Variability in plasma rifampicin concentrations and role of SLCO1B1, ABCB1, AADAC2 and CES2 genotypes in Ethiopian patients with tuberculosis.

BACKGROUND: Rifampicin, a key drug against tuberculosis (TB), displays wide between-patient pharmacokinetics variability and concentration-dependent antimicrobial effect. We investigated variability in plasma rifampicin concentrations and the role of SLCO1B1, ABCB1, arylacetamide deacetylase (AADAC) and carboxylesterase 2 (CES-2) genotypes in Ethiopian patients with TB. METHODS: We enrolled adult patients with newly diagnosed TB (n = 119) who had received 2 weeks of rifampicin-based anti-TB therapy. Venous blood samples were obtained at three time points post-dose. Genotypes for SLCO1B1 (c.388A > G, c.521T > C), ABCB1 (c.3435C > T, c.4036A > G), AADACc.841G > A and CES-2 (c.269-965A > G) were determined. Rifampicin plasma concentration was quantified using LC-MS/MS. Predictors of rifampicin Cmax and AUC0-7 h were analysed. RESULTS: The median rifampicin Cmax and AUC0-7 were 6.76 µg/mL (IQR 5.37-8.48) and 17.05 µg·h/mL (IQR 13.87-22.26), respectively. Only 30.3% of patients achieved the therapeutic efficacy threshold (Cmax>8 µg/mL). The allele frequency for SLCO1B1*1B (c.388A > G), SLCO1B1*5 (c.521T > C), ABCB1 c.3435C > T, ABCB1c.4036A > G, AADAC c.841G > A and CES-2 c.269-965A > G were 2.2%, 20.2%, 24.4%, 14.6%, 86.1% and 30.6%, respectively. Sex, rifampicin dose and ABCB1c.4036A > G, genotypes were significant predictors of rifampicin Cmax and AUC0-7. AADACc.841G > A genotypes were significant predictors of rifampicin Cmax. There was no significant influence of SLCO1B1 (c.388A > G, c.521T > C), ABCB1c.3435C > T and CES-2 c.269-965A > G on rifampicin plasma exposure variability. CONCLUSIONS: Subtherapeutic rifampicin plasma concentrations occurred in two-thirds of Ethiopian TB patients. Rifampicin exposure varied with sex, dose and genotypes. AADACc.841G/G and ABCB1c.4036A/A genotypes and male patients are at higher risk of lower rifampicin plasma exposure. The impact on TB treatment outcomes and whether high-dose rifampicin is required to improve therapeutic efficacy requires further investigation.

Clinical and genetic characteristics of CEL-MODY (MODY8): a literature review and screening in Chinese individuals diagnosed with early-onset type 2 diabetes.

OBJECTIVE: CEL-related maturity-onset diabetes of the young (CEL-MODY, MODY8) is a special type of monogenetic diabetes caused by mutations in the carboxyl-ester lipase (CEL) gene. This study aimed to summarize the genetic and clinical characteristics of CEL-MODY patients and to determine the prevalence of the disease among Chinese patients with early-onset type 2 diabetes (EOD). METHODS: We systematically reviewed the literature associated with CEL-MODY in PubMed, Embase, Web of Science, China National Knowledge Infrastructure and Wanfang Data to analyze the features of patients with CEL-MODY. We screened and evaluated rare variants of the CEL gene in a cohort of 679 Chinese patients with EOD to estimate the prevalence of CEL-MODY in China. RESULTS: In total, 21 individuals reported in previous studies were diagnosed with CEL-MODY based on the combination of diabetes and pancreatic exocrine dysfunction as well as frameshift mutations in exon 11 of the CEL gene. CEL-MODY patients were nonobese and presented with exocrine pancreatic affection (e.g., chronic pancreatitis, low fecal elastase levels, pancreas atrophy and lipomatosis) followed by insulin-dependent diabetes. No carriers of CEL missense mutations were reported with exocrine pancreatic dysfunction. Sequencing of CEL in Chinese EOD patients led to the identification of the variant p.Val736Cysfs*22 in two patients. However, these patients could not be diagnosed with CEL-MODY because there were no signs that the exocrine pancreas was afflicted. CONCLUSION: CEL-MODY is a very rare disease caused by frameshift mutations affecting the proximal VNTR segments of the CEL gene. Signs of exocrine pancreatic dysfunction provide diagnostic clues for CEL-MODY, and genetic testing is vital for proper diagnosis. Further research in larger cohorts is needed to investigate the characteristics and prevalence of CEL-MODY in the Chinese population.

Characteristics of CXE family of Salvia miltiorrhiza and identification of interactions between SmGID1s and SmDELLAs.

