Fatty Acid Synthesis Pathway Research Articles

Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9.

Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.

Engineering the Lipid and Fatty Acid Metabolism in Yarrowia lipolytica for Sustainable Production of High Oleic Oils

Oleic acid is widely applied in the chemical, material, nutritional, and pharmaceutical industries. However, the current production of oleic acid via high oleic plant oils is limited by the long growth cycle and climatic constraints. Moreover, the global demand for high oleic plant oils, especially the palm oil, has emerged as the driver of tropical deforestation causing tropical rainforest destruction, climate change, and biodiversity loss. In the present study, an alternative and sustainable strategy for high oleic oil production was established by reprogramming the metabolism of the oleaginous yeast Yarrowia lipolytica using a two-layer "push-pull-block" strategy. Specifically, the fatty acid synthesis pathway was first engineered to increase oleic acid proportion by altering the fatty acid profiles. Then, the content of storage oils containing oleic acid was boosted by engineering the synthesis and degradation pathways of triacylglycerides. The strain resulting from this two-layer engineering strategy produced the highest titer of high oleic microbial oil reaching 56 g/L with 84% oleic acid in fed-batch fermentation, representing a remarkable improvement of a 110-fold oil titer and 2.24-fold oleic acid proportion compared with the starting strain. This alternative and sustainable method for high oleic oil production shows the potential of substitute planting.

Effects of glycerol and glucose on docosahexaenoic acid synthesis in Aurantiochyrium limacinum SFD-1502 by transcriptome analysis

Docosahexaenoic acid (DHA) has numerous functions in adjusting the organic health and pragmatic value in medicine and food field. In this study, we compared glycerol and glucose as the only carbon source for DHA production by Aurantiochytrium. When the glycerol concentration was 120 g/L, the maximum DHA yield was 11.08 g/L, and the DHA yield increased significantly, reaching 47.67% of the total lipid content. When the cells grew in glucose, the DHA proportion was 37.39%. Transcriptome data showed that the glycolysis pathway and tricarboxylic acid cycle in Aurantiochytrium were significantly inhibited during glycerol culture, which promoted the tricarboxylic acid transport system and was conducive to the synthesis of fatty acids by acetyl coenzyme A; glucose as substrate activated fatty acid synthesis (FAS)pathway and produced more saturated fatty acids, while glycerol as substrate activated polyketide synthase (PKS)pathway and produced more long-chain polyunsaturated fatty acids. This laid a foundation for fermentation metabolism regulation and molecular transformation.

Acetyl-CoA Carboxylases and Diseases.

Acetyl-CoA carboxylases (ACCs) are enzymes that catalyze the carboxylation of acetyl-CoA to produce malonyl-CoA. In mammals, ACC1 and ACC2 are two members of ACCs. ACC1 localizes in the cytosol and acts as the first and rate-limiting enzyme in the de novo fatty acid synthesis pathway. ACC2 localizes on the outer membrane of mitochondria and produces malonyl-CoA to regulate the activity of carnitine palmitoyltransferase 1 (CPT1) that involves in the β-oxidation of fatty acid. Fatty acid synthesis is central in a myriad of physiological and pathological conditions. ACC1 is the major member of ACCs in mammalian, mountains of documents record the roles of ACC1 in various diseases, such as cancer, diabetes, obesity. Besides, acetyl-CoA and malonyl-CoA are cofactors in protein acetylation and malonylation, respectively, so that the manipulation of acetyl-CoA and malonyl-CoA by ACC1 can also markedly influence the profile of protein post-translational modifications, resulting in alternated biological processes in mammalian cells. In the review, we summarize our understandings of ACCs, including their structural features, regulatory mechanisms, and roles in diseases. ACC1 has emerged as a promising target for diseases treatment, so that the specific inhibitors of ACC1 for diseases treatment are also discussed.

Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis.

