Quantitative morphokinetic parameters identify novel dynamics of oocyte meiotic maturation and cumulus expansion†.

Meiotic maturation and cumulus expansion are essential for the generation of a developmentally competent gamete, and both processes can be recapitulated in vitro. We used a closed time-lapse incubator (EmbryoScope+™) to establish morphokinetic parameters of meiotic progression and cumulus expansion in mice and correlated these outcomes with egg ploidy. The average time to germinal vesicle breakdown (GVBD), time to first polar body extrusion (PBE), and duration of meiosis I were 0.91 ± 0.01, 8.82 ± 0.06, and 7.93 ± 0.06 h, respectively. The overall rate of cumulus layer expansion was 0.091 ± 0.002 µm/min, and the velocity of expansion peaked during the first 8 h of in vitro maturation (IVM) and then slowed. IVM of oocytes exposed to Nocodazole, a microtubule disrupting agent, and cumulus oocyte complexes (COCs) to 4-methylumbelliferone, a hyaluronan synthesis inhibitor, resulted in a dose-dependent perturbation of morphokinetics, thereby validating the system. The incidence of euploidy following IVM was >90% for both denuded oocytes and intact COCs. No differences were observed between euploid and aneuploid eggs with respect to time to GVBD (0.90 ± 0.22 vs. 0.97 ± 0.19 h), time to PBE (8.89 ± 0.98 vs. 9.10 ± 1.42 h), duration of meiosis I (8.01 ± 0.91 vs. 8.13 ± 1.38 h), and overall rate and kinetics of cumulus expansion (0.089 ± 0.02 vs 0.088 ± 0.03 µm/min) (P > 0.05). These morphokinetic parameters provide novel quantitative and non-invasive metrics for the evaluation of meiotic maturation and cumulus expansion and will enable screening compounds that modulate these processes.

Inverting glucuronidation of hymecromone in situ by catalytic nanocompartments.

Glucuronidation is a metabolic pathway that inactivates many drugs including hymecromone. Adverse effects of glucuronide metabolites include a reduction of half-life circulation times and rapid elimination from the body. Herein, we developed synthetic catalytic nanocompartments able to cleave the glucuronide moiety from the metabolized form of hymecromone in order to convert it to the active drug. By shielding enzymes from their surroundings, catalytic nanocompartments favor prolonged activity and lower immunogenicity as key aspects to improve the therapeutic solution. The catalytic nanocompartments (CNCs) consist of self-assembled poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) diblock copolymer polymersomes encapsulating ß-glucuronidase. Insertion of melittin in the synthetic membrane of these polymersomes provided pores for the diffusion of the hydrophilic hymecromone-glucuronide conjugate to the compartment inside where the encapsulated ß-glucuronidase catalyzed its conversion to hymecromone. Our system successfully produced hymecromone from its glucuronide conjugate in both phosphate buffered solution and cell culture medium. CNCs were non-cytotoxic when incubated with HepG2 cells. After being taken up by cells, CNCs produced the drug in situ over 24 hours. Such catalytic platforms, which locally revert a drug metabolite into its active form, open new avenues in the design of therapeutics that aim at prolonging the residence time of a drug.

Hymecromone: a clinical prescription hyaluronan inhibitor for efficiently blocking COVID-19 progression.

Currently, there is no effective drugs for treating clinically COVID-19 except dexamethasone. We previously revealed that human identical sequences of SARS-CoV-2 promote the COVID-19 progression by upregulating hyaluronic acid (HA). As the inhibitor of HA synthesis, hymecromone is an approved prescription drug used for treating biliary spasm. Here, we aimed to investigate the relation between HA and COVID-19, and evaluate the therapeutic effects of hymecromone on COVID-19. Firstly, HA was closely relevant to clinical parameters, including lymphocytes (n = 158; r = -0.50; P < 0.0001), C-reactive protein (n = 156; r = 0.55; P < 0.0001), D-dimer (n = 154; r = 0.38; P < 0.0001), and fibrinogen (n = 152; r = 0.37; P < 0.0001), as well as the mass (n = 78; r = 0.43; P < 0.0001) and volume (n = 78; r = 0.41; P = 0.0002) of ground-glass opacity, the mass (n = 78; r = 0.48; P < 0.0001) and volume (n = 78; r = 0.47; P < 0.0001) of consolidation in patient with low level of hyaluronan (HA < 48.43 ng/mL). Furthermore, hyaluronan could directly cause mouse pulmonary lesions. Besides, hymecromone remarkably reduced HA via downregulating HAS2/HAS3 expression. Moreover, 89% patients with hymecromone treatment had pulmonary lesion absorption while only 42% patients in control group had pulmonary lesion absorption (P < 0.0001). In addition, lymphocytes recovered more quickly in hymecromone-treated patients (n = 8) than control group (n = 5) (P < 0.05). These findings suggest that hymecromone is a promising drug for COVID-19 and deserves our further efforts to determine its effect in a larger cohort.

Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence.

BACKGROUND: Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. RESULTS: A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5'-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of ß-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. CONCLUSIONS: EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience.

Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

A Reusable Column Method Using Glycopolymer-Functionalized Resins for Capture-Detection of Proteins and Escherichia coli.

The use of glycopolymer-functionalized resins (Resin-Glc), as a solid support, in column mode for bacterial/protein capture and quantification is explored. The Resin-Glc is synthesized from commercially available chloromethylated polystyrene resin and glycopolymer, and is characterized by fourier transform infrared spectroscopy, thermogravimetry, and elemental analysis. The percentage of glycopolymer functionalized on Resin-Glc is accounted to be 5 wt%. The ability of Resin-Glc to selectively capture lectin, Concanavalin A, over Peanut Agglutinin, reversibly, is demonstrated for six cycles of experiments. The bacterial sequestration study using SYBR (Synergy Brands, Inc.) Green I tagged Escherichia coli/Staphylococcus aureus reveals the ability of Resin-Glc to selectively capture E. coli over S. aureus. The quantification of captured cells in the column is carried out by enzymatic colorimetric assay using methylumbelliferyl glucuronide as the substrate. The E. coli capture studies reveal a consistent capture efficiency of 105  CFU (Colony Forming Units) g-1 over six cycles. Studies with spiked tap water samples show satisfactory results for E. coli cell densities ranging from 102 to 107  CFU mL-1 . The method portrayed can serve as a basis for the development of a reusable solid support in capture and detection of proteins and bacteria.

Arginine-259 of UGT2B7 Confers UDP-Sugar Selectivity.

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.

Understanding thermal and organic solvent stability of thermoalkalophilic lipases: insights from computational predictions and experiments.

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

Inhibition of UDP-glucuronosyltransferases by different furoquinoline alkaloids.

Herbs are often administered in combination with therapeutic drugs, raising the possibility for herb-drug interactions (HDIs). Furoquinoline alkaloids are found in Rutaceae plants, which are structurally similar and have many medicinal properties. This study aims to investigate the inhibition of four furoquinoline alkaloids on the activity of UDP-glucuronosyltransferases (UGTs).The recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition type and parameters were determined, and in silico docking was employed to elucidate the inhibition difference of furoquinoline alkaloids towards UGTs.Dictamine, haplopine, γ-fagarine and skimmianine strongly inhibited UGT1A3, UGT1A7, UGT1A9 and UGT2B4, respectively. Among them, dictamnine inhibited more than 70% of the four UGTs. Inhibition kinetics determination showed that they all exerted competitive inhibition, and the inhibition kinetic constant (Ki) was determined to be 8.3, 7.2, 3.7 and 33.9 µM, respectively. In vitro-in vivo extrapolation (IVIVE) was employed to demonstrate the inhibition possibility for four alkaloids. Skimmianine was proved to be more suitable for clinical application. In silico docking study indicated that the hydrophobic interactions played a key role in the inhibition of furoquinoline alkaloids towards three of the four UGTs. In conclusion, monitoring the interactions between furoquinoline alkaloids and drugs mainly undergoing UGTs-catalyzed metabolism is necessary.

Mutations in GAA Gene in Tunisian Families with Infantile Onset Pompe Disease: Novel Mutation and Structural Modeling Investigations.