Carboxylesterase (CXE) is a class of hydrolases that contain an α/ß folding domain, which plays critical roles in plant growth, development, and stress responses. Based on the genomic and transcriptomic data of Salvia miltiorrhiza, the SmCXE family was systematically analyzed using bioinformatics. The results revealed 34 SmCXE family members in S. miltiorrhiza, and the SmCXE family could be divided into five groups (Group I, Group II, Group III, Group IV, and Group V). Cis-regulatory elements indicated that the SmCXE promoter region contained tissue-specific and development-related, hormone-related, stress-related, and photoresponsive elements. Transcriptome analysis revealed that the expression levels of SmCXE2 were highest in roots and flowers (SmCXE8 was highest in stems and SmCXE19 was highest in leaves). Further, two GA receptors SmCXE1 (SmGID1A) and SmCXE2 (SmGID1B) were isolated from the SmCXE family, which are homologous to other plants. SmGID1A and SmGID1B have conserved HGGSF motifs and active amino acid sites (Ser-Asp-Val/IIe), which are required to maintain their GA-binding activities. SmGID1A and SmGID1B were significantly responsive to gibberellic acid (GA3) and methyl jasmonate (MeJA) treatment. A subcellular assay revealed that SmCXE1 and SmCXE2 resided within the nucleus. SmGID1B can interact with SmDELLAs regardless of whether GA3 exists, whereas SmGID1A can only interact with SmDELLAs in the presence of GA3. A Further assay showed that the GRAS domain mediated the interactions between SmGID1s and SmDELLAs. This study lays a foundation for further elucidating the role of SmCXE in the growth and development of S. miltiorrhiza.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41 kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

The effects of carvacrol on development and gene expression profiles in Spodoptera frugiperda.

The fall armyworm, Spodoptera frugiperda, is a highly polyphagous agricultural pest that is widely distributed around the world and causes severe crop yield loss. Carvacrol showed adverse effects on many pests, such as larval death and growth inhibition. While the effects of carvacrol on S. frugiperda larvae are not yet known. In this study, the effects of carvacrol on S. frugiperda, including larval growth inhibition and mortality induction, were observed. The detoxification and digestive enzyme activities of larvae with 1.0 and 2.0 g/kg carvacrol treatments were analyzed. Carvacrol boosted the enzyme activities of carboxylesterase (CarE) and glutathione S-transferase (GST) while decreasing the activities of α-amylase (AMS), lipase (LIP), and trypsin. A total of 3422 differentially expressed genes were identified in the larvae treated with 2.0 g/kg carvacrol, of which the DEGs involved in xenobiotic detoxification, food digestion, and insecticidal targets were further examined. These results suggest that carvacrol could regulate growth and development by affecting the process of food digestion, and exert its toxicity on the larvae through interaction with a variety of insecticidal targets. While the altered expressions of detoxification enzymes might be related to the detoxification and metabolism of carvacrol. Our findings offer a theoretical foundation for the use of carvacrol for S. frugiperda control in the field.

The effect of chlorogenic acid, a potential botanical insecticide, on gene transcription and protein expression of carboxylesterases in the armyworm (Mythimna separata).

Chlorogenic acid (CGA) is a potential botanical insecticide metabolite that naturally occurs in various plants. Our previous studies revealed CGA is sufficient to control the armyworm Mythimna separata. In this study, we conducted a proteomic analysis of saliva collected from M. separata following exposure to CGA and found that differentially expressed proteins (DEPs) treated with CGA for 6 h and 24 h were primarily enriched in glutathione metabolism and the pentose phosphate pathway. Notably, we observed six carboxylesterase (CarE) proteins that were enriched at both time points. Additionally, these corresponding genes were expressed at levels 5.05 to 130.25 times higher in our laboratory-selected resistance strains. We also noted a significant increase in the enzyme activity of carboxylesterase following treatments with varying CGA concentrations. Finally, we confirmed that knockdown of MsCarE14, MsCarE28, and MsCCE001h decreased the susceptibility to CGA in resistance strain, indicating three CarE genes play crucial roles in CGA detoxification. This study presents the first report on the salivary proteomics of M. separata, offering valuable insights into the role of salivary proteins. Moreover, the determination of CarE mediated susceptibility change to CGA provides new targets for agricultural pest control and highlights the potential insecticide resistance mechanism for pest resistance management.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c.

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0-8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Ferulic acid production from wheat bran by integration of enzymatic pretreatment and a cold-adapted carboxylesterase catalysis.