Dietary bioactive lipids, one of the three primary nutrients, is not only essential for growth and provides nutrients and energy for life's activities but can also help to guard against disease, such as Alzheimer's and cardiovascular diseases, which further strengthen the immune system and maintain many body functions. Many microorganisms, such as yeast, algae, and marine fungi, have been widely developed for dietary bioactive lipids production. These biosynthetic processes were not limited by the climate and ground, which are also responsible for superiority of shorter periods and high conversion rate. However, the production process was also exposed to the challenges of low stability, concentration, and productivity, which was derived from the limited knowledge about the critical enzyme in the metabolic pathway. Fortunately, the development of enzymatic research methods provides powerful tools to understand the catalytic process, including site-specific mutagenesis, protein dynamic simulation, and metabolic engineering technology. Thus, we review the characteristics of critical desaturase and elongase involved in the fatty acids' synthesis metabolic pathway, which aims to not only provide extensive data for enzyme rational design and modification but also provides a more profound and comprehensive understanding of the dietary bioactive lipids' synthetic process.

Low Temperature Affects Fatty Acids Profiling and Key Synthesis Genes Expression Patterns in Zanthoxylum bungeanum Maxim.

Zanthoxylum bungeanum is one of the most important medicinal and edible homologous plants because of its potential health benefits and unique flavors. The chemical components in compositions and contents vary with plant genotype variations and various environmental stress conditions. Fatty acids participate in various important metabolic pathways in organisms to resist biotic and abiotic stresses. To determine the variations in metabolic profiling and genotypes, the fatty acid profiling and key differential genes under low temperature stress in two Z. bungeanum varieties, cold-tolerant (FG) and sensitive (FX), were investigated. Twelve main fatty acids were found in two Z. bungeanum varieties under cold stress. Results showed that the contents of total fatty acids and unsaturated fatty acids in FG were higher than those in FX, which made FG more resistant to low temperature. Based on the result of orthogonal partial least squares discriminant analysis, palmitic acid, isostearic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FG under cold stress, while isomyristic acid, palmitic acid, isostearic acid, stearic acid, oleic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FX. Furthermore, fatty acid synthesis pathway genes fatty acyl-ACP thioesterase A (FATA), Delta (8)-fatty-acid desaturase 2 (SLD2), protein ECERIFERUM 3 (CER3), fatty acid desaturase 3 (FAD3) and fatty acid desaturase 5 (FAD5) played key roles in FG, and SLD2, FAD5, 3-oxoacyl-[acyl-carrier-protein] synthase I (KAS I), fatty acyl-ACP thioesterase B (FATB) and acetyl-CoA carboxylase (ACC) were the key genes responding to low temperature in FX. The variation and strategies of fatty acids in two varieties of Z. bungeanum were revealed at the metabolic and molecular level. This work provides a reference for the study of chemical components in plant stress resistance.

A "Weird" Mitochondrial Fatty Acid Oxidation as a Metabolic "Secret" of Cancer.

Cancer metabolism is an extensively studied field since the discovery of the Warburg effect about 100 years ago and continues to be increasingly intriguing and enigmatic so far. It has become clear that glycolysis is not the only abnormally activated metabolic pathway in the cancer cells, but the same is true for the fatty acid synthesis (FAS) and mevalonate pathway. In the last decade, a lot of data have been accumulated on the pronounced mitochondrial fatty acid oxidation (mFAO) in many types of cancer cells. In this article, we discuss how mFAO can escape normal regulation under certain conditions and be overactivated. Such abnormal activation of mitochondrial β-oxidation can also be combined with mutations in certain enzymes of the Krebs cycle that are common in cancer. If overactivated β-oxidation is combined with other common cancer conditions, such as dysfunctions in the electron transport complexes, and/or hypoxia, this may alter the redox state of the mitochondrial matrix. We propose the idea that the altered mitochondrial redox state and/or inhibited Krebs cycle at certain segments may link mitochondrial β-oxidation to the citrate-malate shuttle instead to the Krebs cycle. We call this abnormal metabolic condition “β-oxidation shuttle”. It is unconventional mFAO, a separate metabolic pathway, unexplored so far as a source of energy, as well as a source of cataplerosis, leading to biomass accumulation, accelerated oxygen consumption, and ultimately a source of proliferation. It is inefficient as an energy source and must consume significantly more oxygen per mole of ATP produced when combined with acetyl-CoA consuming pathways, such as the FAS and mevalonate pathway.

Evaluation of the expression of fatty acid synthase and O-GlcNAc transferase in patients with liver cancer by exploration of transcriptome databases and experimental approaches.