Pompe disease, a rare, autosomal, recessive, inherited, lysosomal storage disorder, is caused by mutations in the acid α-glucosidase (GAA) gene leading to a deficiency of the lysosomal GAA enzyme. Some GAA mutations eliminate all enzymatic activities, causing severe infantile Pompe disease; others allow residual GAA activity and lead to middle adulthood forms. Here, we report a cohort of 12 patients, belonging to 11 unrelated families, with infantile Pompe disease. The mutational analysis of GAA gene revealed a novel c.1494G > A (p.Trp498X) mutation in one patient and three known mutatio,ns including the c.1497G > A (p.Trp499X) mutation, in two patients, the c.1927G > A (p.Gly643Arg) mutation in one patient and the common c.236_246del (p.Pro79ArgfsX13) mutation in eight patients. The high prevalence of c.236_246del mutation in our cohort (58%) was supported by the existence of a common founder ancestor that was confirmed by its segregation of similar SNPs haplotype, including four intragenic SNPs of GAA gene. In addition, a 3D structure model and a docking were generated for the mutant p.Gly643Arg using the crystal structure of human GAA as template and the 4-methylumbelliferyl-α-D-glucopyranoside as substrate. The results showed that the arginine at position 643 caused electrostatic changes in neighboring regions, leading to the repulsion between the amino acids located in the catalytic cavity of the GAA enzyme, thus restricting access to its substrate. These structural defects could cause the impairment of the transport and maturation previously reported for p.Gly643Arg mutation.

Lysosomal acid lipase does not have a propeptide and should not be considered being a proprotein.

Lysosomal acid lipase (LAL) plays an important role in lipid metabolism by performing hydrolysis of triglycerides and cholesteryl esters in the lysosome. Based upon characteristics of LAL purified from human liver, it has been proposed that LAL is a proprotein with a 55 residue propeptide that may be essential for proper folding, intracellular transport, or enzymatic function. However, the biological significance of such a propeptide has not been fully elucidated. In this study, we have performed a series of studies in cultured HepG2 and HeLa cells to determine the role of the putative propeptide. However, by Western blot analysis and subcellular fractionation, we have not been able to identify a cleaved LAL lacking the N-terminal 55 residues. Moreover, mutating residues surrounding the putative cleavage site at Lys76 ↓ in order to disrupt a proteinase recognition sequence, did not affect LAL activity. Furthermore, forcing cleavage at Lys76 ↓ by introducing the optimal furin cleavage site RRRR↓EL between residues 76 and 77, did not affect LAL activity. These data, in addition to bioinformatics analyses, indicate that LAL is not a proprotein. Thus, it is possible that the previously reported cleavage at Lys76 ↓ could have resulted from exposure to proteolytic enzymes during the multistep purification procedure.

A comparative study of acetylcholinesterase and general-esterase activity assays using different substrates, in vitro and in vivo exposures and model organisms.

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC50 values for the general inhibitor tetraisopropyl pyrophosphoramide (iso-OMPA) were similar, confirming that both substrates targeted the same group of enzymes. The GE substrate 4-MUB was applicable both in vitro and in vivo, while 1-NA was only applicable in vitro due to its high acute toxicity. When comparing the zooplankton crustacean Daphnia magna and the sediment dwelling Chironomus riparius, the latter had a four-fold higher maximal AChE activity (Vmax) and a higher susceptibility to the AChE inhibitor BW284c51 (four-fold lower 50% inhibitory concentration, IC50), but a lower maximal GE activity and lower susceptibility to iso-OMPA (higher IC50), indicating significant species differences between in C. riparius and D. magna. We conclude that both choice of substrate and exposure method matters for the outcome of esterase assays and that esterase compositions between species may vary significantly.

A disclosure gel for visual detection of live Bacillus anthracis spores.

AIMS: To develop a gel formulation to trigger a visual signal for rapid disclosure of the location and extent of surface contamination with viable Bacillus anthracis spores. METHODS AND RESULTS: Methylumbelliferyl-α-d-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating. It remained hydrated for a sufficient time for α-glucosidase activity present in intact B. anthracis spores to cleave the substrate and release the fluorescent product, methylumbelliferone. The presence of B. anthracis spores could be disclosed at 5 × 104 CFU per reaction test well (0·32 cm2 ) both visually and using fluorescence detection equipment. CONCLUSIONS: The disclosure gel provides a rapid, visual response to the presence of B. anthracis spores on a surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The disclosure gel demonstrates the first steps towards the development of a formulation that can provide nonspecialist users with a visual alert to the presence of B. anthracis spores on a surface. It is envisioned that such a formulation would be beneficial in scenarios where exposure to spore release is a risk, and could be used in the initial assessment of equipment to aid prioritization and localized execution of a decontamination strategy.

