Size-distribution and driving factors of aerosol oxidative potential in rural kitchen microenvironments of northeastern India.

This study reports size-resolved dithiothreitol (DTT)-based oxidative potential (OP: total and water-soluble) in rural kitchens using liquefied petroleum gas (LPG), firewood (FW), and mixed biomass (MB) fuels in northeastern (NE) India. In comparison to LPG, volume-normalized total OP (OPtotal(v)DTT) was enhanced by a factor of ∼5 in biomass-using kitchens (74 ± 35 to 78 ± 42 nmol min-1 m-3); however, mass-normalized total OP (OPtotal(m)DTT) was similar between LPG and FW users and higher by a factor of 2 in MB-using kitchens. The water-insoluble OP (OPwi(v, m)DTT) fraction in OPtotal(v, m)DTT was greater than 50% across kitchens. Size distributions across kitchens and OPDTT categories ranged from unimodal to trimodal. OPws(v)DTT was driven by metals as well as organics across size fractions while OPwi(v)DTT was majorly constrained by metals with an increasing importance of organics in fine particles of biomass-using kitchens. Multiple linear regression analysis revealed that Cu and Ba explained 71% of the OPtotal(v)DTT variability in LPG-using kitchens, while water-soluble organic carbon (WSOC) and Ba were responsible for 44% variability in FW-using kitchens. Finally, the high internal dose of OPtotal(v)DTT (28-31 nmol min-1 m-3) in biomass-using kitchens established the severity of oxidative stress on the exposed population.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

A saponin-polybromophenol antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA polymerase.

BACKGROUND: Gradual increase of multidrug resistant infections is a threat to the human race as MDR plasmids have acquired.>10 mdr and drug efflux genes to inactivate antibiotics. Plants secret anti-metabolites to retard growth of soil and water bacteria and are ideal source of antibiotics. PURPOSE: Purpose of the study is to discover an alternate phyto-drug from medicinal plants of India that selectively kills MDR bacteria. METHODS: MDR bacteria isolated from Ganga river water, milk, chicken meat and human hair for testing phyto-extracts. Eighty medicinal plants were searched and six phyto-extracts were selected having good antibacterial activities as demonstrated by agar-hole assays giving 15 â€‹mm or greater lysis zone. Phyto-extracts were made in ethanol or methanol (1:5 w/v) for overnight and were concentrated. Preparative TLC and HPLC were performed to purify phytochemical. MASS, NMR, FTIR methods were used for chemical analysis of CU1. In vitro RNA polymerase and DNA polymerase assays were performed for target identification. RESULTS: CU1 belongs to a saponin bromo-polyphenol compound with a large structure that purified on HPLC C18 column at 3min. CU1 is bacteriocidal but three times less active than rifampicin in Agar-hole assay. While in LB medium it shows greater than fifteen times poor inhibitor due to solubility problem. CU1 inhibited transcription from Escherichia coli as well as Mycobacterium tuberculosis RNA Polymerases. Gel shift assays demonstrated that CU1 interferes at the open promoter complex formation step. On the other hand CU1 did not inhibit DNA polymerase. CONCLUSION: Phyto-chemicals from Cassia fistula bark are abundant, less toxic, target specific and may be a safer low cost drug against MDR bacterial diseases.

Remote loading paclitaxel-doxorubicin prodrug into liposomes for cancer combination therapy.

The combination of paclitaxel (PTX) and doxorubicin (DOX) has been widely used in the clinic. However, it remains unsatisfied due to the generation of severe toxicity. Previously, we have successfully synthesized a prodrug PTX-S-DOX (PSD). The prodrug displayed comparable in vitro cytotoxicity compared with the mixture of free PTX and DOX. Thus, we speculated that it could be promising to improve the anti-cancer effect and reduce adverse effects by improving the pharmacokinetics behavior of PSD and enhancing tumor accumulation. Due to the fact that copper ions (Cu2+) could coordinate with the anthracene nucleus of DOX, we speculate that the prodrug PSD could be actively loaded into liposomes by Cu2+ gradient. Hence, we designed a remote loading liposomal formulation of PSD (PSD LPs) for combination chemotherapy. The prepared PSD LPs displayed extended blood circulation, improved tumor accumulation, and more significant anti-tumor efficacy compared with PSD NPs. Furthermore, PSD LPs exhibited reduced cardiotoxicity and kidney damage compared with the physical mixture of Taxol and Doxil, indicating better safety. Therefore, this novel nano-platform provides a strategy to deliver doxorubicin with other poorly soluble antineoplastic drugs for combination therapy with high efficacy and low toxicity.

