Carboxyl Ester Research Articles

Generation of β Cells from iPSC of a MODY8 Patient with a Novel Mutation in the Carboxyl Ester Lipase (CEL) Gene.

Maturity-onset diabetes of the young (MODY) 8 is a rare form of monogenic diabetes characterized by a mutation in CEL (carboxyl ester lipase) gene, which leads to exocrine pancreas dysfunction, followed by β cell failure. Induced pluripotent stem cells can differentiate into functional β cells. Thus, β cells from MODY8 patients can be generated in vitro and used for disease modelling and cell replacement therapy. A genetic study was performed in a patient suspected of monogenic diabetes. A novel heterozygous pathogenic variant in CEL (c.1818delC) was identified in the proband, allowing diagnosis of MODY8. Three MODY8-iPSC (induced pluripotent stem cell) clones were reprogrammed from skin fibroblasts of the patient, and their pluripotency and genomic stability confirmed. All 3 MODY8-iPSC differentiated into β cells following developmental stages. MODY8-iPSC-derived β cells were able to secrete insulin upon glucose dynamic perifusion. The CEL gene was not expressed in iPSCs nor during any steps of endocrine differentiation. iPSC lines from a MODY8 patient with a novel pathogenic variant in the CEL gene were generated; they are capable of differentiation into endocrine cells, and β cell function is preserved in mutated cells. These results set the basis for in vitro modelling of the disease and potentially for autologous β cell replacement.

The position of single-base deletions in the VNTR sequence of the carboxyl ester lipase (CEL) gene determines proteotoxicity

Variable number of tandem repeat (VNTR) sequences in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which is specifically expressed in pancreatic acinar cells and encodes the digestive enzyme carboxyl ester lipase. Rare single-base deletions (DELs) within the first (DEL1) or fourth (DEL4) VNTR segment of CEL cause maturity-onset diabetes of the young, type 8 (MODY8), an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Studies on the DEL1 variant have suggested that MODY8 is initiated by CEL protein misfolding and aggregation. However, it is unclear how the position of single-base deletions within the CEL VNTR affects pathogenic properties of the protein. Here, we investigated four naturally occurring CEL variants, arising from single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, and DEL13). When the four variants were expressed in human embryonic kidney 293 cells, only DEL1 and DEL4 led to significantly reduced secretion, increased intracellular aggregation, and increased endoplasmic reticulum stress compared with normal CEL protein. The level of O-glycosylation was affected in all DEL variants. Moreover, all variants had enzymatic activity comparable with that of normal CEL. We conclude that the longest aberrant protein tails, resulting from single-base deletions in the proximal VNTR segments, have highest pathogenic potential, explaining why DEL1 and DEL4 but not DEL9 and DEL13 have been observed in patients with MODY8. These findings further support the view that CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to endoplasmic reticulum stress and activation of the unfolded protein response.

(Keynote) DT-Diaphorase As a Promising Catalytic Label for Rapid and Sensitive Immunosensors

The most common enzyme labels in biosensors are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), which, however, have some limitations for use in electrochemical immunosensors. DT-diaphorase (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH. Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal and long-term stability. Nevertheless, DT-D has never been employed as a catalytic label in biosensors. In this presentation, we report that DT-D can be used as a bifunctional enzyme label for rapid and sensitive electrochemical immunosensors: DT-D can convert an electrochemically inactive substrate into an electrochemically active product and DT-D can participate in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling induced by the product. We found that DT-D has high catalytic activities for nitroso reduction, phosphoester hydrolysis, and carboxyl ester hydrolysis as well as quinone reduction in the presence of NADH. 4-Nitroso-1-naphthol, 1-amino-2-naphthyl phosphate, and 4-aminonaphthalene-1-yl acetate are used as electrochemically inactive substrates for corresponding enzymatic reactions, which are converted into electrochemically active aminohydroxy-naphthalenes. This combination of DT-D and the substrates allows for high electrochemical signal-to-background ratios, because (i) the catalytic reaction by DT-D is fast, (ii) the electrochemical oxidation of the aminohydroxy-naphthalenes is rapid, even at an indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The electrochemical immunosensor using DT-D and 4-nitroso-1-naphthol detects parathyroid hormone with a low detection limit of 2 pg/mL, the immunosensor using DT-D and 1-amino-2-naphthyl phosphate detects outer membrane vesicles from Escherichia coli with a detection limit of 8 ng/mL, and the immunosensor using DT-D and 4-aminonaphthalene-1-yl acetate detects thyroid-stimulating hormone with a detection limit of ~2 pg/mL. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common ALP and HRP.

