Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part II): In Silico Prediction in Antidiabetic Extracts

Background

Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV.

Methodology/Principal Findings

Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012) [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity). Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors.

Conclusions/Significance

Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.     

Moderate red-wine consumption partially prevents body weight gain in rats fed a hyperlipidic diet

Red wine is a beverage that can exert a broad spectrum of health-promoting actions both in humans and laboratory animal models if consumed moderately. However, information about its effect on body weight is scarce. We have evaluated the effect of moderate red wine consumption on body weight and energy intake in male Zucker lean rats fed a hypercaloric diet for 8 weeks. For this purpose, we used three 5-animal groups: a high-fat diet group (HFD), a high-fat-diet red-wine-drinking group (HFRWD), and a standard diet group (SD). After 8 weeks, the HFRWD group had a lower body weight gain (175.66 +/- 2.78% vs 188.22 +/- 4.83%; P<.05) and lower energy intake (269.45 +/- 4.02 KJ/animal.day vs day vs 300.81 +/- 4.52 KJ/animal.day; P<.05) and had less fat mass at epididymal location respect to the whole body weight (0.014 +/- 0.001 vs 0.017 +/- 0.001; P<.05) than the HFD group. However, the red wine didn't modified the fed efficiency 0.012 +/- 0.001 g/KJ for HFRWD group versus 0.013 +/- 0.001 g/KJ for the HFD one (P=.080). These findings, though preliminary, show that moderate red wine intake can prevent the increase of body weight by modulating energy intake in a rat diet-induced model of obesity.     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

Dispersion of the HIV-1 Epidemic in Men Who Have Sex with Men in the Netherlands: A Combined Mathematical Model and Phylogenetic Analysis

Background

The HIV-1 subtype B epidemic amongst men who have sex with men (MSM) is resurgent in many countries despite the widespread use of effective combination antiretroviral therapy (cART). In this combined mathematical and phylogenetic study of observational data, we aimed to find out the extent to which the resurgent epidemic is the result of newly introduced strains or of growth of already circulating strains.

Methods and Findings

As of November 2011, the ATHENA observational HIV cohort of all patients in care in the Netherlands since 1996 included HIV-1 subtype B polymerase sequences from 5,852 patients. Patients who were diagnosed between 1981 and 1995 were included in the cohort if they were still alive in 1996. The ten most similar sequences to each ATHENA sequence were selected from the Los Alamos HIV Sequence Database, and a phylogenetic tree was created of a total of 8,320 sequences. Large transmission clusters that included ≥10 ATHENA sequences were selected, with a local support value ≥ 0.9 and median pairwise patristic distance below the fifth percentile of distances in the whole tree. Time-varying reproduction numbers of the large MSM-majority clusters were estimated through mathematical modeling. We identified 106 large transmission clusters, including 3,061 (52%) ATHENA and 652 Los Alamos sequences. Half of the HIV sequences from MSM registered in the cohort in the Netherlands (2,128 of 4,288) were included in 91 large MSM-majority clusters. Strikingly, at least 54 (59%) of these 91 MSM-majority clusters were already circulating before 1996, when cART was introduced, and have persisted to the present. Overall, 1,226 (35%) of the 3,460 diagnoses among MSM since 1996 were found in these 54 long-standing clusters. The reproduction numbers of all large MSM-majority clusters were around the epidemic threshold value of one over the whole study period. A tendency towards higher numbers was visible in recent years, especially in the more recently introduced clusters. The mean age of MSM at diagnosis increased by 0.45 years/year within clusters, but new clusters appeared with lower mean age. Major strengths of this study are the high proportion of HIV-positive MSM with a sequence in this study and the combined application of phylogenetic and modeling approaches. Main limitations are the assumption that the sampled population is representative of the overall HIV-positive population and the assumption that the diagnosis interval distribution is similar between clusters.

Conclusions

The resurgent HIV epidemic amongst MSM in the Netherlands is driven by several large, persistent, self-sustaining, and, in many cases, growing sub-epidemics shifting towards new generations of MSM. Many of the sub-epidemics have been present since the early epidemic, to which new sub-epidemics are being added.     

On the biophysics and kinetics of toehold-mediated DNA strand displacement

Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.     

Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

Background  

For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.     

