Lipase Gene Research Articles

Cinnamaldehyde Decreases the Pathogenesis of Aeromonas hydrophila by Inhibiting Quorum Sensing and Biofilm Formation

Antibiotics were the main fishery drugs for treating Aeromonas hydrophila (A. hydrophila) infection, which would generate selective pressure and result in the appearance of antibiotic-resistant bacteria. The bacterial quorum sensing (QS) system provides a new alternative strategy against A. hydrophila infection. QS inhibitors can reduce bacterial virulence behaviors by disrupting QS, which has no effect on bacterial growth. Therefore, we studied the effect of cinnamaldehyde from a natural plant extract on the QS of A. hydrophila aiming to reduce its pathogenicity. The efficacy of cinnamaldehyde against A. hydrophila was evaluated from various aspects, including the effects on aerolysin, lipase, protease, swarming motility, biofilm formation, acyl-homoserine lactones (AHLs), and QS-related genes. Moreover, the therapeutic effect of cinnamaldehyde in vitro and in vivo was studied. The results showed that cinnamaldehyde could decrease the virulence phenotypes of A. hydrophila regulated by QS. Moreover, the transcriptions of related genes (aerA, ahyR, and ahyI) were downregulated following the addition of cinnamaldehyde. The in vitro and in vivo therapeutic assays show that cinnamaldehyde could reduce the aerolysin-mediated A549 cell injury and increase the survival rate of crucian carp infected with A. hydrophila. These results indicate that cinnamaldehyde would be a candidate QS inhibitor against A. hydrophila infection.

The Interplay among Glucocorticoid Therapy, Platelet-Activating Factor and Endocannabinoid Release Influences the Inflammatory Response to COVID-19.

COVID-19 is associated with a dysregulated immune response. Currently, several medicines are licensed for the treatment of this disease. Due to their significant role in inhibiting pro-inflammatory cytokines and lipid mediators, glucocorticoids (GCs) have attracted a great deal of attention. Similarly, the endocannabinoid (eCB) system regulates various physiological processes including the immunological response. Additionally, during inflammatory and thrombotic processes, phospholipids from cell membranes are cleaved to produce platelet-activating factor (PAF), another lipid mediator. Nonetheless, the effect of GCs on this lipid pathway during COVID-19 therapy is still unknown. This is a cross-sectional study involving COVID-19 patients (n = 200) and healthy controls (n = 35). Target tandem mass spectrometry of plasma lipid mediators demonstrated that COVID-19 severity affected eCBs and PAF synthesis. This increased synthesis of eCB was adversely linked with systemic inflammatory markers IL-6 and sTREM-1 levels and neutrophil counts. The use of GCs altered these lipid pathways by reducing PAF and increasing 2-AG production. Corroborating this, transcriptome analysis of GC-treated patients blood leukocytes showed differential modulation of monoacylglycerol lipase and phospholipase A2 gene expression. Altogether, these findings offer a breakthrough in our understanding of COVID-19 pathophysiology, indicating that GCs may promote additional protective pharmacological effects by influencing the eCB and PAF pathways involved in the disease course.

Comparative Genomic Analysis of a Thermophilic Protease-Producing Strain Geobacillus stearothermophilus H6.

The genus Geobacillus comprises thermophilic gram-positive bacteria which are widely distributed, and their ability to withstand high temperatures makes them suitable for various applications in biotechnology and industrial production. Geobacillus stearothermophilus H6 is an extremely thermophilic Geobacillus strain isolated from hyperthermophilic compost at 80 °C. Through whole-genome sequencing and genome annotation analysis of the strain, the gene functions of G. stearothermophilus H6 were predicted and the thermophilic enzyme in the strain was mined. The G. stearothermophilus H6 draft genome consisted of 3,054,993 bp, with a genome GC content of 51.66%, and it was predicted to contain 3750 coding genes. The analysis showed that strain H6 contained a variety of enzyme-coding genes, including protease, glycoside hydrolase, xylanase, amylase and lipase genes. A skimmed milk plate experiment showed that G. stearothermophilus H6 could produce extracellular protease that functioned at 60 °C, and the genome predictions included 18 secreted proteases with signal peptides. By analyzing the sequence of the strain genome, a protease gene gs-sp1 was successfully screened. The gene sequence was analyzed and heterologously expressed, and the protease was successfully expressed in Escherichia coli. These results could provide a theoretical basis for the development and application of industrial strains.