The inhibition of glycosaminoglycan incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

The extracellular matrix (ECM) contains a variety of complex macromolecules including proteoglycans (PGs) and glycosaminoglycans (GAGs). PG consists of a protein core with covalently attached carbohydrate side chains called GAGs. Several PGs, including versican, biglycan, decorin and syndecan are involved in odontogenesis while the role of GAGs in those PGs in this process remains unclarified. The purpose of this study was to investigate the influence of GAGs on tooth development. The mandibular first molars at early bell stage were cultivated with or without 4-methylumbelliferyl-ß-D-xyloside (Xyl-MU). The cultured tooth germs were metabolically labelled with [35S] Na2SO4, then PGs in tooth germs and cultured medium were extracted separately and analyzed by gel filtration. Morphological changes were evaluated on days 2, 4, 6, and histological changes were examined by hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). Related proteins and genes of cytodifferentiation were further examined by immunohistochemistry (IHC) and quantitive real-time PCR (qPCR) respectively. Meanwhile, BrdU incorporation assay was used to explore the effect of Xyl-MU on the cell proliferation of cultured tooth germs. The results demonstrated that the incorporation of GAGs to PGs in cultured tooth germs was heavily inhibited by Xyl-MU. Accompanied by the inhibition of GAGs incorporation, Xyl-MU altered tooth morphogenesis and delayed the differentiation of ameloblasts and odontoblasts. Proliferation of inner enamel epithelium (IEE) was also inhibited. Therefore, we draw a conclusion that the inhibition of GAGs incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases.

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 µM to 16.81 µM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 µM to 11.51 µM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

The inhibition of UDP-glucuronosyltransferases (UGTs) by tetraiodothyronine (T4) and triiodothyronine (T3).

1. UDP-glucuronosyltransferases (UGTs) are important drug-metabolizing enzymes (DMEs) catalyzing the glucuronidation elimination of various xenobiotics and endogenous substances. Endogenous substances are important regulators for the activity of various UGT isoforms. Triiodothyronine (T3) and thyroxine (T4) are important thyroid hormones essential for normal cellular differentiation and growth. The present study aims to elucidate the inhibition behavior of T3 and T4 on the activity of UGT isoforms. 2. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to screen the inhibition potential of T3 and T4 on the activity of various UGT isoforms. Initial screening results showed that T4 exerted stronger inhibition potential than T3 on the activity of various UGT isoforms at 100 µM. Inhibition kinetics was determined for the inhibition of T4 on the representative UGT isoforms, including UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7. The results showed that T4 competitively inhibited the activity of UGT1A1, -1A3, -1A7, 1A10 and -2B7, and noncompetitively inhibited the activity of UGT1A8. The inhibition kinetic parameters were calculated to be 1.5, 2.4, 11, 9.6, 4.8 and 3.0 µM for UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7, respectively. In silico docking method was employed to demonstrate why T4 exerted stronger inhibition than T3 towards UGT1A1. Stronger hydrogen bonds and hydrophobic interaction between T4 and activity cavity of UGT1A1 than T3 contributed to stronger inhibition of T4 towards UGT1A1. 3. In conclusion, more clinical monitoring should be given for the patients with the elevation of T4 level due to stronger inhibition of UGT isoforms-catalyzed metabolism of drugs or endogenous substances by T4.

Thermoactivation of a cellobiohydrolase.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

Kinetics study on recombinant alkaline phosphatase and correlation with the generated fluorescent signal.

Alkaline phosphatase (AP) (EC 3.1.3.1) is one of the most commonly used enzymes in immunoassays. In VIDAS® assays (bioMérieux, Marcy l'Etoile, France), AP catalyzes the hydrolysis of 4-methylumbelliferyl phosphate (4-MUP) in 4-methylumbelliferone (4-MU) producing a fluorescent signal. This work introduces an original method of characterization of the kinetic parameters Km, Vmax, and Kcat of AP embedded in VIDAS® assays. Assessment of such constants allows us to predict the fluorescent signal generated for given amounts of enzyme and its associated substrate; in the particular case of VIDAS®, it has been estimated that 0.06 nmol/L of AP produces 3144 Relative Fluorescent Values (RFV). ABBREVIATIONS: 4-MUP, 4-Methylumbelliferyl phosphate; 4-MU, 4-Methylumbelliferone; RFV, Relative Fluorescent Values; RFU, Relative Fluorescent Units; QDs, Quantum Dots; LoD, Limit of Detection.

Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides.

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain.