetic variability for trait improvement and gene discovery. In this study, forward screening of a soybean fast neutron (FN) mutant population identified an individual that produced seed with nearly twice the amount of sucrose (8.1% on dry matter basis) and less than half the amount of oil (8.5% on dry matter basis) as compared to wild type. Bulked segregant analysis (BSA), comparative genomic hybridization, and genome resequencing were used to associate the seed composition phenotype with a reciprocal translocation between chromosomes 8 and 13. In a backcross population, the translocation perfectly cosegregated with the seed composition phenotype and exhibited non-Mendelian segregation patterns. We hypothesize that the translocation is responsible for the altered seed composition by disrupting a β-ketoacyl-[acyl carrier protein] synthase 1 (KASI) ortholog. KASI is a core fatty acid synthesis enzyme that is involved in the conversion of sucrose into oil in developing seeds. This finding may lead to new research directions for developing soybean cultivars with modified carbohydrate and oil seed composition.

A Genetic Screen Reveals Novel Targets to Render Pseudomonas aeruginosa Sensitive to Lysozyme and Cell Wall-Targeting Antibiotics.

Pseudomonas aeruginosa is capable of establishing airway infections. Human airway mucus contains a large amount of lysozyme, which hydrolyzes bacterial cell walls. P. aeruginosa, however, is known to be resistant to lysozyme. Here, we performed a genetic screen using a mutant library of PAO1, a prototype P. aeruginosa strain, and identified two mutants (ΔbamB and ΔfabY) that exhibited decrease in survival after lysozyme treatment. The bamB and fabY genes encode an outer membrane assembly protein and a fatty acid synthesis enzyme, respectively. These two mutants displayed retarded growth in the airway mucus secretion (AMS). In addition, these mutants exhibited reduced virulence and compromised survival fitness in two different in vivo infection models. The mutants also showed susceptibility to several antibiotics. Especially, ΔbamB mutant was very sensitive to vancomycin, ampicillin, and ceftazidime that target cell wall synthesis. The ΔfabY displayed compromised membrane integrity. In conclusion, this study uncovered a common aspect of two different P. aeruginosa mutants with pleiotropic phenotypes, and suggests that BamB and FabY could be novel potential drug targets for the treatment of P. aeruginosa infection.

Abstract A27: Systematic discovery of mutation-specific synthetic lethals by mining pan-cancer primary tumor data

Abstract Synthetic lethality, in which a single gene defect leads to dependency on a second gene that is otherwise not essential, is an attractive paradigm to identify targeted therapies for somatic mutations. Current methods to detect synthetic lethal (SL) partners for somatic mutations use large-scale shRNA screens in cell lines, combine shRNA data with tumor genomic data or use human orthologs of yeast SL interactions. These approaches are limited as they rely on cell line or yeast data, which are not representative of primary tumors. We have developed MiSL, a novel computational algorithm that utilizes large pan-cancer patient datasets (mutation, copy number and gene expression) to identify SL partners for specific mutations in specific cancer types. The underlying assumption of our approach is that, across multiple cancers, SL partners of a mutation will be amplified more frequently or deleted less frequently, with concordant changes in expression, in primary tumor samples harboring the mutation. Application of MiSL produced candidate SL partners for 30-80% of recurrent mutations in 12 cancers. Importantly, MiSL identified candidate SL partners for mutations (mut) in genes such as IDH1 that are not well-represented in existing cell lines. This is a distinct advantage over recent computational methods that combine shRNA data along with genomic data to make their predictions. Since MiSL uses only genomic and gene expression data, it allows assessment of a wide range of primary human tumors and mutations found in large primary tumor data sets such as TCGA. We validated MiSL using existing data and large-scale shRNA experiments we performed in doxycycline-inducible expression systems. We found that IDH1mut MiSL candidates in acute myeloid leukemia (AML) were enriched (p=0.004) for essential genes specific to IDH1mut but not IDH1 wildtype cells determined by a DECIPHER shRNA screen covering 9,965 human genes performed in doxycycline-inducible IDH1 (R132) THP-1 cells. Importantly, 1 out of 5 MiSL candidates was a SL partner of IDH1mut in AML cells as per the shRNA screen, indicating MiSL's strong predictive power. Also, for multiple mutations in colorectal cancer, MiSL candidates were enriched (p&amp;lt;0.05) with genes that were selectively essential in the mutated colorectal cell-lines in Achilles data. Next, we used MiSL to identify novel and druggable SL partners in (i) AML and (ii) breast cancer. MiSL predicted a novel SL interaction in AML between IDH1mut and ACACA, the rate-limiting enzyme of fatty acid synthesis. Consistent with our prediction, pharmacologic or genetic blockade of ACACA prevented cell proliferation in the presence of IDH1mut, but not with IDH1 wildtype, in AML cell lines. Furthermore, when transduced with lentivirus encoding RFP-marked shRNA to ACACA, primary IDH1mut AML cells exhibited markedly reduced engraftment of RFP-positive human CD45+CD33+ leukemic cells compared to scrambled non-targeting shRNA (p&amp;lt;0.05) at 12 weeks post-engraftment, validating the SL interaction between mutant IDH1 and ACACA. This vulnerability indicates a novel role for IDH1mut in reprogramming lipid metabolism. MiSL also predicted that AKT1 is a SL partner of PIK3CAmut in breast cancer which we experimentally confirmed using 8 breast cancer lines. All four PIK3CAmut (but not wildtype) breast cancers were sensitive to AKT1 inhibition in viability and colony assays. In summary, MiSL is a general computational solution that finds novel SL interactions. Specifically, IDH1mut-ACACA is the first in vivo validated synthetic lethal in human tumor cells discovered purely by computational analysis of tumor genomic data. MiSL can greatly accelerate identification of pharmacologic targets associated with specific somatic mutations in specific tumor types for all kinds of mutations, thereby making it directly translatable to clinical applications. MiSL can also pinpoint predictive genetic biomarkers that can identify/extend indications for targeted therapies. Citation Format: Subarna Sinha, Daniel Thomas, Steven Chan, Yang Gao, Diede Brunen, Damoun Torabi, Andreas Reinisch, Rene Bernards, Ravindra Majeti, David L. Dill. Systematic discovery of mutation-specific synthetic lethals by mining pan-cancer primary tumor data. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A27.