Biodegradation of Oil by a Newly Isolated Strain Acinetobacter junii WCO-9 and Its Comparative Pan-Genome Analysis.

Waste oil pollution and the treatment of oily waste present a challenge, and the exploitation of microbial resources is a safe and efficient method to resolve these problems. Lipase-producing microorganisms can directly degrade waste oil and promote the degradation of oily waste and, therefore, have very significant research and application value. The isolation of efficient oil-degrading strains is of great practical significance in research into microbial remediation in oil-contaminated environments and for the enrichment of the microbial lipase resource library. In this study, Acinetobacter junii WCO-9, an efficient oil-degrading bacterium, was isolated from an oil-contaminated soil using olive oil as the sole carbon source, and its enzyme activity of ρ-nitrophenyl decanoate (ρ-NPD) decomposition was 3000 U/L. The WCO-9 strain could degrade a variety of edible oils, and its degradation capability was significantly better than that of the control strain, A junii ATCC 17908. Comparative pan-genome and lipid degradation pathway analyses indicated that A. junii isolated from the same environment shared a similar set of core genes and that the species accumulated more specific genes that facilitated resistance to environmental stresses under different environmental conditions. WCO-9 has accumulated a complete set of oil metabolism genes under a long-term oil-contamination environment, and the compact arrangement of abundant lipase and lipase chaperones has further strengthened the ability of the strain to survive in such environments. This is the main reason why WCO-9 is able to degrade oil significantly more effectively than ATCC 17908. In addition, WCO-9 possesses a specific lipase that is not found in homologous strains. In summary, A. junii WCO-9, with a complete triglyceride degradation pathway and the specific lipase gene, has great potential in environmental remediation and lipase for industry.

Acupoint Catgut Embedding Improves Lipid Metabolism in Exercise-Induced Fatigue Rats via the PPAR Signaling Pathway

To improve the phenomenon of exercise-induced fatigue that often occurs during horse racing, we previously studied the improvement in exercise tolerance by acupoint catgut embedding preconditioning in an exercise-induced fatigue rat model. We found that acupoint catgut embedding pretreatment effectively improved animal exercise tolerance. Here, by combining transcriptomics and metabolomics, we aimed to explore the underlying mechanisms of this improvement. We used blood biochemical detection combined with ELISA to detect triglyceride (TG), total cholesterol (TC), lactate dehydrogenase (LDH), high-density lipoprotein (HDL), alanine transaminase (ALT), aspartate aminotransferase (AST), and glucose (GLU), arachidonic acid (AA), and free fatty acid (FFA) content and found that acupoint embedding can correct FFA, AA, TG, LDH, and AST in the blood. We used RT-qPCR to measure the expression of genes in tissue from the quadriceps femoris muscle. We found that solute carrier family 27 member 2 (Slc27a2), fatty acid binding protein 1 (Fabp1), apolipoprotein C3 (Apoc3), and lipoprotein lipase (Lpl) genes in the peroxisome proliferator-activated receptor (PPAR) signaling pathway were important. The regulation of lipid metabolism through the PPAR signaling pathway was important for improving the exercise endurance of rats in our exercise-induced fatigue model. Therefore, we conclude that acupoint catgut embedding can not only promote body fat decomposition and reduce lactic acid accumulation but also promote the repair of tissue damage and liver damage caused by exercise fatigue. Acupoint catgut embedding regulates the PPAR signaling pathway by upregulating Lpl expression and downregulating Slc27a2, Fabp1, and Apoc3 expression to further improve body fat metabolism.

Impact of surface pre-reacted glass ionomer filler eluate on lipase gene expression in Candida albicans: An in vitro study.