Tumor occurrence and development are closely related to metabolism abnormalities. One of the metabolic networks that is dysregulated during carcinogenesis is the fatty acid synthesis pathway, which is mainly controlled by fatty acid synthase (FASN). We previously demonstrated in proliferating HepG2 liver cancer cells that FASN expression depends on the catalytic activity of O-GlcNAc transferase (OGT) and the activation of the mechanistic/mammalian target of rapamycin (mTOR) pathway. The aim of the present study was to go further in these investigations by analyzing datasets and tissues of patients with liver cancer. To that purpose, transcriptome databases were explored, and reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used. Database analyses revealed that FASN and OGT gene expression was higher in certain cancer tissues, including liver hepatocellular carcinoma, compared with that in non-cancerous tissues. At the protein level, FASN expression was higher in the liver cancer-derived cell lines HepG2 and Hep3B compared with the immortalized human hepatocytes IHH cell line. However, neither the expression of OGT nor of its product O-GlcNAcylation showed any significant difference among the three hepatic cell lines. Subsequently, the expression of FASN and OGT at the protein and mRNA levels was evaluated in human liver cancer and non-tumoral tissues from the same patients with different liver lesions. The results from western blotting demonstrated a significant increase in OGT ands O-GlcNAcylation expression in liver cancer tissues independently of the type of lesion characterizing the non-tumoral counterpart. As previously reported for HepG2 proliferating cells, the protein level of FASN was positively correlated with the activation of mTOR and, although a rather upward trend, a high variability in its expression was monitored between patients. However, the results from immunohistochemistry showed no particular modification for OGT and O-GlcNAcylation expression and a significant increase in FASN expression in cancer tissues compared with that in adjacent non-tumoral tissues. Non-significant changes were observed for FASN and OGT mRNA levels between tumoral and non-tumoral samples, with a high variability between patients. Taken together, these results demonstrated that FASN expression was higher in hepatic cancer tissues in comparison with non-tumoral tissues. Furthermore, OGT expression and activity were shown to vary greatly between cell or cancer type, making any generalization difficult.

Metabolomics analysis of freeze–thaw tolerance enhancement mechanism of ε-poly-l-lysine on industrial yeast

Antimicrobial polycationic peptide ε-poly-l-lysine (ε-PL) enhanced the freeze–thaw tolerance of industrial yeast; the enhancement mechanism of ε-PL on yeast was studied. Results showed that a ε-PL coating was observed in ε-PL-treated yeast. After 4 times of freeze–thaw, the cell viability, glycerol content, and CO2 production of 0.6 mg/mL ε-PL-treated yeast were higher than those of untreated yeast, specifically, the cell viability of ε-PL-treated yeast was 87.6%, and that of untreated yeast was 68.5%. Metabolomic results showed that the enhancement mechanism of ε-PL on yeast was related to the promotion of cell membrane-related fatty acid synthesis pathways before freeze–thaw treatment, and the promotion of trehalose biosynthesis and glycerophospholipid metabolism pathways after freeze–thaw. Furthermore, ε-PL induced inhibition of the tricarboxylic acid cycle, resulting in a longer stationary phase at the beginning of the freeze–thaw and ultimately providing a higher level of freeze–thaw stress tolerance than untreated yeast.

Extracellular vesicles released by steatotic hepatocytes alter adipocyte metabolism

AbstractThe composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3‐L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes‐secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3‐L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic‐EVs in the development of pathologies associated with MetS.

Comparative Study of Bacillus amyloliquefaciens X030 on the Intestinal Flora and Antibacterial Activity Against Aeromonas of Grass Carp.

Beneficial microorganisms to control bacterial diseases has been widely used in aquaculture, Bacillus amyloliquefaciens (BaX030) as a probiotic feed additive was a commonly biological control method. Added sucrose promoted the growth of BaX030, and the yield of its antibacterial substance macrolactin A was enhanced by 1.46-fold. A total of 2055 proteins were screened through proteomics, with 143 upregulated and 307 downregulated. Differential protein expression analysis and qRT-PCR verification showed that the pentose phosphate pathway and the fatty acid synthesis pathway were upregulated, thereby providing sufficient energy and precursors for the synthesis of macrolactin A. The influence of some potential regulatory factors (SecG, LiaI, MecG and ComG) on macrolactin A was discovered. After grass carp were fed with BaX030, the abundance of probiotics (Fusobacterium, Proteobacteria, Gemmobacter) were higher than the control group, and the abundance of potential pathogenic bacteria (Planctomycetes, Aeromonas) were significantly lower than the control group. The cell and challenge experiments showed that BaX030 can significantly increase the expression of C3 and IL8 in the liver and kidney, which decreases the risk of immune organ disease. Moreover, BaX030 effectively reduced the mortality of grass carp. The results revealed that BaX030 can significantly improve the structure of the intestinal flora, enhance immunity and it is beneficial to the control of grass carp Aeromonas.

PHLPP1 promotes neutral lipid accumulation through AMPK/ChREBP-dependent lipid uptake and fatty acid synthesis pathways

SummaryInfiltration of arterial intima by foamy macrophages is a hallmark of early atherosclerotic lesions. Here, we investigated the potential role of Ser/Thr phosphatase PHLPP1 in foam cell development. PHLPP1 levels were elevated in OxLDL-exposed macrophages and high-fat diet (HFD)-fed zebrafish larvae. Using overexpression and knockdown approaches, we show that PHLPP1 promotes the accumulation of neutral lipids, and augments cellular total cholesterol and free fatty acid (FFA) levels. RNA-Seq analysis uncovered PHLPP1 role in lipid metabolism pathways. PHLPP1 interacted with and modestly increased ChREBP recruitment to Fasn promoter. PHLPP1-mediated lipid accumulation was attenuated by AMPK activation. Pharmacological inhibition or CRISPR/Cas9-mediated disruption of PHLPP1 resulted in lower lipid accumulation in the intersegmental vessels of HFD-fed zebrafish larvae along with a reduction in total cholesterol and triglyceride levels. Deficiency of phlp-2, C. elegans PHLPP1/2 ortholog, abolished lipid accumulation in high cholesterol-fed worms. We conclude that PHLPP1 exerts a significant effect on lipid buildup.

Expression of Membrane Bound O-Acyltransferase Domain Containing 7 after Myocardial Infarction and its Role in Lipid Metabolism in vitro.

Background: Previous microarray analysis on peripheral blood leukocytes from three patients with acute myocardial infarction (AMI) showed that elevated expression of membrane bound o-acyltransferase domain containing 7(MBOAT7) relative to control. To further verify these findings, we investigated more patients and explored the possible mechanisms in vitro.Objective: To study alterations in MBOAT7 expression in leukocytes after AMI, and to explore the relationship between MBOAT7 and lipid metabolism pathways in hepatocytes in vitro.Methods: Ninety patients with AMI and 90 controls were recruited from the Han population in Northeast China. RT-fluorescent PCR was used to measure MBOAT7 mRNA levels. MBOAT7 interference and overexpression vectors were constructed and transfected into L-02 hepatocytes and expression was examined by RT-qPCR and western blotting. The expression of SCAP, LDLR, HMGCR, ACAT1, ABCA1, SREBP1, ACC, FAS, SCD, and PPARγ in the lipid metabolism pathway were investigated by RT-qPCR. Triglyceride and cholesterol levels were measured by ELISA.Results: It was found that MBOAT7 mRNA levels were elevated in the leukocytes of patients with AMI. Hepatocytes were successfully transfected, shown by attenuated MBOAT7 mRNA levels in the silenced group (0.41±0.04 vs 1.01±0.07 for control, P=0.0019 <0.01) and raised levels in the overexpressing cells (23.29±0.39 vs 1.00±0.06 for control, P <0.0001). These results were confirmed by western blotting. Expression of the lipid metabolism-related genes was altered in response to MBOAT7 expression. Triglyceride levels increased after MBOAT7 silencing (118.40 ± 2.26 vs 70.54 ± 0.25 for control, P<0.0001), as did those of cholesterol (628.30 ± 8.89 vs 544.70 ± 11.04, P = 0.0041) but were not altered on MBOAT7 overexpression.Conclusion: MBOAT7 did not affect the metabolism of triglycerides in hepatocytes through fatty acid synthesis and decomposition pathways. The MBOAT7 level in the peripheral blood can be used as a marker for acute myocardial infarction but cannot be used as a single therapeutic target to regulate lipid metabolism.

