Pharmacokinetic–pharmacodynamic modelling of the analgesic effects of lumiracoxib, a selective inhibitor of cyclooxygenase-2, in rats

Background and purpose: This study establishes a pharmacokinetic/pharmacodynamic (PK/PD) model to describe the time course and in vivo mechanisms of action of the antinociceptive effects of lumiracoxib, evaluated by the thermal hyperalgesia test in rats. Experimental approach: Female Wistar fasted rats were injected s.c. with saline or carrageenan in the right hind paw, followed by either 0, 1, 3, 10 or 30 mg·kg-1 of oral lumiracoxib at the time of carrageenan injection (experiment I), or 0, 10 or 30 mg·kg-1 oral lumiracoxib at 4 h after carrageenan injection (experiment II). Antihyperalgesic responses were measured as latency time (LT) to a thermal stimulus. PK/PD modelling of the antinociceptive response was performed using the population approach with NONMEM VI. Results: A two-compartment model described the plasma disposition. A first-order model, including lag time and decreased relative bioavailability as a function of the dose, described the absorption process. The response model was: LT = LT0/(1 + MED). LT0 is the baseline response, and MED represents the level of inflammatory mediators. The time course of MED was assumed to be equivalent to the predicted profile of COX-2 activity and was modelled according to an indirect response model with a time variant synthesis rate. Drug effects were described as a reversible inhibition of the COX-2 activity. The in vivo estimate of the dissociation equilibrium constant of the COX-2-lumiracoxib complex was 0.24 mg·mL-1. Conclusions: The model developed appropriately described the time course of pharmacological responses to lumiracoxib, in terms of its mechanism of action and pharmacokinetics

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5- meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1) / DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short aminoterminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA, and is required for efficient activity on 5-meC:G, but not for T:G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5- meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly-paired base such as 5- meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases

DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine-DNA glycosylases

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA-methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown if they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-meC from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process, and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants

Opportunities, Constraints and Perceptions of Rural Communities Regarding Their Potential to Contribute to Forest Landscape Transitions Under REDD+: Case Studies from Mexico

In Mexico, REDD+ is being presented as a win-win policy enabling forest communities to benefit financially and diversify their income sources while preserving and increasing their forest carbon stocks through more sustainable management. Under the national programme, it is expected that forest communities will have opportunities to tailor their own approaches. However, to date there is little understanding about what opportunities and constraints exist in reality for forest communities to contribute to REDD+, and even less about how their members perceive these opportunities. We assess potential and constraints at community level and investigate perceptions about opportunities in REDD+ and strategies that communities are currently envisaging for participation, in seven communities in the Ayuquila River Basin and around the Chamela-Cuixmala Biosphere Reserve in Jalisco, and in the area surrounding the Monarch Butterfly Reserve in Michoacan. We find that there is more opportunity for reduced degradation and forest enhancement than for reduced deforestation, in all the communities; that it may be difficult to establish additionality for REDD+ activities in some communities; that the amount of forest resource per community may greatly affect the potential to participate; that the presence of people with no land rights may complicate the distribution of benefits; that communities expect REDD+ in general to follow the Payment for Environmental Services model, and that lack of information about what activities may count as REDD+ activities and what level of financial rewards may be expected mean that communities cannot at present adequately appraise whether REDD+ will be worth their while or no

The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair

Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER

A DNA 3′ Phosphatase Functions in Active DNA Demethylation in Arabidopsis

DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′ phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway

On-line tools to improve the presentation skills of scientific results

[EN] In experimental sciences and engineering it is essential to communicate and present the results effectively. The authors have participated in several educational innovation projects since 2016, aimed at developing of materials to improve the communication skills of scientific results. An exhaustive and updated compilation of the international rules that constitute the basis for the writted and oral scientific presentations was carried out. The good teaching practices in these fields were also identified. The results of those previous projects have shown the need to incorporate web questionnaires and other interactive content into the educational program. These are adapted to the demands of the students and provide a training feeback. In this contribution, the new materials that are being developed within the innovation project UV-SFPIE_PID19-1096780, funded by the University of Valencia, are presented. They are devoted to facilitate the acquisition of communication skills of scientific results. In particular, these tools combine ICT self-learning environments with traditional classroom teaching (blended learning). The project methodology includes educational data mining aimed at identifying the most effective materials and activities to achieve its objectives. The aim of these mixed learning tools is to facilitate the acquisition by the students of the necessary skills of oral and written communication, improve their presentation skills and, consequently, also their employability as university graduates.This work has been supported by the University of Valencia through project SFPIE_PID19-1096780.Campos-Taberner, M.; Gilabert, M.; Manzanares, J.; Mafé, S.; Cervera, J.; García-Haro, F.; Martínez, B.... (2020). On-line tools to improve the presentation skills of scientific results. IATED. 4907-4910. https://doi.org/10.21125/inted.2020.1342S4907491

Contributions of amino acid, acylcarnitine and sphingolipid profiles to type 2 diabetes risk among South-Asian Surinamese and Dutch adults

Introduction: People of South Asian origin are at high risk of type 2 diabetes (T2D), but the underpinning mechanisms are not fully understood. We determined ethnic differences in acylcarnitine, amino acid and sphingolipid concentrations and determined the associations with T2D. Research design and methods: Associations between these metabolites and incident T2D among Dutch and South-Asian Surinamese were determined in participants from the Healthy Life in an Urban Setting (HELIUS) study (Amsterdam, the Netherlands) using Prentice-weighted Cox regression. The HELIUS study includes 95 incident T2D cases and a representative subcohort of 700 people from a cohort of 5977 participants with a mean follow-up of 4 years. Results: Concentrations of acylcarnitines were comparable between both ethnic groups. Amino acid and lactosylceramide concentrations were higher among South-Asian Surinamese than Dutch (eg, isoleucine 65.7 (SD 16.3) vs 60.7 (SD 15.6) µmol/L). Ceramide concentrations were lower among South-Asian Surinamese than Dutch (eg, Cer d18:1 8.48 (SD 2.04) vs 9.08 (SD 2.29) µmol/L). Metabolic dysregulation preceded T2D without evidence for a multiplicative interaction by ethnicity. Most amino acids and (dihydro)ceramides were associated with increased risk (eg, Cer d18:1 HR 2.38, 95% CI 1.81 to 3.12) while acylcarnitines, glycine, glutamine and lactosylceramides were associated with decreased risk for T2D (eg, LacCer d18:2 HR 0.56, 95% CI 0.42 to 0.77). Conclusions: Overall, these data suggest that the disturbances underlying amino acid and sphingolipid metabolism may be predictive of T2D risk in populations of both South Asian and European background. These observations may be used as starting point to unravel the underlying metabolic disturbances