Although a surface pre-reacted glass ionomer (S-PRG) exerts a suppressive effect on Candida albicans (C. albicans) activity and growth, its influence on the expression of the lipase gene (LIP) family including LIP1-LIP10, an indicator of clinical infection, has not yet been investigated. Therefore, in this study, we evaluated the effect of S-PRG filler eluates on LIP expression in C. albicans using real-time reverse-transcription polymerase chain reaction. Candida albicans was treated with an S-PRG filler diluted at ratios of 1:32 and 1:64 for 24 h at 37°C. The diluted S-PRG filler eluates (1:32) suppressed lipase activity in C. albicans by downregulating LIP5 (0.54±0.25 relative to that of the control) and LIP8 (0.35±0.074) expression after 24 h, which corresponded with decreased lipase activity. At a dilution factor of 1:64, there was no significant difference in LIP expression. Thus, the S-PRG filler eluate has potential to suppress fungal activity by downregulating LIP expression.

High producer variant of lipoprotein lipase may protect from hepatocellular carcinoma in alcohol-associated cirrhosis

Progression of alcohol-associated liver disease (ALD) is driven by genetic predisposition. The rs13702 variant in the lipoprotein lipase (LPL) gene is linked to non-alcoholic fatty liver disease. We aimed at clarifying its role in ALD. Patients with alcohol-associated cirrhosis, with (n= 385) and without hepatocellular carcinoma (HCC) (n= 656), with HCC attributable to viral hepatitis C (n= 280), controls with alcohol abuse without liver damage (n= 366), and healthy controls (n= 277) were genotyped regarding the LPL rs13702 polymorphism. Furthermore, the UK Biobank cohort was analysed. LPL expression was investigated in human liver specimens and in liver cell lines. Frequency of the LPL rs13702 CC genotype was lower in ALD with HCC in comparison to ALD without HCC both in the initial (3.9% vs. 9.3%) and the validation cohort (4.7% vs. 9.5%; p <0.05 each) and compared with patients with viral HCC (11.4%), alcohol misuse without cirrhosis (8.7%), or healthy controls (9.0%). This protective effect (odds ratio [OR]= 0.5) was confirmed in multivariate analysis including age (OR= 1.1/year), male sex (OR= 3.0), diabetes (OR= 1.8), and carriage of the PNPLA3 I148M risk variant (OR= 2.0). In the UK Biobank cohort, the LPL rs13702C allele was replicated as a risk factor for HCC. Liver expression of LPL mRNA was dependent on LPL rs13702 genotype and significantly higher in patients with ALD cirrhosis compared with controls and alcohol-associated HCC. Although hepatocyte cell lines showed negligible LPL protein expression, hepatic stellate cells and liver sinusoidal endothelial cells expressed LPL. LPL is upregulated in the liver of patients with alcohol-associated cirrhosis. The LPL rs13702 high producer variant confers protection against HCC in ALD, which might help to stratify people for HCC risk. Hepatocellular carcinoma is a severe complication of liver cirrhosis influenced by genetic predisposition. We found that a genetic variant in the gene encoding lipoprotein lipase reduces the risk for hepatocellular carcinoma in alcohol-associated cirrhosis. This genetic variation may directly affect the liver, because, unlike in healthy adult liver, lipoprotein lipase is produced from liver cells in alcohol-associated cirrhosis.

Y12F mutation in Pseudomonas plecoglossicida S7 lipase enhances its thermal and pH stability for industrial applications: a combination of in silico and in vitro study.

Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55°C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.

CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae.

Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins.

Identification and characterization of novel carboxyl ester lipase gene variants in patients with different subtypes of diabetes

IntroductionMutations of CEL gene were first reported to cause a new type of maturity-onset diabetes of the young (MODY) denoted as MODY8 and then were also found in patients with...

LIPG is a novel prognostic biomarker and correlated with immune infiltrates in lung adenocarcinoma.