Independent mutational events in the hypervariable region of the carboxyl ester lipase gene: Identification of two new CEL-MODY families from Sweden and the Czech Republic

Independent mutational events in the hypervariable region of the carboxyl ester lipase gene: Identification of two new CEL-MODY families from Sweden and the Czech Republic

Protein misfolding in combination with other risk factors in CEL-HYB1-mediated chronic pancreatitis.

The hybrid allele of the carboxyl ester lipase gene (CEL-HYB1) is a genetic risk factor for chronic pancreatitis (CP) although the mechanism promoting disease development is largely unknown. Here, we aimed to clinically describe subjects carrying the CEL-HYB1 allele and to elucidate why the protein product is pathogenic by analyzing pancreatic secretions and cellular models. Norwegian cases (n = 154) diagnosed with recurrent acute pancreatitis or CP were subjected to genetic screening by a CEL-HYB1-specific PCR assay followed by Sanger sequencing. For investigation of CEL-HYB1 protein secretion, duodenal juice samples from cases and controls were analyzed by western blotting. HEK293cells were transfected with constructs expressing CEL-HYB1 or the normal CEL protein (CEL-WT) and analyzed by qPCR, cell fractionation and western blotting. Two CEL-HYB1-positive families were identified. In both pedigrees, CEL-HYB1 did not fully co-segregate with disease. One proband had recurrent acute pancreatitis and was an active smoker. Her mother was a CEL-HYB1 carrier who had suffered from several attacks of acute pancreatitis until she stopped smoking. The other proband was diagnosed with CP and pancreas divisum. Her CEL-HYB1-positive parent was symptom-free but exhibited pancreatic imaging changes. When analyzing the CEL protein in duodenal juice, CEL-WT was readily detectable but no band corresponding to the risk variant was seen. In CEL-HYB1-transfected cells, we observed impaired protein secretion, protein aggregation and endoplasmic reticulum stress. Our data suggest that CEL-HYB1, in combination with well-known pancreatitis risk factors, causes disease through the misfolding-dependent pathway of genetic CP risk.

Single nucleotide polymorphisms in CEL-HYB1 increase risk for chronic pancreatitis through proteotoxic misfolding.

Genetic variants contribute to the risk of chronic pancreatitis (CP) in adults and children. The risk variant CEL-HYB1, a recombinant hybrid allele of CEL and its neighboring pseudogene (CELP), encodes a pathogenic variant of the pancreatic digestive enzyme carboxyl ester lipase (CEL). We previously identified combinations of two non-synonymous SNPs, c.1463T>C (p. Ile488Thr) and c.1643C>T (p. Thr548Ile), in the break point region of CEL-HYB1. Herein, we tested whether these missense variants alter CP risk and their impact on functional properties of the CEL-HYB1 protein. Examination of CEL-HYB1 haplotypes in European patients and controls revealed that the combinationThr488-Ile548 was present only in cases (p ≤ .001). The lipase activity of purified recombinant CEL-HYB1 variants showed normal or near normal activity. CEL-HYB variants expressed in HEK293T cells all had decreased secretion compared with CEL, formed intracellular protein aggregates, and triggered endoplasmic reticulum stress. Thus, we propose that the presence of missense variants in CEL-HYB increases the pathogenicity of CEL-HYB1 through misfolding and gain-of-function proteotoxicity. Interestingly, Thr488-Ile548 and Thr488-Thr548 were equally pathogenic in the functional assays even though only the Thr488-Ile548 haplotype was significantly enriched in cases. The explanation for the mismatch between genetic and functional data requires further investigation.

Impacts of surface chemistry of functional carbon nanodots on the plant growth

Functional carbon nanodots (FCNs) with multiple chemical groups have great impact on the growth regulation of plants. To understand the role of the chemical groups, FCNs were reduced from the raw material by pyrolysis method and hydrolysis method. The chemical structure of these materials were characterized by using TGA, TEM, FT-IR, XPS, Raman and elementary analysis. The raw and reduced FCNs were used as plants growth regulators in culture medium of Arabidopsis thaliana. Our results indicate there is a strong correlation between the physiological responses of plants and the surface chemistries (especially carboxyl group and ester group) of the nanomaterials. The quantum-sized FCNs with multiple carboxyl groups and ester groups show better aqueous dispersity and can induce various positive physiological responses in Arabidopsis thaliana seedlings compared with the FCNs decorated without carboxyl and ester as well as aggregated FCNs. The raw FCNs present higher promotion capacity in plants biomass and roots length, and the quantum-sized FCNs are easier to be absorbed by plants and generate more positive effects on plants.