High-value chemical production from natural lignocellulose transformation is a reliable waste utilization approach. A gene encoding cold-adapted carboxylesterase in Arthrobacter soli Em07 was identified. The gene was cloned and expressed in Escherichia coli to obtain a carboxylesterase enzyme with a molecular weight of 37.2 KDa. The activity of the enzyme was determined using α-naphthyl acetate as substrate. Results showed that the optimum enzyme activity of carboxylesterase was at 10 °C and pH 7.0. It was also found that the enzyme could degrade 20 mg enzymatic pretreated de-starched wheat bran (DSWB) to produce 235.8 µg of ferulic acid under the same conditions, which was 5.6 times more than the control. Compared to the chemical strategy, enzymatic pretreatment is advantageous because it is environmentally friendly, and the by-products can be easily treated. Therefore, this strategy provides an effective method for high-value utilization of biomass waste in agriculture and industry.

[Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure].

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in ß-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

Identification, expression profiles and involvement in insecticides tolerance and detoxification of carboxylesterase genes in Bactrocera dorsalis.

Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis. Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α-esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes. With the exception of BdCarE21, all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis.

Generation of Caco-2 cells with predictable metabolism by CYP3A4, UGT1A1 and CES using the PITCh system.

Caco-2 cells are widely used as an in vitro intestinal model. However, the expression levels of the drug-metabolizing enzymes CYP3A4 and UGT1A1 are lower in these cells than in intestinal cells. Furthermore, the majority of prodrugs in use today are ester-containing, and carboxylesterase (CES) 1 and CES2 are among the enzymes that process the prodrugs into drugs. In the human small intestine, CES1 is hardly expressed while CES2 is highly expressed, but the CES expression pattern in Caco-2 cells is the opposite. In this study, we generated CYP3A4-POR-UGT1A1-CES2 knock-in (KI) and CES1 knock-out (KO) Caco-2 (genome-edited Caco-2) cells using a PITCh system. Genome-edited Caco-2 cells were shown to express functional CYP3A4, POR, UGT1A1 and CES2 while the expression of the CES1 protein was completely knocked out. We performed transport assays using temocapril. The Papp value of temocapril in genome-edited Caco-2 cells was higher than that in WT Caco-2 cells. Interestingly, the amount of temocaprilat on the apical side in genome-edited Caco-2 cells was lower than that in WT Caco-2 cells. These results suggest that genome-edited Caco-2 cells are more suitable than WT Caco-2 cells as a model for predicting intestinal drug absorption and metabolism.

Inducible Gut-Specific Carboxylesterase SlCOE030 in Polyphagous Pests of Spodoptera litura Conferring Tolerance between Nicotine and Cyantraniliprole.

Insecticides tolerance in herbivorous arthropods is associated with preadaptation to host plant allelochemicals. However, how plant secondary metabolites activate detoxifying metabolic genes to develop tolerance remains unclear. Herein, the tolerance of Spodoptera litura larvae to cyantraniliprole was increased after nicotine exposure. An S. litura α esterase, SlCOE030, was predominantly expressed in the midgut and induced after exposure to cyantraniliprole, nicotine, and cyantraniliprole plus nicotine. Drosophila melanogaster with ectopically overexpressed SlCOE030 enhanced cyantraniliprole and nicotine tolerance by 4.91- and 2.12-fold, respectively. Compared to UAS-SlCOE030 and Esg-GAL4 lines, the Esg > SlCOE030 line laid more eggs after nicotine exposure. SlCOE030 knockdown decreased the sensitivity of nicotine-treated S. litura larvae to cyantraniliprole. Metabolism assays indicated that recombinant SlCOE030 protein metabolizes cyantraniliprole. Homology modeling and molecular docking analysis demonstrated that SlCOE030 exhibits effective affinities for cyantraniliprole and nicotine. Thus, insect CarEs may result in the development of cross-tolerance between synthetic insecticides and plant secondary metabolites.

Molecular identification of carboxylesterase genes and their potential roles in the insecticides susceptibility of Grapholita molesta.

Grapholita molesta is one of the most damaging pests worldwide in stone and pome fruits. Application of chemical pesticides is still the main method to control this pest, which results in resistance to several types of insecticides. Carboxylesterase (CarE) is one of the important enzymes involved in the detoxification metabolism and tolerance of xenobiotics and insecticides. However, the roles of CarEs in insecticides susceptibility of G. molesta are still unclear. In the present study, the enzyme activity of CarEs and the mRNA expression of six CarE genes were consistently elevated after treatment with three insecticides (emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole). According to spatio-temporal expression profiles, six CarE genes expressed differently in different developmental stages, and highly expressed in some detoxification metabolic organs. RNAi-mediated knockdown of these six CarE genes indicated that the susceptibility of G. molesta to all these three insecticides were obviously raised after GmCarE9, GmCarE14, GmCarE16, and GmCarE22 knockdown, respectively. Overall, these results demonstrated that GmCarE9, GmCarE14, GmCarE16, and GmCarE22 play a role in the susceptibility of G. molesta to emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole treatment. This study expands our understanding of CarEs in insects, that the same CarE gene could participate in the susceptibility to different insecticides.