Although many biomarkers for lung adenocarcinoma (LUAD) have been identified, their specificity and sensitivity remain unsatisfactory. Endothelial lipase gene (LIPG) plays an important role in a variety of cancers, but its role in lung adenocarcinoma remains unclear. TCGA, GEO, K-M plotter, CIBERSORT, GSEA, HPA, and GDSC were used to analyze LIPG in LUAD. Data analysis was mainly achieved by R 4.0.3. The expression of LIPG in LUAD tissues was higher than that in adjacent normal tissues, especially in women, patients aged >65 years, and those with lymph node metastasis. High expression predicted a poor prognosis. The results of enrichment analysis suggest that LIPG may exert profound effects on the development of LUAD through multiple stages of lipid metabolism and immune system regulation. In addition, LIPG expression was significantly correlated with the expression levels of multiple immune checkpoint genes and the abundance of multiple immune infiltrates, including the activated memory CD4 T cell, M1 macrophage, neutrophil, plasma cells, and T follicular helper (Tfh) cells in the LUAD microenvironment content. At the same time, patients with high LIPG expression respond well to a variety of antitumor drugs and have a low rate of drug resistance. LIPG is a prognostic marker and is associated with lipid metabolism and immune infiltration in LUAD.

Elucidating type 2 diabetes mellitus risk factor by promoting lipid metabolism with gymnemagenin: An in vitro and in silico approach.

Introduction: Adipose tissue functions as a key endocrine organ which releases multiple bioactive substances and regulate obesity-linked complications. Dysregulation of adipocyte differentiation, triglyceride metabolism, adipokines production and lipid transport contributes to impaired lipid metabolism resulting in obesity, insulin resistance and type 2 diabetes. Gymnema sylvestre plant is frequently used in Ayurveda for treatment of diabetes and obesity. Gymnemagenin is a major bioactive compound of Gymnema sylvestre. The present study was undertaken to elucidate the role of gymnemagenin in lipid metabolism by in vitro and computational approaches. Methods: A panel of twelve genes viz., Fasn, Lipe, Lpl, Pparg, Plin2, Cidea, Scd1, Adipoq, Lep, Ccl2, Fabp4, and Slc2a4, essential in lipid metabolism were selected and gene expression pattern and triglyceride content were checked in adipocytes (3T3L1 cells) with/without treatment of gymnemagenin by Real time PCR and colorimetric estimation, respectively. Mode of action of gymnemagenin on Pparg and Fabp4 was accomplished by computational studies. Gene set enrichment and network pharmacology were performed by STRING and Cytoscape. Molecular docking was performed by AutoDock vina by POAP pipeline. Molecular dynamics, MM-PBSA were done by Gromacs tool. Results: In vitro study showed that gymnemagenin improved triglyceride metabolism by up regulating the expression of lipase genes viz., Lipe and Lpl which hydrolyse triglyceride. Gymnemagenin also up regulated the expression of anti-inflammatory gene Adipoq. Importantly, gymnemagenin treatment up regulated the expression of Pparg gene and the downstream target genes (Plin2, Cidea, and Scd1) which are associated with adipogenesis. However, gymnemagenin has no effect on expression of Fabp4, codes for a lipid transport protein. In silico study revealed that gymnemagenin targeted 12 genes were modulating 6 molecular pathways involved in diabetes and obesity. Molecular docking and dynamics revealed that gymnemagenin stably bind to active site residue of Pparg and failed to bind to Fabp4 active site compared to its standard molecules throughout 100ns MD production run. Gymnemagenin scored binding free energy of -177.94 and -25.406kJ/mol with Pparg and Fabp4, respectively. Conclusion: Gymnemagenin improved lipid metabolism by increasing triglyceride hydrolysis (lipolysis), up regulating the crucial gene of adipogenesis and increasing the expression of anti-inflammatory adipokine proving its therapeutic importance as anti-obesity and anti-diabetic phytocompound.

Production and Quantification of Virulence Factors in Malassezia Species.

Seventy-seven strains of Malassezia were included in this study. Biofilm and hydrolytic enzyme production were studied by using specific solid media. The Real-Time reverse transcriptase qPCR method was applied to determine the overexpression of genes encoding the extracellular enzymes. All included Malassezia species produced biofilms. No statistically significant difference was observed between Malassezia species in biofilm formation (p = 0.567). All Malassezia species produced lipase, and 95% of Malassezia globosa showed a strong enzymatic activity (Pz = 0.55 ± 0.02). A statistically significant difference was observed between the mean keratinase indices of Malassezia slooffiae and the other Malassezia species (p = 0.005). The overexpression of one or more genes was observed in 100% of strains isolated from patients with folliculitis, 87.5% - with pityriasis versicolor, and 57.14% of the control group isolates. A statistically significant difference in the lipase gene expression (p = 0.042) was between the strains from patients with folliculitis and the control group. This investigation provides more information about the frequency of the production of the major enzymes considered virulence factors of Malassezia species. Interestingly, the overexpression of one or more genes was observed in strains isolated from patients with Malassezia disorders.

Editorial introductions.

Current Opinion in Lipidology was launched in 1990. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of lipidology is divided into six sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editor for this issue. SECTION EDITOR Erik StroesErik StroesErik S. G. Stroes is Chair of the Department of Vascular Medicine at the Academic Medical Center, Amsterdam, The Netherlands. After gaining his Doctor of Medicine degree from Erasmus University, Rotterdam, The Netherlands, he then specialized in internal medicine at University Medical Center, Utrecht, The Netherlands, and later in vascular medicine at the Academic Medical Center, Amsterdam. Professor Stroes’ research interests include the role of the vessel wall in atherosclerosis development and lipid disorders in relation to atherogenesis. He has been an investigator in numerous lipid-lowering trials using various surrogate markers, such as intima-media thickness and flow-mediated dilation, and more recently 3T magnetic resonance imaging as a surrogate marker for vascular disease progression. He has also been involved in the development of novel therapeutic moieties for dyslipidemia, such as lipoprotein lipase gene therapy, antisense oligonucleotide inhibition of apolipoprotein B synthesis, and reconstituted high-density lipoprotein infusion. He has been the recipient of numerous research grants and several awards. Professor Stroes is Chair of the Dutch Atherosclerosis Society, member of the Vascular Biology Working Group, member of the American Heart Association Council on Arteriosclerosis, Thrombosis and Vascular Biology, and former Chair of the Dutch Internist Vascular Society. He has published more than 300 papers in various peer-reviewed journals and authored several book chapters in the field. Gerald F WattsGerald F WattsGerald F Watts (DSc DM PhD FRCP FRACP) trained at Imperial College (St Mary's Hospital) and King's College (St Thomas’ Hospital) in the University of London. He is a consultant physician, specializing in lipid disorders and cardiovascular medicine, within the department of cardiology at Royal Perth Hospital, as well as Winthrop Professor of Cardiometabolic Medicine in the University of Western Australia. He is Chair of The Familial Hypercholesterolaemia (FH)-Australasia Network and executive board member of the International Atherosclerosis Society. His research interests include lipid disorders and cardiovascular prevention, with clinical interest focusing on delivering improved health care for patients and families with FH. Professor Watts is actively involved in undergraduate medical education and supervisors PhD students and post-doctoral fellows. He has authored over 500 published works, including scientific articles, reviews, book chapters and monographs, and is associate editor of Atherosclerosis, Journal Clinical Lipidology and Metabolism.