83-OR: Pancreatic Exocrine to Endocrine Cell Cross Talk Mediates Endoplasmic Reticulum Stress and Beta-Cell Dysfunction in MODY8

MODY8 (maturity onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes. This disease is associated with frameshift mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar tissue. Patients with the mutation develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes in MODY8 patients. In this study, we investigated the potential crosstalk between exocrine acinar cells and endocrine beta cells in vitro. Co-culture of beta cells (EndoC-bH1 beta cells) with donor cells (HEK293 embryonic kidney cells or 266-6 acinar cells) overexpressing mutant or wild type CEL gene in an in vitro transwell system demonstrated transfer of mutant protein from donor cells to beta cells. While both the wild type and mutant CEL proteins were observed to be taken up by the beta cells there was a significantly higher amount of the mutant (11.4±2.3% cells) compared to the wild type protein (1.8±0.7% cells) (n=3, p<0.01 mutant vs. wild type). The mutant protein formed intracellular aggregates represented as punctuate structures within the cytoplasm of beta cells. Culturing beta cells in mutant donor cell-conditioned media for 12 days activated the unfolded protein response, and increased endoplasmic reticulum stress by promoting the gene expression of BiP, spliced XBP1, and caused beta cell secretory dysfunction (insulin stimulation index 16.7 mM/3.3 mM: 1.3±0.1 (mutant) vs. 2.1±0.3 fold (wild type), n=8, p=0.05). Our data indicate a unique crosstalk between pancreatic acinar and beta cells, which adversely affects the latter by increasing ER stress and impairing insulin secretion. This study provides one possible explanation of how a mutant gene expressed specifically in acinar cells causes endocrine cell dysfunction and diabetes in MODY8 patients. Disclosure S. Kahraman: None. D. Diegisser: None. B.B. Johansson: None. A. Molven: None. R. Kulkarni: None. Funding National Institutes of Health (R01DK67536)

Carboxylated cellulose cryogel beads via a one-step ester crosslinking of maleic anhydride for copper ions removal

In this study, we developed a one step protocol to prepare highly carboxylated and chemically crosslinked cellulose nanofibril (CNF) cryogel beads using maleic anhydride (MA). Fourier transform infrared spectroscopy (FTIR) and conductometric-potentiometric titration results confirmed the presence of carboxyl groups and ester linkages produced simultaneously during the ring open reaction of MA, yielding a carboxylic content of up to 2.78 mmol/g. The effect of CNF concentration on the morphology and wet mechanical strength of the crosslinked cryogel beads were also investigated, and results suggested that higher CNF concentration yielded a compact network that displayed a maximum compressive stress of 2800 Pa at 60 % strain. In addition, the heavy metal ions (i.e., Cu (II)) removal capacity, kinetics, mechanism as well as the recyclability of the resulted CNF-MA cryogel beads were examined.

Principles of lipid–enzyme interactions in the limbus region of the catalytic site of Candida antarctica Lipase B

Lipases (E.C. 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates such as triacylglycerols and phospholipids. Candida antarctica Lipase B (CALB) acts in aqueous as well as in low-water media, thus being of considerable biochemical significance with high interest also for its industrial applications.The hydrolysis reaction follows a two-step mechanism, or ‘interfacial activation’, with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Once positioned within the catalytic triad, substrates are then hydrolysed, and products released. However, the intermediate steps of substrate transfer from the lipidic-aqueous phase to the enzyme surface and then down to the catalytic site are still unclear.By inhibiting CALB with ethyl phosphonate and incubating with glyceryl tributyrate (2,3-di(butanoyloxy)propyl butanoate), the crystal structure of the lipid-enzyme complex, at 1.55 Å resolution, shows the tributyrin in the limbus region of active site. The substrate is found 10 Å above the catalytic Ser, with the glycerol backbone pre-aligned for further processing by key interactions via an extended water network with α-helix10 and α-helix5.The findings offer new elements to elucidate the mechanism of substrate recognition, transfer and catalysis of Candida antarctica Lipase B (CALB) and lipases in general.

DT-Diaphorase As a Promising Catalytic Label for Rapid and Sensitive Immunosensors

The most common enzyme labels in biosensors are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), which, however, have some limitations for use in electrochemical immunosensors. DT-diaphorase (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH. Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal and long-term stability. Nevertheless, DT-D has never been employed as a catalytic label in biosensors. In this presentation, we report that DT-D can be used as a bifunctional enzyme label for rapid and sensitive electrochemical immunosensors: DT-D can convert an electrochemically inactive substrate into an electrochemically active product and DT-D can participate in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling induced by the product. We found that DT-D has high catalytic activities for nitroso reduction, phosphoester hydrolysis, and carboxyl ester hydrolysis as well as quinone reduction in the presence of NADH. 4-Nitroso-1-naphthol, 1-amino-2-naphthyl phosphate, and 4-aminonaphthalene-1-yl acetate are used as electrochemically inactive substrates for corresponding enzymatic reactions, which are converted into electrochemically active aminohydroxy-naphthalenes. This combination of DT-D and the substrates allows for high electrochemical signal-to-background ratios, because (i) the catalytic reaction by DT-D is fast, (ii) the electrochemical oxidation of the aminohydroxy-naphthalenes is rapid, even at an indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The electrochemical immunosensor using DT-D and 4-nitroso-1-naphthol detects parathyroid hormone with a low detection limit of 2 pg/mL, the immunosensor using DT-D and 1-amino-2-naphthyl phosphate detects outer membrane vesicles from Escherichia coli with a detection limit of 8 ng/mL, and the immunosensor using DT-D and 4-aminonaphthalene-1-yl acetate detects thyroid-stimulating hormone with a detection limit of ~2 pg/mL. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common ALP and HRP.

Association of a new 99-bp indel of the CEL gene promoter region with phenotypic traits in chickens

Carboxyl ester lipase (CEL) encodes a cholesterol ester hydrolase that is secreted into the duodenum as a component of pancreatic juice. The objective of this study was to characterize the CEL gene, investigate the association between the CEL promoter variants and chicken phenotypic traits, and explore the CEL gene regulatory mechanism. An insertion/deletion (indel) caused by a 99-bp insertion fragment was shown for the first time in the chicken CEL promoter, and large differences in allelic frequency were found among commercial breeds, indigenous and feral birds. Association analysis demonstrated that this indel site had significant effects on shank length, shank girth, chest breadth at 8 weeks (p < 0.01), evisceration weight, sebum weight, breast muscle weight, and leg weight (p < 0.05). Tissue expression profiles showed extremely high levels of the CEL gene in pancreatic tissue. Moreover, the expression levels of the genes APOB, MTTP, APOV1 and SREBF1, which are involved in lipid transport, were significantly reduced by adding a 4% oxidized soybean oil diet treatment at the individual level and transfecting the embryonic primary hepatocytes with a CEL-overexpression vector. Interestingly, the results showed that the expression level of the II homozygous genotype was significantly higher than that of the ID and DD genotypes, while individuals with DD genotypes had higher phenotypic values. Therefore, these data suggested that the CEL gene might affect body growth by participating in hepatic lipoprotein metabolism and that the 99-bp indel polymorphism could be a potentially useful genetic marker for improving the economically important traits of chickens.

Genomic and biochemical evidence of dietary adaptation in a marine herbivorous fish.

Adopting a new diet is a significant evolutionary change, and can profoundly affect an animal's physiology, biochemistry, ecology and genome. To study this evolutionary transition, we investigated the physiology and genomics of digestion of a derived herbivorous fish, Cebidichthys violaceus. We sequenced and assembled its genome (N50 = 6.7 Mb) and digestive transcriptome, and revealed the molecular changes related to digestive enzymes (carbohydrases, proteases and lipases), finding abundant evidence of molecular adaptation. Specifically, two gene families experienced expansion in copy number and adaptive amino acid substitutions: amylase and carboxyl ester lipase (cel), which are involved in the digestion of carbohydrates and lipids, respectively. Both show elevated levels of gene expression and increased enzyme activity. Because carbohydrates are abundant in the prickleback's diet and lipids are rare, these findings suggest that such dietary specialization involves both exploiting abundant resources and scavenging rare ones, especially essential nutrients, like essential fatty acids.

