Enzymatic synthesis of pharmacologically relevant chiral sulfoxides by improved CbBVMO variants.

Baeyer-Villiger monooxygenases (BVMOs) are able to catalyse the asymmetric oxidation of sulfides. This property has made them attractive catalysts for the synthesis of chiral sulfoxide drugs. Here, we have designed and synthesised an exhaustive combinatorial mutant library of the previously identified lansoprazole sulfide monooxygenase CbBVMOV1. From this synthetic combinatorial mutant library, the best mutant, CbBVMOV3, was selected with a specific activity of approximately 1 U mg-1 for lansoprazole sulfoxides. We then optimised the reaction conditions of a two-phase system, achieving the enzymatic asymmetric synthesis of (R)-lansoprazole in a space-time yield of 213 g L-1 d-1 and an enantiomeric excess of >99% (R) with no detectable by-products. In addition, CbBVMOV3 showed higher activity towards other prazole sulfides. These results indicate the potential application of CbBVMO in the chiral sulfoxide drug industry.

Continuous photometric activity assays for lytic polysaccharide monooxygenase-Critical assessment and practical considerations.

Lytic polysaccharide monooxygenase (LPMO) is a monocopper-dependent enzyme that cleaves glycosidic bonds by using an oxidative mechanism. In nature, they act in concert with cellobiohydrolases to facilitate the efficient degradation of lignocellulosic biomass. After more than a decade of LPMO research, it has become evident that LPMOs are abundant in all domains of life and fulfill a diverse range of biological functions. Independent of their biological function and the preferred polysaccharide substrate, studying and characterizing LPMOs is tedious and so far mostly relied on the discontinuous analysis of the solubilized reaction products by HPLC/MS-based methods. In the absence of appropriate substrates, LPMOs can engage in two off-pathway reactions, i.e., an oxidase and a peroxidase-like activity. These futile reactions have been exploited to set up easy-to-use continuous spectroscopic assays. As the natural substrates of newly discovered LPMOs are often unknown, widely applicable, simple, reliable, and robust spectroscopic assays are required to monitor LPMO expression and to perform initial biochemical characterizations, e.g., thermal stability measurements. Here we provide detailed descriptions and practical protocols to perform continuous photometric assays using either 2,6-dimethoxyphenol (2,6-DMP) or hydrocoerulignone as colorimetric substrates as a broadly applicable assay for a range of LPMOs. In addition, a turbidimetric measurement is described as the currently only method available to continuously monitor LPMOs acting on amorphous cellulose.

Assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility.

Endometriosis is the cause of infertility. The eutopic endometrium of women with endometriosis showed an aberrant expression pattern of multitude genes. The role of TET1 protein in the pathogenesis of endometriosis and related infertility is not sufficiently known. Further, knowledge on TET1 transcriptional control still remains incomplete. The aim of the study was assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility. The study included 44 infertile patients with endometriosis (IWE) and 77 infertile (IW) and fertile (FW) patients without endometriosis. The research material was eutopic endometrium. The TET1 mRNA level was analyzed by qPCR. Western blot was used to evaluate the level of TET1 protein. The level of DNA methylation and H3K27me3 level of TET1 gene's promoter region were assessed using HRM and ChIP qPCR, respectively. The level of TET1 expression (TET1 mRNA; TET1 protein level) was lower in IWE during the implantation window (p < 0.001; p = 0.0329). The level of TET1 DNA methylation was higher in the secretory endometrium in mild and advanced IWE (p < 0.004; p < 0.008). H3K27me3 level did not differ between the study groups. The diminished expression of TET1 gene during the secretory phase, may account for the aberrant process of embryonic implantation in infertile endometriosis patients. DNA hypermethylation of TET1 gene is a potential relevant regulator of its expression. H3K27me3 occupancy does not affect the expression of TET1 gene in our study group.

Sequential Assessment of Multiple Epigenetic Modifications of Cytosine in Whole Genomic DNA by Surface Plasmon Resonance.