Епідеміологія метаболічного синдрому та концепції механізмів його розвитку

The purpose of the study was to analyze literary sources with the study of modern views on the epidemiology of the metabolic syndrome and pro-inflammatory concepts of the mechanisms of the development of insulin resistance and type 2 diabetes mellitus, as one of its components. Materials and methods. Analytical and bibliosemantic methods were used in the research. During the scientific search, 53 sources of modern domestic and foreign literature were reviewed and analyzed. Results and discussion. Metabolic syndrome is the most common disorder of endocrine regulation, which is one of the threatening health care problems of the 21st century. In recent years, it remains almost the most important problem of endocrinology, cardiology, dietology, internal and family medicine. In recent decades, foreign researchers assign the most important role in the mechanism of metabolic syndrome development to adipocytes of adipose tissue. Most scientists consider insulin resistance to be the pathogenetic basis of metabolic syndrome. Insulin resistance manifests itself as the resistance of body cells to the action of insulin and occurs as a result of a violation of the insulin signal in these cells, the implementation of which is carried out by a membrane-mediated mechanism. Molecular manifestations of insulin resistance should be sought among the components of the insulin cascade, which includes the receptor, the substrate of the insulin receptor (IRS protein), the PI3-kinase cascade, and the GLUT-4 glucose transporter activation system. It should be noted that within the framework of the relationship between obesity, inflammation and insulin resistance, the following cytokines, TNF-α and IL-6, deserve more attention. TNF-α reduces the tyrosine protein kinase activity of the insulin receptor, the insulin-stimulated phosphorylation of its substrates (serine phosphorylation) and the expression of matrix ribonucleic acid GLUT-4 in muscle and adipose tissues, which is accompanied by impaired glucose transport. Also, TNF-α reduces the expression of the lipoprotein lipase gene, stimulates lipogenesis and synthesis of fatty acids, which additionally increases the degree of IP and obesity. IL-6 reduces the expression of the transporter GLUT-4 and the substrate of the insulin receptor, while also having an anti-inflammatory effect due to the reduction of TNF-α and interferon. Under the influence of this cytokine, the level of glycerol and free fatty acids in blood serum increases, which is accompanied by a lipotoxic effect on β-cells of the pancreas. Conclusion. Metabolic syndrome is a pathological process, the spread of which has become epidemic both in developed foreign countries and in Ukraine. One of the important components of the pathogenesis of the metabolic syndrome is insulin resistance, the mechanisms of which, in particular, are related to pro-inflammatory cytokine-mediated mechanisms affecting insulin signaling, glucose transport, involved in pathological changes in lipid synthesis and metabolism, as well as pro-oxidant and cytotoxic processes

Virulence genes contributing to Aeromonas veronii pathogenicity in Nile tilapia (Oreochromis niloticus): approaching the development of live and inactivated vaccines

This study aimed to develop and evaluate live and inactivated vaccines to Aeromonas veronii pathogenicity in Nile tilapia. Therefore, five well-identified Aeromonas veronii isolates, including A (HY1), A (HY2), A (HY3), A (HY4), and A (HY6) isolated from diseased Nile tilapia (Oreochromis niloticus), were used for vaccine preparation. Virulence genes detected by a polymerase chain reaction (PCR) and lethal dose determination were conducted. Nile tilapia, each with a body weight of 25 ± 0.5 g were divided into six experimental groups (each of 20): T1 group (control), fish were injected with saline as a negative control, T2 group (formalin-killed vaccine) for the A (HY2) strain, T3 group ( formalized killed vaccine) for the A (HY4), T4 group (autoclaved vaccine) for the A (HY2), T5 group (autoclaved vaccine) for A (HY4), and T6 (live vaccine) for A (HY1), triplicate. At the end of the immunization period, all groups were challenged by A. veronii, A (HY2). Blood samples were drawn 21 days post-immunization and 3 days after the challenge test for antibody titer assay. The results showed that the pathogenicity of strains A (HY2) and A (HY4) was the strongest, as the lethality rates (LR) were 100% and 90%, respectively, whereas the pathogenicity was moderate for strains A (HY3) and A (HY6) (LR 60% for each). A (AY1) was the weakest strain as no dead fish was found for this strain. The presence of alt, act, aerolysin, lipase, and fla genes as the main cause of the pathogenesis. The best protective efficacy was obtained from the live vaccine, A (HY1) with a protective rate of about 94.12% (relative percentage of survival, RPS), compared to autoclaved killed vaccines and formalin-killed vaccines. Based on immunoglobulin estimation (IgM) and RPS%, our data concluded that A (HY1) live vaccine had the best vaccine prophylactic effect against the highly pathogenic strain A(HY2).