Assessment of the PM2.5 oxidative potential in a coastal industrial city in Northern France: Relationships with chemical composition, local emissions and long range sources.

The objective of this work was to relate PM2.5 Oxidative Potential (OP) data to PM composition and PM local and distant source contributions. PM2.5 collected in Dunkerque, a coastal industrial city in North of France, was extensively characterized for major and minor chemical species. PM2.5 filters were extracted using a synthetic pulmonary fluid to achieve OP estimation based on Ascorbic Acid (AA) and dithiothreitol (DTT) depletion assays. In order to evidence relationships between OP values, chemical composition and local and distant source contributions, correlation coefficient, Principal Component Analysis (PCA), concentration roses, polar plots and concentration weighted trajectories were used. Heterogeneous conclusions were drawn using the three first methods as the bivariate polar plots lead to dismiss some of the correlations evidenced using correlation coefficient and PCA. Both AA and DTT tests appeared complementary as they were not sensitive to the same species/source contribution. The bivariate polar plot representation of OP values versus wind direction and wind speed revealed that PM2.5 concentration and combustion sources were linked to OP-AA, whereas emissions from integrated steelworks, electric steelworks, heavy fuel oil combustion and traffic non-exhaust significantly contribute to OP-DTT. Sea-salts, aged sea-salts, crustal, secondary sulfates and secondary nitrates sources were not found to contribute to OP values. Constant weighted trajectories evidenced several source regions responsible for high OP values with Belgium, Germany, Netherlands and France at the leader position. Contribution of inland regions appeared possibly related to the biomass and traffic related combustion while heavy fuel oil combustion could also be involved in the contribution of marine and coastal areas.

Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.

The Neural Tract Between the Hypothalamus and Basal Forebrain in the Ascending Reticular Activating System: A Diffusion Tensor Tractography Study.

OBJECTIVE: Ascending Reticular Activating System (ARAS) has a key role in consciousness. The ARAS is a complex network consisting of a portion of the brainstem reticular formation, nonspecific thalamic nuclei, hypothalamus, Basal Forebrain (BF), and cerebral cortex. We examined the reconstruction method and features of the neural tract between the hypothalamus and the BF in normal subjects, using Diffusion Tensor Tractography (DTT). METHODS: Twenty-three healthy subjects were recruited. The ARAS between the hypothalamus and the BF was reconstructed by two Regions of Interest (ROIs): 1) seed ROI - the isolated green portion for the BF on the color map, 2) target ROI - the hypothalamus on the axial image. DTT parameters of the ARAS between the hypothalamus and the BF were examined. RESULTS: Among 46 hemispheres in 23 normal subjects, 24 hemispheres (52.2 %) were identified in the ARAS between the hypothalamus and the BF. The reconstructed ARAS between the hypothalamus and the BF connected from the hypothalamus to the commissural level and anteriorly through the anterior commissure and then reached the BF. CONCLUSION: Using DTT, the ARAS between the hypothalamus and the BF was identified in normal subjects. Because the hypothalamus and BF are related to the regulation of wakefulness and sleep, our reconstruction method and results would be useful in the research on sleep and wakefulness aspects of consciousness.

Tissue specific effects of feeds supplemented with grape pomace or olive oil mill wastewater on detoxification enzymes in sheep.

The aim of the present study was to investigate the effects of livestock feed supplemented with grape pomace (GP) or olive oil mill wastewater (OMW) byproducts on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheep. Thus, 36 male sheep of Chios breed were divided into 3 homogeneous groups, control group (n = 12), GP group (n = 12) and OMW group (n = 12), receiving standard or experimental feed. Liver and spleen tissues were collected at 42 and 70 days post-birth. The enzymatic activity of superoxide dismutase (SOD) and glutathione-s-transferase (GST) and also the protein expression of γ-synthase glutamyl custeine (γ-GCS) were determined in these tissues. The results showed GP group exhibited increased enzymatic activity of GST and protein expression of γ-GCS in liver compared to control group. In GP group's spleen, GST activity was increased compared to control but γ-GCS expression was not affected. In OMW group's liver, GST activity was increased and γ-GCS expression was reduced compared to control. In OMW group's spleen, GST activity was increased but GCS expression was not affected. SOD activity was not affected in both tissues either in GP or OMW group.