Carboxyl ester lipase is highly conserved in utilizing maternal supplied lipids during early development of zebrafish and human

Carboxyl ester lipase (Cel), is a lipolytic enzyme secreted by the pancreas, which hydrolyzes various species of lipids in the gut. Cel is also secreted by mammary gland during lactation and exists in breast milk. It facilitates dietary fat digestion and absorption, thus contributing to normal infant development. This study aimed to examine whether the Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants, and how Cel contributes to the utilization of yolk lipids in zebrafish. The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf, and in the exocrine pancreas after 72 hpf. The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention. The total cholesterol, cholesterol ester, free cholesterol, and triglyceride were reduced in the morphants' body while accumulated in the yolk (except triglyceride). The FFA content of whole embryos was much lower in morphants than in standard controls. Moreover, the delayed development in cel (cel1/cel2) double morphants was partially rescued by FFA and cholesterol supplementation. Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants. Correspondingly, shrunken midbrain tectum, microphthalmia, pigmentation-delayed eyes as well as down-regulated Shh target genes were observed in the CNS of double morphants. Interestingly, cholesterol injections reversed these CNS alterations. Our findings suggested that cel genes participate in the lipid releasing from yolk sac to developing body, thereby contributing to the normal growth rate and CNS development in zebrafish.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis

Background/objectivesCarboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. MethodsThe structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. ResultsCEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. ConclusionsOur results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein’s tail region.

Pathogenic Carboxyl Ester Lipase (CEL) Variants Interact with the Normal CEL Protein in Pancreatic Cells.

Mutations in the gene encoding the digestive enzyme carboxyl ester lipase (CEL) are linked to pancreatic disease. The CEL variant denoted CEL-HYB predisposes to chronic pancreatitis, whereas the CEL-MODY variant causes MODY8, an inherited disorder of endocrine and exocrine pancreatic dysfunction. Both pathogenic variants exhibit altered biochemical and cellular properties compared with the normal CEL protein (CEL-WT, wild type). We here aimed to investigate effects of CEL variants on pancreatic acinar and ductal cell lines. Following extracellular exposure, CEL-HYB, CEL-MODY, and CEL-WT were endocytosed. The two pathogenic CEL proteins significantly reduced cell viability compared with CEL-WT. We also found evidence of CEL uptake in primary human pancreatic acinar cells and in native ductal tissue. Moreover, coexpression of CEL-HYB or CEL-MODY with CEL-WT affected secretion of the latter, as CEL-WT was observed to accumulate intracellularly to a higher degree in the presence of either pathogenic variant. Notably, in coendocytosis experiments, both pathogenic variants displayed a modest effect on cell viability when CEL-WT was present, indicating that the normal protein might diminish toxic effects conferred by CEL-HYB and CEL-MODY. Taken together, our findings provide valuable insight into how the pathogenic CEL variants predispose to pancreatic disease and why these disorders develop slowly over time.

A consecutive chlorination and alkaline hydrolysis process for rapid degradation and detoxication of malathion in aqueous solution

Although oxidation and advanced oxidation can degrade malathion from water, unfortunately it is generally transformed into more toxic products, such as malaoxon. In the present study, a new approach has been proposed for the degradation and detoxication of malathion in aqueous solution using a combined process of chlorination followed by alkaline hydrolysis. The possible transformation pathway was proposed after identification of the main intermediate products using ultra-performance liquid chromatography – electrospray tandem mass spectrometry. The results showed that on average 90.3% of malathion was transformed into malaoxon during chlorination via a desulfurization reaction. After that, in the subsequent hydrolysis reaction, malaoxon was decomposed to malaoxon monoacid and diethyl 2-mercaptosuccinate via a carboxyl ester hydrolysis and a competing elimination reaction, respectively. At the same time, toxicity assessment of the treated solutions during the whole treatment process was performed based on the inhibition of acetylcholinesterase. It was found that during chlorination, the toxicity index HAChE of the malathion aqueous solution increased sharply to a maximum within 5 min due to the formation of malaoxon in solution. In the subsequent hydrolysis process, the toxicity index of the treated solution was almost unchanged during the first 10 min, but then quickly decreased to about zero within the next 10 min. This indicated a complete detoxication of the newly formed malaoxon and the whole malathion solution.

Carboxyl Esterase-Like Activity of DT-Diaphorase and Its Use for Signal Amplification.