Chitosan nanocarriers mediated dsRNA delivery in gene silencing for Helicoverpa armigera biocontrol

Specific gene silencing by RNA interference (RNAi) involving exogenous double stranded RNA (dsRNA) delivery has potential in Helicoverpa armigera control, a resistant insect pest. Here, ionotropically synthesized cationic chitosan nanoparticles (CNPs, 95 nm size, +36 mV charge) showed efficient dsRNA loading (95 %) and effective protection from insect gut nucleases and pH degradation. The CNPs were tagged with fluorescence and found to be stable on leaf surface (24 h) and were internalized by columnar insect gut cells. A single dose of CNPs:dsRNA complex (containing 0.1 μg dsRNA) ingested by H. armigera larvae via artificial/leaf feed effectively silenced lipase and chitinase target genes (2–2.7 fold downregulation) and suppressed their respective enzyme activities (2–5.3 fold). RNAi caused reduced pupation (5-fold) and impaired moth emergence. RNAi effects correlated significantly with 100% insect mortality (PCA 0.97–0.99). Furthermore, specific dsRNA did not affect non-target insects Spodoptera litura and Drosophila melanogaster. Developed CNPs:dsRNA complexes towards RNAi targets can serve as a safe, targeted insecticide for sustainable crop protection.

Identification and characterization of two types of triacylglycerol lipase genes from Neocaridina denticulata sinensis

Triacylglycerol lipases (TGLs) can catalyze the hydrolysis reaction of triacylglycerol serving multiple functions in most organisms. Based on the genomic and transcriptomic databases of Neocaridina denticulata sinensis, two TGL genes from N. denticulata sinensis designated NdTGL1 and NdTGL2 were identified and characterized. NdTGL1 showed the highest expression in the stomach, followed by the testis and hepatopancreas, and NdTGL2 exhibited the maximum expression in the hepatopancreas, followed by the stomach and heart. Under the stimulation of copper ion, the expression of NdTGL1 peaked at 12 h and the expression of NdTGL2 elevated significantly at 24 h after stimulation (P < 0.05). It is speculated that NdTGLs may play an important role in the stress response of N. denticulata sinensis. Challenged with Vibrio parahaemolyticus, the expression profiles of NdTGL1 and NdTGL2 in the hepatopancreas was different, which indicates that the immune response of the V. parahaemolyticus challenge might lead to changes in triglyceride metabolism. The recombinant NdTGL (recNdTGL1 and recNdTGL2) were obtained and the enzymatic characterization of recNdTGL1 and recNdTGL2 were determined. The common maximum activity and stability of the recNdTGL1 and recNdTGL2 were observed at 45 °C and 10 °C, respectively. Both recNdTGL1 and recNdTGL2 exhibited the highest activity at pH 10.0. Furthermore, the recNdTGL1 and recNdTGL2 displayed the maximum stability at pH 5.0 and pH 8.0, respectively. In presence of different metal ions, the enzyme activity of recNdTGL1 and recNdTGL2 were inhibited by Cu2+ and Zn2+, and decreased by about 25%. Studies on the triacylglycerol lipases of N. denticulata sinensis provide theoretical support for studies related to fat metabolism in crustaceans and studies on response mechanism of digestive enzymes to microbial pathogens.

Abstract 12042: A Mouse Model of Heart Failure With Preserved Ejection Fraction Driven by Hyperlipidemia and Enhanced Cardiac LDLR Expression