Nitric oxide overcomes Cd and Cu toxicity in in vitro-grown tobacco plants through increasing contents and activities of rubisco and rubisco activase.

Toxic heavy metals such as cadmium (Cd) and copper (Cu) are global problems that are a growing threat to the environment. Despite some heavy metals are required for plant growth and development, others are considered toxic elements and do not play any known physiological role in plant cells. Elevated doses of Cd or Cu cause toxicity in plants and generate damages due to the stress condition and eventually cause a significant reduction in quantity and quality of crop plants. The nitric oxide (NO) donor sodium nitroprusside (SNP) is reported to alleviate the toxicity of some heavy metals like Cd and Cu. In the current study, the role of NO in alleviating stresses of Cd and Cu was investigated in in vitro-grown tobacco (Nicotiana tabacum) Based on plant growth, total chlorophyll contents, contents and activities of rubisco and rubisco activase. According to the results of this study, the growth and total chlorophyll contents of Cd/Cu stressed plants were hugely decreased in the absence of SNP, while the supplementation of SNP resulted in a significant increase of both fresh weight and total chlorophyll contents. Remarkable reductions of Rubisco and rubisco activase contents and activities were observed in Cd and Cu-induced plants. SNP supplementation showed the highest contents and activities of rubisco and rubisco activase compared to the control and Cu/Cd-stressed plants. Taken together, our findings suggest that SNP could play a protective role in regulation of plant responses to abiotic stresses such as Cd and Cu by enhancing Rubisco and Rubisco activase.

Development and implementation of an HPLC-ECD method for analysis of vitamin C in plasma using single column and automatic alternating dual column regeneration.

OBJECTIVES: Vitamin C (l-ascorbic acid) is a water-soluble micronutrient necessary for human life. Inadequate intake can lead to the fatal disease scurvy. Measurement of vitamin C is used to assess nutritional status and to monitor supplementation. The goal of this study was to develop a chromatographic method for the quantitation of vitamin C in human plasma. DESIGN AND METHODS: Samples were prepared by protein precipitation, addition of internal standard, and reduction with dithiothreitol. Separation of ascorbic acid was accomplished by isocratic elution on a reverse-phase column; concentration was determined by coulometry. The method was validated through studies of assay linearity, sensitivity, imprecision, accuracy, analytical specificity, and carryover. RESULTS: The new assay was developed using a single pump/single analytical column HPLC system. Results correlated well with our previously used spectrophotometric method. The analytical measurement range was 1.0-2500 µmol/L. The injection-to-injection time was 13 min. Subsequently, to increase method throughput and shorten turnaround time, a dual LC pump system with a 2-position/10-port switching valve capable of performing automatic alternating column regeneration was validated and implemented. The injection-to-injection time was reduced 2-fold to 6 min. The method was linear to 5000 µmol/L; limit of quantification was 1.9 µmol/L. Total imprecision was less than 5%. CONCLUSIONS: We have developed a robust method suitable for routine clinical measurement of vitamin C in plasma specimens. The method incorporates a simplified sample preparation and a stable, non-endogenous internal standard to specifically quantify vitamin C. Faster throughput was achieved by employing an automatic alternating column regeneration system.

Integrating virtual and biochemical screening for protein tyrosine phosphatase inhibitor discovery.

Protein tyrosine phosphatases (PTPs) represent an important class of enzymes that mediate signal transduction and control diverse aspects of cell behavior. The importance of their activity is exemplified by their significant contribution to disease etiology with over half of all human PTP genes implicated in at least one disease. Small molecule inhibitors targeting individual PTPs are important biological tools, and are needed to fully characterize the function of these enzymes. Moreover, potent and selective PTP inhibitors hold the promise to transform the treatment of many diseases. While numerous methods exist to develop PTP-directed small molecules, we have found that complimentary use of both virtual (in silico) and biochemical (in vitro) screening approaches expedite compound identification and drug development. Here, we summarize methods pertinent to our work and others. Focusing on specific challenges and successes we have experienced, we discuss the considerable caution that must be taken to avoid enrichment of inhibitors that function by non-selective oxidation. We also discuss the utility of using "open" PTP structures to identify active-site directed compounds, a rather unconventional choice for virtual screening. When integrated closely, virtual and biochemical screening can be used in a productive workflow to identify small molecules targeting PTPs.

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro.