Since the discovery of the active DNA demethylation pathway in mammals, numerous efforts have been made to distinguish epigenetic cytosine variants, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). However, the rapid discrimination of multiple cytosine variants in DNA remains challenging because the conventional assays require time-consuming DNA pretreatments, such as enzymatical digestion and chemical conversion. Here we demonstrated the high-throughput discrimination of four cytosine variants in DNA by using a sequential surface-plasmon-resonance (SPR)-based immunochemical assay. The target DNAs were biotinylated in one step with a bifunctional linker 1 and robustly immobilized on a streptavidin-coated sensor surface to hold them in place during an alkali washing designed to remove residual antibodies. By repeating the injection of antibodies and washing, we achieved a sequential assessment of cytosine variants in identical DNA and identified the yield of in vitro 5mC oxidation in genomic DNA by the ten-eleven translocation 1 (TET1) enzyme. These results demonstrated that our sequential SPR-based immunochemical assay was effective for evaluating multiple epigenetic modifications in a whole genome with a single row operation without time-consuming DNA pretreatments.

Characterization of inheritance and preliminary biochemical mechanisms of spirotetramat resistance in Phenacoccus solenopsis Tinsley: An economic pest from Pakistan.

Phenacoccus solenopsis is an economically important insect pest of different agronomic and horticultural field crops. In Pakistan, the cotton crop was severely attacked by P. solenopsis during 2007 and since then a varied group of insecticides are used by farmers to manage this pest. As a result, insecticide resistance has become a barrier in control of P. solenopsis. The current study was designed to explore the basics of genetics, realized heritability and possible genetic mechanisms of resistance against spirotetramat in P. solenopsis. Before selection, the wild population (Wild-Pop) showed 5.97-fold resistance when compared with lab-reared susceptible strain (Susceptible Lab-Pop). The P. solenopsis was selected with spirotetramat to 21 generations, called Spiro-SEL Pop, which showed 463.21-fold resistance as compared with the Susceptible Lab-Pop. The values of LC50 for F1 (Spiro-SEL Pop ♂ × Susceptible Lab-Pop ♀) and F1 (Spiro-SEL Pop ♀ × Susceptible Lab-Pop ♂) populations were statistically similar and values of dominance level were 0.42 and 0.54, respectively. Reciprocal crosses between Susceptible Lab-Pop and Spiro-SEL Pop showed that resistance was of autosomal in nature with incomplete dominant traits. According to the fit test, monogenic model estimation of the number of genes, which are responsible for the development of spirotetramat resistance in a population of P. solenopsis, showed that multiple genes are involved in controlling the resistance levels in tested strains of P. solenopsis. The value of heritability for resistance against spirotetramat was 0.13 in P. solenopsis. Our results suggested the presence of a metabolic-based resistance mechanism associated with the monooxygenases in P. solenopsis, while testing the synergism mechanism. These results will provide the baseline to design an effective control strategy to manage P. solenopsis in the field.

Functional assessment of rat pulmonary flavin-containing monooxygenase activity.

The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model. Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible. The predicted lung clearance (CLlung) estimated from microsomes and slices was 16 ± 0.6 and 2.1 ± 0.3 mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CLlung using rat lung microsomes corresponded to approximately 35% of rat CLliver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine. Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

Lactate biosensing: The emerging point-of-care and personal health monitoring.

Lactate plays a crucial role in the anaerobic metabolic pathway of humans. In situations of oxygen deficit, its production increases; leading to several life-threatening conditions such as hemorrhage, respiratory failure, trauma or ischemia from lactate acidosis. Lactate level detection and point-of-care (POC) monitoring in a fast, accurate and non-invasive manner is ultimately important for many health care applications. Optical and electrochemical techniques are employed in lactate sensing to achieve high sensitivity and selectivity, miniaturization, portability, simplicity, and low cost. To improve the selectivity and sensitivity, two important enzymes, lactate oxidase (LOx) and lactate dehydrogenese (LDH) are employed. Conventional methods for lactate detection are not fast enough to be used in point-of-care or personal health monitoring settings. Moreover, the existing point-of-care lactate sensing tools follow invasive or partially invasive sampling protocols such as finger pricking. In this review, a comprehensive overview of different lactate biosensing devices is presented. Particularly, the state-of-the-art and prospects of wearable, non-invasive lactate sensing from different biofluids are discussed.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Role of warfarin pharmacogenetic testing in clinical practice.

Chronic oral anticoagulation with warfarin is difficult to maintain within the therapeutic range and requires frequent monitoring and dose adjustments. Variations in two genes, VKORC1 and CYP2C9, have been associated with variation in warfarin metabolism among individuals. Patients with CYP2C9*2 and *3 variants have longer times to dose stabilization and are at higher risk of serious and life-threatening bleeding. VKORC1 polymorphisms significantly influence time to first therapeutic warfarin range, and variants in this gene determine low-, intermediate- and high-warfarin dose requirements. The prevalence of CYP2C9 and VKORC1 polymorphisms vary among different ethnic groups, and can account for over 30% of variance in warfarin dose. Recent studies suggest that the pharmacogenomics-guided dosing algorithm can accurately predict warfarin dosage and might reduce adverse events. We aim to review the pharmacogenetics of warfarin metabolism and the clinical role of genetic testing for warfarin therapy.

Accuracy assessment of pharmacogenetically predictive warfarin dosing algorithms in patients of an academic medical center anticoagulation clinic.

The objectives of this retrospective cohort study are to evaluate the accuracy of pharmacogenetic warfarin dosing algorithms in predicting therapeutic dose and to determine if this degree of accuracy warrants the routine use of genotyping to prospectively dose patients newly started on warfarin. Seventy-one patients of an outpatient anticoagulation clinic at an academic medical center who were age 18 years or older on a stable, therapeutic warfarin dose with international normalized ratio (INR) goal between 2.0 and 3.0, and cytochrome P450 isoenzyme 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genotypes available between January 1, 2007 and September 30, 2008 were included. Six pharmacogenetic warfarin dosing algorithms were identified from the medical literature. Additionally, a 5 mg fixed dose approach was evaluated. Three algorithms, Zhu et al. (Clin Chem 53:1199-1205, 2007), Gage et al. (J Clin Ther 84:326-331, 2008), and International Warfarin Pharmacogenetic Consortium (IWPC) (N Engl J Med 360:753-764, 2009) were similar in the primary accuracy endpoints with mean absolute error (MAE) ranging from 1.7 to 1.8 mg/day and coefficient of determination R (2) from 0.61 to 0.66. However, the Zhu et al. algorithm severely over-predicted dose (defined as >or=2x or >or=2 mg/day more than actual dose) in twice as many (14 vs. 7%) patients as Gage et al. 2008 and IWPC 2009. In conclusion, the algorithms published by Gage et al. 2008 and the IWPC 2009 were the two most accurate pharmacogenetically based equations available in the medical literature in predicting therapeutic warfarin dose in our study population. However, the degree of accuracy demonstrated does not support the routine use of genotyping to prospectively dose all patients newly started on warfarin.

Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.

Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.

Pharmacogenomics of 4-hydroxycoumarin anticoagulants.

Oral anticoagulants of the 4-hydroxycoumarin class, typified by warfarin, are used worldwide to treat thromboembolic disease. These drugs show the beneficial attributes of high efficacy and low cost, but patient management can be complicated by their narrow therapeutic index and wide inter-individual variability in dosing. Our understanding of the latter complication has improved significantly in recent years due to intense investigation of genetic factors influencing drug pharmacokinetics (CYP2C9) and pharmacodynamic response (VKORC1). In particular, the discovery of polymorphisms in the VKORC1 gene that strongly impact oral anticoagulant dose has heightened expectations that genetic testing for a relatively small cadre of warfarin-response genes might substantially enhance patient care in this area, especially during the initiation phase of therapy. However, enthusiasm for genotype-based dosing of oral anticoagulants must be balanced against the ready availability of both a simple phenotypic test (prothrombin time) and an antidote to over-anticoagulation (vitamin K). Wide-spread acceptance of genetically based tests for establishing therapy with warfarin and its congeners will likely require additional evidence that such an approach offers protection against a variety of negative anticoagulation outcomes, especially severe bleeding, as well as offering utility across many racial populations. This article will review recent events in these and other related areas.

Quantitative assessment of phenol hydroxylase diversity in bioreactors using a functional gene analysis.

We describe a quantitative analysis of the genetic diversity of phenol-degrading potential in bacterial communities from laboratory-scale activated sludge. Genomic DNA extracted from activated sludge from two sequential batch reactors fed with synthetic sewage plus phenol was amplified using conserved primers for the major subunit of the phenol hydroxylase (LmPH) gene and used to generate clone libraries. Following phylogenetic analysis, 59 sequences containing a 470-bp fragment clustered into six distinct subgroups with a genetic distance of 8%, most likely representing ecologically relevant variants of the enzyme. Seven sets of primers were designed to target the six clusters and used to obtain quantitative information on the dynamics of LmPH gene diversity using real-time PCR assays throughout 9 months of bioreactors operation. Total LmPH gene copy number remained approximately steady in phenol-amended and control reactors. However, a significant increase in phenol-degrading activity in the phenol-amended sludge was accompanied by a parallel increase in LmPH gene diversity, suggesting that phenol degradation in the activated sludge depends on the combined activity of a number of redundant species.

Translation of pharmacogenetics into clinically relevant testing modalities.

Pharmacogenetics (PGx) relies on the genetic makeup of an individual to predict drug response and efficacy, as well as potential adverse drug events. Significant advances in PGx research have been made since inherited differences in response to such drugs as isoniazid and succinylcholine were explored in the 1950s, and the clinical utility and application of PGx are especially apparent in some subspecialty areas of chemotherapeutic, psychotropic drug, and anticoagulant therapies.

Pharmacogenomics-based tailored versus standard therapeutic regimen for eradication of H. pylori.

Helicobacter pylori eradication rates by triple therapy with a proton pump inhibitor, amoxicillin, and clarithromycin at standard doses depend on bacterial susceptibility to clarithromycin and patient CYP2C19 genotypes. We examined the usefulness of a personalized therapy for H. pylori infection based on these factors as determined by genetic testing. First, optimal lansoprazole dosing schedules that would achieve sufficient acid inhibition to allow H. pylori eradication therapy in each of different CYP2C19 genotype groups were determined by a 24-h intragastric pH monitoring. Next, 300 H. pylori-positive patients were randomly assigned to the standard regimen group (lansoprazole 30 mg twice daily (b.i.d.)), clarithromycin 400 mg b.i.d., and amoxicillin 750 mg b.i.d. for 1 week) or the tailored regimen group based on CYP2C19 status and bacterial susceptibility to clarithromycin assessed by genetic testing. Patients with failure of eradication underwent the second-line regimen. The per-patient cost required for successful eradication was calculated for each of the groups. In the first-line therapy, the intention-to-treat eradication rate in the tailored regimen group was 96.0% (95% CI=91.5-98.2%, 144/150), significantly higher than that in the standard regimen group (70.0%: 95% CI=62.2-77.2%, 105/150) (P<0.001). Final costs per successful eradication in the tailored and standard regimen groups were $669 and $657, respectively. In conclusion, the pharmacogenomics-based tailored treatment for H. pylori infection allowed a higher eradication rate by the initial treatment without an increase of the final per-patient cost for successful eradication. However, the precise cost-effectiveness of this strategy remains to be determined.

Genotype and phenotype relationship in drug metabolism.

Pharmacogenetics, one of the fields of clinical pharmacology, studies how genetic factors influence drug response. If hereditary traits are taken into account appropriately before starting drug treatment, the type of drug and its dosage can be tailored to the individual patient's needs. Today, the relationships between dosage requirements and genetic variations in drug-metabolizing enzymes such as cytochrome P450 (CYP) 2D6, CYP2C9, and CYP2C19 or in drug transporters such as p-glycoprotein (ABCB1) and OATP-C (SLC21A6) are substantiated best. A standard dose will bring about more adverse effects than usual if enzymatic activity is lacking or feeble. Sometimes, however, therapeutic response might be better because of higher concentrations: proton pump inhibitors for eradication of Helicobacter pylori are more efficacious in carriers of a deficient CYP2C19 variant. In some cases, genetic tests can help distinguish between responders and nonresponders of a specific drug treatment, and genotype-based dosage is possible.

In vitro and in vivo assessment of herb drug interactions.

Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions.

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis.

The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts alpha-phenylalanine to beta-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield beta-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both beta-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of beta-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of beta-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.