Dietary Supplementation with Sodium Butyrate Reduces High-sucrose Diet-induced Hepatic Accumulation of Triacylglycerols and Expression of Fatty Acid Synthesis Enzymes in Rats.

We investigated the effects of dietary supplementation with sodium butyrate (NaB) on the lipid levels, gene expression, and proteins related to lipid metabolism in nonalcoholic fatty liver disease (NAFLD) rat models fed a high-sucrose diet for 3 weeks. Supplementation with 1% and 3% NaB reduced high-sucrose-induced hepatic triacylglycerol levels and expression of genes and proteins related to fatty acid synthesis, such as fatty acid synthase and malic enzyme, in a dose-dependent manner. NaB supplementation did not affect hepatic cholesterol levels or expression of genes related to β-oxidation. NaB may prevent high-sucrose-induced NAFLD by repressing the fatty acid synthesis pathway.

Transcriptomic and Proteomic Characterizations of the Molecular Response to Blue Light and Salicylic Acid in Haematococcus pluvialis

Haematococcus pluvialis accumulates a large amount of astaxanthin under various stresses, e.g., blue light and salicylic acid (SA). However, the metabolic response of H. pluvialis to blue light and SA is still unclear. We investigate the effects of blue light and SA on the metabolic response in H. pluvialis using both transcriptomic and proteomic sequencing analyses. The largest numbers of differentially expressed proteins (DEPs; 324) and differentially expressed genes (DEGs; 13,555) were identified on day 2 and day 7 of the treatment with blue light irradiation (150 μmol photons m−2s−1), respectively. With the addition of SA (2.5 mg/L), a total of 63 DEPs and 11,638 DEGs were revealed on day 2 and day 7, respectively. We further analyzed the molecular response in five metabolic pathways related to astaxanthin synthesis, including the astaxanthin synthesis pathway, the fatty acid synthesis pathway, the heme synthesis pathway, the reactive oxygen species (ROS) clearance pathway, and the cell wall biosynthesis pathway. Results show that blue light causes a significant down-regulation of the expression of key genes involved in astaxanthin synthesis and significantly increases the expression of heme oxygenase, which shows decreased expression by the treatment with SA. Our study provides novel insights into the production of astaxanthin by H. pluvialis treated with blue light and SA.

Cloning and characterisation of a Δ9 fatty acyl desaturase-like gene from the red claw crayfish (Cherax quadricarinatus) and its expression analysis under cold stress.

Desaturase is one of the key enzymes in the unsaturated fatty acid synthesis pathway. Δ9 desaturase catalyzes the synthesis of oleic acid from stearic acid by introducing double bonds in the 9th and 10th carbon chains, thereby increasing the content of MUFAs in the body. In order to explore the main function of the Δ9 desaturase gene under low temperature stress, RACE-PCR technology was used in this study to clone the full-length sequence of the CqFAD9-like from the hepatopancreas of red claw crayfish, Cherax quadricarinatus. The full length of the sequence is 1236 bp, and the open reading frame is 1041 bp, encoding 346amino acid residues. The 5 'UTR is 116 bp, the 3' UTR is 79 bp, and the 3 'UTR contains a PloyA tail. The predicted theoretical isoelectric point and molecular weight are 8.68 and 40.28kDa, respectively. Homology analysis showed that the sequence had the highest similarity with FAD9 from crustaceans. The results of real-time PCR showed that the expression level of this gene was highest in the hepatopancreas, which was significantly higher than other tissues, followed by the ovaries, brain ganglion and stomach. At the same time, the expression of the CqFAD9-like in hepatopancreas of crayfish cultured at 25, 20, 15 and 9°C for four weeks was detected. The results showed that expression of the FAD9 gene increased gradually with decreasing temperature, indicating that metabolic desaturation might play a regulatory role during cold stress.

Identification and Analysis of Genes Involved in Double Fertilization in Rice.