Background: HFpEF prevalence has surpassed that of HFrEF. Elevated accumulation of myocardial lipids is a prominent feature of HFpEF patients and the gene for lipoprotein lipase (LPL) in cardiac biopsies is decreased compared with HFrEF or healthy controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and increasing plasma triglycerides (TG) and LDL cholesterol. We hypothesized that hyperlipidemia driven by p407 and overexpression (OE) of cardiac LDLR in mice mimics a subset of human HFpEF. Methods: WT-129 mice were subject to 4 wks of biweekly i.p. p407-injections with or without a single i.v. injection of AAV9-cTnT-LDLR (n=31); AAV9-cTnT-Luciferase control (n=11), or single i.v. injection with AAV9-cTnT-LDLR alone (n=10). Treatment groups were compared to an untreated group (n=26). Echo, blood pressure (BP), whole-body plethysmography (WBP), ECG telemetry, activity wheel monitoring (AWM), biochemical tests, and histological changes were assessed at 4-8 wks. Results: At 4 wks, p407 and LDLR OE led to diastolic dysfunction (DD) (prolonged IVRT, decreased E/A (p&lt;0.05 and p&lt;0.001 respectively)), preserved EF and increased LV wall thickness (LVID and LVAW, p&lt;0.0001). BP and WBP were normal at 4 wks but respiration (breaths per minute) decreased at 8 wks (p&lt;0.01). ECG and AWM respectively indicated heart block and decreased exercise activity (p&lt;0.001). With the double treatment, total cholesterol increased in heart, skeletal muscle and liver (p&lt;0.05), while TG was decreased only in hearts (p&lt;0.001) and liver (p&lt;0.05). Double treatment also promoted elevated myocardial lipids, fibrosis, increased Wet/Dry lung (p&lt;0.001) and HW/BW (p&lt;0.05). Xanthelasma, ascites, and cardiac ischemia were noted in the double and single, p407 + luciferase groups. Sudden death (SD) occurred between 6-12 wks in double and p407+Luciferase treatment groups. Conclusion: We present a unique model of early onset, rapidly evolving HFpEF where double treatment mice acquire DD, arrhythmia, cardiac hypertrophy, fibrosis, pulmonary congestion, exercise intolerance, and preserved EF in the absence of obesity, HTN, CKD or DM. The model may represent a subset of HFpEF wherein hyperlipidemia drives a severe phenotype with SD as a prominent endpoint.

Abstract 10922: Maternal Hypercholesterolemia Induces Changes in DNA Methylation Pattern of Lipid Metabolism Genes in the Human Placenta

Introduction: Maternal hypercholesterolemia (MHC) during gestation is reported to enhance formation of fatty streaks in human fetuses leading to atherosclerosis in their adulthood. As maternal lipid levels increase during gestation as a normal physiological response to growing fetus, cholesterol levels are not routinely monitored in the outpatient clinic. The mechanisms mediating the stimulus transferred from hypercholesterolemic mothers to the fetus resulting in atherogenesis remains to be delineated. Hypothesis: We hypothesize that high lipid levels of hypercholesterolemic mother program the fetus by causing changes in DNA methylation patterns of lipid transport and metabolism genes in the placenta. Methods: Pregnant females within 100 days of first pregnancy were recruited. Total cholesterol (TC) and Low Density Lipoprotein (LDL) levels above 200mg/dL and 130mg/dL respectively were considered as hypercholesterolemic (MHC; n=50). Pregnant mothers with normal cholesterol levels were also recruited (NC; n=50). Lipid profiling of maternal blood was performed in all trimesters. Cord blood at the time of birth and neonatal blood on the 2 nd day post birth were also profiled. DNA methylation array was performed in the placenta of MHC and NC mothers post delivery. Data were analyzed using RnBeads and Limma package. Data were validated by qRT PCR and immunohistochemistry of placental tissues. Results: Lipid profiling revealed high TC and LDL levels in MHC mothers and high triglycerides in their neonates on the 2 nd day post birth. There were 3789 differentially methylated gene sets reported in the placenta of hypercholesterolemic mothers of which, 167 genes were hypomethylated and 94 genes were hypermethylated in the CpG islands. The hypermethylation and downregulation of lysosomal lipase genes (LIPA) was observed in the placenta of MHC mothers. This was complemented by upregulated expression of PEX11A, a peroxisomal biogenesis factor and ABCD1 which is a fatty acid transporter. Conclusions: We report the hypermethylation of lysosomal lipid degradation genes and hypomethylation of peroxisomal genes in the placenta of MHC mothers. This results in high triglycerides in neonates with a risk for early atherogenesis in adulthood.