Homology Modeling and Structural Validation Studies of 3 Oxoacyl (Acyl Carrier Protein) Synthase II and Dihydropteroate Synthase Protein of Neisseria meningitides

Abstract Objective: Neisseria meningitidis (NM) is foremost causative agent of bacterial meningitis. The two very important protein has been selected for the study of homology modeling and structural validation in Neisseria meningitis. The 3 oxoacyl (acyl carrier protein) synthase II (3OASII) enzyme which involved in fatty acid biosynthesis of Neisseria meningitidis. This enzyme in fatty acid synthesis and target for discovery of novel antibacterial agent. Dihydropteroate synthase (DHPS), an enzyme plays an acute role in the bacterial folate metabolism which catalyzes the development of 7, 8- dihydropteroate from p-aminobenzoic acid (pABA) and 6- hydroxymethyl-7, 8-dihydropterin-pyrophosphate (DHPP). The absence of the folate pathway in higher eukaryotes makes it an attractive drug target. In current study, a complete 3 oxoacyl (acyl carrier protein) synthase II and Dihydropteroate synthase protein structure model was generated using Modeller V9.12 software so as to understand the mechanistic detail of its functioning. The energy minimization analysis using YASARA and the stereo chemical quality of the models was checked by Rampage, Verfy3D, Errat, ProSA and QMEAN servers. The analysis of present studies provide three dimensional (3D) structure of 3 oxoacyl synthase II and dihydropteroate synthase protein will be helpful for the advance study of drug designing in meningitis disease.Conclusions: The current study showed that 3 oxoacyl synthase II and Dihydropteroate synthase modeled protein is highly reliable and stable, so it will use to identifying the drug designing research of meningitis disease.

Anti-Cancer Activity of Methanol Extracts of Cichorium Intybus on Human Breast Cancer SKBR3 Cell Line

Abstract Background: Breast cancer is themost prevalent cancer and the second cause of death among women around the world. Inmany cancers, including breast cancer, Fatty acid synthase (FASN) gene expression is increased significantly. In breast cancer cell lines, expression of FASN is higher in HER2 positive cell line like SKBR3 than the others. FASN is the key enzyme for fatty acid synthesis de novo pathway and it is producing palmitate which is necessary for cellmembrane formation. Cichoriumintybus is amedicinal plant that effectively leads to inhibition of fatty acid synthase and thus reduces the percentage of survival of cancer cell lines. Objectives: The aimof this studywas to evaluate the effect ofmethanol extract of Chicoriumintybus root on percentage of survival in SKBR3 cell line. Methods: Human breast cancer SKBR3 cell line was cultured in DMEM medium. Methanol extract of Cichorium intybus root was extracted and different dilutions (200, 300, 400, 500 and 600g/mL) were added to cell culture. Cell viability was quantitated by MTT assay after 24, 48 and 72 hours. Results: Cichorium intybus could decrease cell viability. The effects of extract on cell viability were observed after 24, 48 and 72 hours on SKBR3 cell line and IC50 was 800, 400 and 300 after 24, 48 and 72 hours of treatment, respectively. Conclusions: Our study shows thatmethanol extract of Cichoriumintybus has cytotoxic effects on tumor cells. This is a pilotwork for further evaluation in the future.

Testosterone differentially regulates targets of lipid and glucose metabolism in liver, muscle and adipose tissues of the testicular feminised mouse.

Testosterone deficiency is commonly associated with obesity, metabolic syndrome, type 2 diabetes and their clinical consequences—hepatic steatosis and atherosclerosis. The testicular feminised mouse (non-functional androgen receptor and low testosterone) develops fatty liver and aortic lipid streaks on a high-fat diet, whereas androgen-replete XY littermate controls do not. Testosterone treatment ameliorates these effects, although the underlying mechanisms remain unknown. We compared the influence of testosterone on the expression of regulatory targets of glucose, cholesterol and lipid metabolism in muscle, liver, abdominal subcutaneous and visceral adipose tissue. Testicular feminised mice displayed significantly reduced GLUT4 in muscle and glycolytic enzymes in muscle, liver and abdominal subcutaneous but not visceral adipose tissue. Lipoprotein lipase required for fatty acid uptake was only reduced in subcutaneous adipose tissue; enzymes of fatty acid synthesis were increased in liver and subcutaneous tissue. Stearoyl-CoA desaturase-1 that catalyses oleic acid synthesis and is associated with insulin resistance was increased in visceral adipose tissue and cholesterol efflux components (ABCA1, apoE) were decreased in subcutaneous and liver tissue. Master regulator nuclear receptors involved in metabolism—Liver X receptor expression was suppressed in all tissues except visceral adipose tissue, whereas PPARγ was lower in abdominal subcutaneous and visceral adipose tissue and PPARα only in abdominal subcutaneous. Testosterone treatment improved the expression (androgen receptor independent) of some targets but not all. These exploratory data suggest that androgen deficiency may reduce the buffering capability for glucose uptake and utilisation in abdominal subcutaneous and muscle and fatty acids in abdominal subcutaneous. This would lead to an overspill and uptake of excess glucose and triglycerides into visceral adipose tissue, liver and arterial walls.