Identification of PPARgamma Partial Agonists of Natural Origin (I): Development of a Virtual Screening Procedure and In Vitro Validation

Background

Although there are successful examples of the discovery of new PPARγ agonists, it has recently been of great interest to identify new PPARγ partial agonists that do not present the adverse side effects caused by PPARγ full agonists. Consequently, the goal of this work was to design, apply and validate a virtual screening workflow to identify novel PPARγ partial agonists among natural products.

Methodology/Principal Findings

We have developed a virtual screening procedure based on structure-based pharmacophore construction, protein-ligand docking and electrostatic/shape similarity to discover novel scaffolds of PPARγ partial agonists. From an initial set of 89,165 natural products and natural product derivatives, 135 compounds were identified as potential PPARγ partial agonists with good ADME properties. Ten compounds that represent ten new chemical scaffolds for PPARγ partial agonists were selected for in vitro biological testing, but two of them were not assayed due to solubility problems. Five out of the remaining eight compounds were confirmed as PPARγ partial agonists: they bind to PPARγ, do not or only moderately stimulate the transactivation activity of PPARγ, do not induce adipogenesis of preadipocyte cells and stimulate the insulin-induced glucose uptake of adipocytes.

Conclusions/Significance

We have demonstrated that our virtual screening protocol was successful in identifying novel scaffolds for PPARγ partial agonists.     

Rainfall-Driven Amplification of Seasonal Acidification in Poorly Buffered Tropical Streams

Acidification in freshwater ecosystems has important ecological and biogeochemical effects. Temperate streams affected by anthropogenic acidification have been extensively studied, but our understanding of natural acidification in tropical streams has been constrained by the lack of long-term datasets. Here, we analyze 14?years of monthly observations from 13 sampling stations in eight tropical streams in lowland Costa Rica. Stream pH increased during the 4-month dry season and declined throughout the wet season. The magnitude of the seasonal pH decline was greatest following the driest dry seasons, including the historically large El Ni?o Southern Oscillation event in 1998 when pH values dropped below 4.0 in some streams. Dissolved CO₂ accounts for the low baseline pH in the poorly buffered study streams, and we hypothesize that an influx of soil-derived CO₂ via subsurface flow paths contributes to the observed seasonal pH declines. Our results show tight coupling between rainfall, terrestrial, and aquatic ecosystems in the tropics. Predicted decreases in dry season rainfall for the tropics may lead to an increased magnitude of seasonal acidification.     

Evolutionary relationships of the carbamoylphosphate synthetase genes

Carbamoylphosphate is a common intermediate in the metabolic pathways leading to the biosynthesis of arginine and pyrimidines. The amino acid sequences of all available proteins that catalyze the formation of carbamoylphosphate were retrieved from Genbank and aligned to estimate their mutual phylogenetic relations. In gram-negative bacteria carbamoylphosphate is synthesized by a two-subunit enzyme with glutamiriase and carbamoylphosphate synthetase (CPS) activity, respectively. In gram-positive bacteria and lower eukaryotes this two-subunit CPS has become dedicated to arginine biosynthesis, while in higher eukaryotes the two subunits fused and subsequently lost the glutaminase activity. The CPS dedicated to pyrimidine synthesis is part of a multifunctional enzyme (CPS II), encoding in addition dihydroorotase and aspartate transcarbamoylase. Evidence is presented to strengthen the hypothesis that the two kinas subdomains of all CPS isozymes arose from a duplication of an ancestral gene in the progenote. A further duplication of the entire CPS gene occurred after the divergence of the plants and before the divergence of the fungi from the eukaryotec root, generating the two isoenzymes involved in either the synthesis of arginine or that of pyrimidines. The mutation rate was found to be five- to tenfold higher after the duplication than before, probably reflecting optimization of the enzymes for their newly acquired specialized function. We hypothesize that this duplication arose from a need for metabolic channeling for pyrimidine biosynthesis as it was accompanied by the tagging of the CPS gene with the genes for dihydroorotase and aspartate transcarbamoylase, and as the duplication occurred independently also in gram-positive bacteria. Analysis of the exon-intron organization of the two kinase subdomains in CPS I and II suggests that ancient exons may have comprised approx. 19 amino acids, in accordance with the prediction of the intron-early theory. Correspondence to: M.J.B. van den Hoff