Carboxyl esterases show limited use as catalytic labels in bioassays because of slow enzymatic reaction. We report that DT-diaphorase from Bacillus stearothermophilus (DT-D, EC 1.6.99.-) shows high carboxyl esterase-like activity in the presence of reduced β-nicotinamide adenine dinucleotide (NADH) and may be used as a better catalytic label than carboxyl esterases. DT-D is a redox enzyme and can participate in signal-amplifying redox cycling. Thus, an electrochemical immunosensor using a DT-D label allows for triple signal amplification based on (i) hydrolysis of a carboxyl ester, (ii) electrochemical-chemical (EC) redox cycling involving an electrode, a hydrolysis product, and NADH, and (iii) electrochemical-enzymatic (EN) redox cycling involving an electrode, a hydrolysis product, DT-D, and NADH. Ester hydrolysis by DT-D is confirmed via spectrophotometric measurement of a chromogenic substrate (4-nitrophenyl acetate) and 1H NMR spectra. Among two phenyl acetates and four naphthyl acetates considered, 4-aminonaphthalene-1-yl acetate (4-NH2-NAc) is chosen as the best acetyl ester substrate because 4-NH2-NAc is stable, its hydrolysis is slow in the absence of DT-D, its hydrolysis is very fast in the presence of DT-D, and EC and EN redox cycling involving the hydrolysis product (4-amino-1-naphthol) is rapid. However, hydrolysis of 4-NH2-NAc by esterase from porcine liver (EC 3.1.1.1.) is very slow. When DT-D is applied to sandwich-type detection of thyroid-stimulating hormone in artificial serum, the detection limit is ∼2 pg/mL, indicating that the developed immunosensor is highly sensitive because of triple signal amplification. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common alkaline phosphatase and horseradish peroxidase.

A new prognostic factor of breast cancer: High carboxyl ester lipase expression related to poor survival

ObjectiveThe enzyme carboxyl ester lipase (CEL), known as bile salt-dependent lipase (BSDL) or bile salt-stimulated lipase (BSSL), is mainly expressed in pancreatic acinar cells and lactating mammary glands. To investigate the link between CEL expression of breast cancer (BC) tissues and the survival of BC patients by analyzing The Cancer Genome Atlas Breast Carcinoma (TCGA-BRCA) level 3 data. MethodsThe clinical information and RNA-sequencing (RNA-Seq) expression data were downloaded from TCGA. Patients were divided into a high CEL expression group and a low CEL expression group using the optimal cutoff value (5.611) identified from the ROC curve. Chi-square test and Fisher exact test were used to find the correlation between the expression of CEL and clinicopathologic features. To assess the diagnostic capability, the receiver operating characteristic (ROC) curve of CEL was drawn. The survival differences between high and low CEL expression groups were compared by Cox regression analysis. Log-rank test was applied to the calculation of p values and the comparison of the Kaplan–Meier curves. Furthermore, Gene Expression Omnibus (GEO) datasets were used for external data validation. ResultsAnalysis of 1104 cases of tumor data showed that CEL was over-expressed in breast cancer. There were relationships between high CEL expression and clinicopathologic features. The high CEL expression group had a lower survival. By analyzing the area under the ROC curve (AUC) of CEL, it was found to have a limited diagnostic capability. CEL expression may be an independent prognostic factor for breast cancer survival through the multivariate analysis. The validation in GEO datasets also showed that CEL expression was higher in breast tumor tissues than in normal breast tissues. High CEL expression was associated with the poor overall survival of breast cancer. ConclusionsHigh CEL expression may be an independent prognostic factor for the poor survival of breast cancer.

Bola-Type Ala-Ala Dipeptides: Odd-Even Effect in Molecular Packing Structures.

A series of bola-type Ala-Ala dipeptides with different alkylene bridges (n = 10-15) were synthesized. In methanol, the molecules with even-numbered carbon bridges self-assembled into twisted nanoribbons, while those with odd-numbered carbon bridges self-assembled into straight belts. The morphology displays a pronounced odd-even dependence upon the number of carbons (n) in the connecting alkylene bridge. The circular dichroism spectra of the self-assemblies showed that molecules with even- and odd-numbered carbon bridges stacked in different structures. FT-IR spectra indicated that the dipeptides with even-numbered carbon bridges formed hydrogen bonds between the amide group and carboxyl ester group, while those with odd-numbered carbon bridges formed hydrogen bonds only between the amide groups. X-ray diffraction patterns revealed that molecules with odd- and even-numbered carbon bridges stacked in monoclinic and triclinic structures, respectively.