Double fertilization is a key determinant of grain yield, and the failure of fertilization during hybridization is one important reason for reproductive isolation. Therefore, fertilization has a very important role in the production of high-yield and well-quality hybrid of rice. Here, we used RNA sequencing technology to study the change of the transcriptome during double fertilization with the help of the mutant fertilization barrier (feb) that failed to finish fertilization process and led to seed abortion. The results showed that 1669 genes were related to the guided growth of pollen tubes, 332 genes were involved in the recognition and fusion of the male–female gametes, and 430 genes were associated with zygote formation and early free endosperm nuclear division. Among them, the genes related to carbohydrate metabolism; signal transduction pathways were enriched in the guided growth of pollen tubes, the genes involved in the photosynthesis; fatty acid synthesis pathways were activated by the recognition and fusion of the male–female gametes; and the cell cycle-related genes might play an essential role in zygote formation and early endosperm nuclear division. Furthermore, among the 1669 pollen tube-related genes, it was found that 7 arabinogalactan proteins (AGPs), 1 cysteine-rich peptide (CRP), and 15 receptor-like kinases (RLKs) were specifically expressed in anther, while 2 AGPs, 7 CRPs, and 5 RLKs in pistil, showing obvious unequal distribution which implied they might play different roles in anther and pistil during fertilization. These studies laid a solid foundation for revealing double fertilization mechanism of rice and for the follow-up investigation.

Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition

BackgroundAcquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.MethodsA cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.ResultsThe GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.ConclusionsNALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

Overview of the Cellular Stress Responses Involved in Fatty Acid Overproduction in E. coli.

Research on microbial fatty acid metabolism started in the late 1960s, and till date, various developments have aided in elucidating the fatty acid metabolism in great depth. Over the years, synthesis of microbial fatty acid has drawn industrial attention due to its diverse applications. However, fatty acid overproduction imparts various stresses on its metabolic pathways causing a bottleneck to further increase the fatty acid yields. Numerous strategies to increase fatty acid titres in Escherichia coli by pathway modulation have already been published, but the stress generated during fatty acid overproduction is relatively less studied. Stresses like pH, osmolarity and oxidative stress, not only lower fatty acid titres, but also alter the cell membrane composition, protein expression and membrane fluidity. This review discusses an overview of fatty acid synthesis pathway and presents a panoramic view of various stresses caused due to fatty acid overproduction in E. coli. It also addresses how certain stresses like high temperature and nitrogen limitation can boost fatty acid production. This review paper also highlights the interconnections that exist between these stresses.

DESIGN, SYNTHESIS AND BUILDING A QSAR MODEL FOR THE INHIBITION OF MYCOBACTERIUM TUBERCULOSIS BY PYRAZOLINE DERIVATIVES

A series of chalcone annulated pyrazoline conjugates have recently been synthesized from our laboratory and identified as possible “LEAD”s against Mycobacterium tuberculosis (MTB) through an in-silico study. The MTB enoyl acyl carrier protein reductase (InhA) plays a vital role in the MTB fatty acid synthesis pathway as essential in the mycolic acid biosynthesis. Therefore, inhibition of InhA could block the biosynthesis of mycolic acids, resulting in mycobacteria death. To further explore the potency of the compounds, a quantitative structure-activity relationship (QSAR) study is carried out. To develop a new pharmacophoric model for this inhibition, a QSAR approach of reported pyrazoline derivatives against tuberculosis has been studied. Multiple linear regression analysis was performed to derive the QSAR models, which were further evaluated internally as well as externally for the prediction of activity. The accurate MIC values (μg/ml) were collected for 23 pyrazoline analogs, and other descriptor parameters, such as log p (o/w), MR, DM, EELE, LUMO, HOMO, and TPSA were compared with these analogs. A training set of 23 analogs, all having a common pyrazoline moiety, provided a cross-validated correlation coefficient (r2) value of 0. 45112 and root mean square error (RMSE) value of 0. 0.27760. Moreover, based on this QSAR study, we have hypothetically estimated the predicted pMIC50 by using trainpred.fit file from Molecular Operating Environment 2009.10 software for the newly synthesized test set i.e., six chalcone based pyrazoline compounds. The resulting QSAR model generated from the present study may be useful in the design of a similar group of pyrazoline analogs as anti-tubercular agents.