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

INTRODUCTION: Mutations 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 type 1 (T1D) and type 2 diabetes (T2D). However, its genotype-phenotype relationship has not been fully determined and how carboxyl ester lipase (CEL) variants result in diabetes remains unclear. The aim of our study was to identify pathogenic variants of CEL in patients with diabetes and confirm their pathogenicity. RESEARCH DESIGN AND METHODS: All five patients enrolled in our study were admitted to Shandong Provincial Hospital and diagnosed with diabetes in the past year. Whole-exome sequencing was performed to identify pathogenic variants in three patients with MODY-like diabetes, one newborn baby with T1D and one patient with atypical T2D, as well as their immediate family members. Then the consequences of the identified variants were predicted by bioinformatic analysis. Furthermore, pathogenic effects of two novel CEL variants were evaluated in HEK293 cells transfected with wild-type and mutant plasmids. Finally, we summarized all CEL gene variants recorded in Human Gene Mutation Database and analyzed the mutation distribution of CEL. RESULTS: Five novel heterozygous variants were identified in CEL gene and they were predicted to be pathogenic by bioinformatic analysis. Moreover, in vitro studies indicated that the expression of CELR540C was remarkably increased, while p.G729_T739del variant did not significantly affect the expression of CEL. Both novel variants obviously abrogated the secretion of CEL. Furthermore, we summarized all reported CEL variants and found that 74.3% of missense mutations were located in exons 1, 3, 4, 10 and 11 and most missense variants clustered near catalytic triad, Arg-83 and Arg-443. CONCLUSION: Our study identified five novel CEL variants in patients with different subtypes of diabetes, expanding the gene mutation spectrum of CEL and confirmed the pathogenicity of several novel variants.

Esterase D interacts with metallothionein 2A and inhibits the migration of A549 lung cancer cells in vitro.

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure / 中华肝脏病杂志

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

Carboxylesterase-overexpressing hTERT-immortalized human adipose stem cells in prostate tumor growth inhibition by irinotecan.

INTRODUCTION: Effective chemotherapy has not yet to be developed for castration-resistant prostate cancer (CRPC). Cell-mediated enzyme prodrug therapy (EPT), including a combination of carboxylesterase (CE) and irinotecan (CPT-11), could be a possible treatment option. This study explored a cell-mediated EPT, including a combination of CE and irinotecan (CPT-11), to inhibit CRPC tumor growth using rabbit CE-overexpressing human TERT-immortalized adipose-derived stem cells (hTERT-ADSC.CE). MATERIALS AND METHODS: An hTERT ADSC.CE cell line was established by transfection with a lentiviral vector (CLV-Ubic) encoding the rabbit CE gene. To determine the in vitro suicide effects of hTERT-ADSC.CE, cell cultures were performed using various concentrations of CPT-11 (0.01-5 µM), and to determine the in vitro cytotoxic effects of hTERT-ADSC.CE cells, PC3 and hTERT-ADSC.CE cells were co-cultured. For the in vivo model, PC3 cells (1 × 106 cells) were injected subcutaneously into the flanks of nude mice and hTERT-ADSC.CE cells were injected via an intracardiac route, followed by the continuous treatment using CPT-11 for 2 weeks. The final change in tumor volume was measured and immunohistochemical analysis was performed. RESULTS: The directional and selective migration of hTERT-ADSC.CE cells toward PC3 cells was significantly stimulated by PC3 cells in vitro. The number of apoptotic PC3 cells significantly increased in the presence of hTERT-ADSC.CE and CPT-11 compared to CPT-11 alone. In the in vivo study, the inhibitory effects of hTERT-ADSC.CE combined with CPT-11 were higher than those of CPT-11 monotherapy. After treatment with CPT-11 alone or ADSC.CE in combination with CPT-11, the removed tumor tissues showed hyperchromatic nuclei and apoptotic bodies. CE-overexpressing ADSCs potentiated the inhibition of tumor growth in CRPC-bearing mice in the presence of CPT-11 prodrugs. CONCLUSIONS: This report suggests that cell-mediated EPT including CE and CPT-11 may be efficacious in treating CRPC.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.