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100-400 µg/mL), arrest cell cycle in the G0/G1 phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.

A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A.

Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation.

Many protein misfolding diseases, for example, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

DHA sensitizes FaO cells to tert-BHP-induced oxidative effects. Protective role of EGCG.

The excessive production of reactive oxygen species has been implicated in several pathologies, such as atherosclerosis, obesity, hypertension and insulin resistance. Docosahexaenoic acid (DHA) may protect against the above mentioned diseases, but paradoxically the main DHA treated pathologies are also associated with increased ROS levels. Therefore, the aim of this study was to explore if in vitro DHA supplementation may increase the sensitivity of cells to tert-BHP induced oxidative stress, and if the green tea polyphenol epigallocatechin-3-gallate (EGCG) is able to correct such detrimental effect. We found that DHA-enriched cells exacerbate ROS generation, decrease cell viability and increase Nrf2 nuclear translocation and HO-1 expression. Interestingly, cellular EGCG is able to counteract oxidative damage from either tert-BHP or DHA-enriched cells. In consequence, our results suggest that in a ROS enriched environment DHA could not always be beneficial for cells and can be considered a double-edged sword in terms of its benefits vs. risks. In this sense, our results propose that the supplementation with potent antioxidant molecules could be an appropriate strategy to reduce the risks related with the DHA supplementation in an oxidative stress-associated condition.

Identification of voltage-gated K(+) channel beta 2 (Kvß2) subunit as a novel interaction partner of the pain transducer Transient Receptor Potential Vanilloid 1 channel (TRPV1).

The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K(+) channel beta 2 subunit (Kvß2), an accessory subunit of voltage-gated K(+) channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvß2 association. Biotinylation assays in the presence of Kvß2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvß subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane.

Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds.

Broccoli (Brassica oleracea L. var. Italica) contains substantial amount of health-promoting compounds such as vitamins, glucosinolates, phenolic compounds, and dietary essential minerals; thus, it benefits health beyond providing just basic nutrition, and consumption of broccoli has been increasing over the years. This review gives an overview on the extraction and separation techniques, as well as the biological activity of some of the above mentioned compounds which have been published in the period January 2008 to January 2013. The work has been distributed according to the different families of health promoting compounds discussing the extraction procedures and the analytical techniques employed for their characterization. Finally, information about the different biological activities of these compounds has been also provided.

Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves.

Senescence-related proteases play important roles in leaf senescence by regulating protein degradation and nutrient recycling. A 98.9kDa senescence-related protease EP3 in wheat leaves was purified by ammonium sulfate precipitation, Q-Sepharose fast flow anion exchange chromatography and gel slicing after gel electrophoresis. Due to its relatively high thermal stability, its protease activity did not decrease after incubation at 40°C for 1-h. EP3 protease was suggested to be a metal-dependent serine protease, because its activity was inhibited by serine protease inhibitors PMSF and AEBSF and metal related protease inhibitor EGTA. It was identified as a subtilisin-like serine protease of the S8A family based on data from both mass spectrometry and the cloned cDNA sequence. Therefore, these data suggest that a serine protease of the S8A subfamily with specific biochemical properties is involved in senescence-associated protein degradation.

A critical comparison of two high-throughput ascorbate analyses methods for plant samples.

Ascorbate (AsA) is an important metabolite involved in stress response and development of plants. Therefore it is necessary to quantify the AsA content in many fields of plant science, including high throughput and critical applications. In this study we compared two different microplate-based AsA assays, which are suitable for high throughput applications: an ascorbate oxidase (AO)-based assay and a dipyridyl (DPD)-based assay. These methods were compared in critical applications, i.e. (i) when AsA concentrations were very low such as in apoplastic extracts, (ii) when plants contained pigments interfering with the spectrometric measurements, and (iii) when plants contained high iron concentration interfering with the color reactions. The precision of measurements was higher with the DPD method, as illustrated by higher recovery rates of internal AsA standards. On the other hand, the AO method was more sensitive to low levels of AsA. This was an advantage in determining apoplastic AsA concentration in rice, which was substantially lower than that of whole tissues. The AO method also had the advantage that plant pigments and high iron concentrations in plants tissues did not interfere with the analysis, as opposed to the DPD assay. In conclusion, both assays had advantages and the choice of a suitable method depends on the specific application.

Development of specific dengue virus 2'-O- and N7-methyltransferase assays for antiviral drug screening.

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA74, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.