Shockwave Treatment vs Surgery for Proximal Fifth Metatarsal Stress Fractures in Soccer Players: A Pilot Study.

BACKGROUND: To compare the clinical, radiologic, and functional outcomes between shockwave and operative treatments for proximal fifth metatarsal stress fractures in soccer players in a pilot study. METHODS: Between 2017 and 2019, 18 soccer players with fifth metatarsal stress fractures attended at Mutualidad de Futbolistas Españoles-Delegación Catalana were included. Patients were randomly assigned into 2 groups receiving either surgery with an intramedullary screw (group 1) or high-energy focused extracorporeal shockwave treatment (group 2 performed once a week for 3 weeks using 2000 impulses at an energy flux density of 0.21 mJ/mm2 and 4 Hz frequency). Clinical (pain), radiologic (bone healing), and functional (Tegner Activity Scale and American Orthopaedic Foot & Ankle Society [AOFAS] ankle-hindfoot scales) outcomes before and after receiving the treatment were compared between both groups. In addition, ability and time to return to play was also compared between groups. RESULTS: No patients were lost to follow-up. There were no statistically significant differences at last follow-up between surgery and extracorporeal shockwave treatment for bone healing, pain relief, AOFAS ankle-hindfoot score, Tegner score, and time return to play. No complications were reported in either of the 2 groups. CONCLUSION: In this pilot study, extracorporeal shockwave treatment and operative treatment were found to be equally effective at reducing pain, achieving bone healing, and allowing the soccer players to return to play after proximal fifth metatarsal stress fractures. This study suggests that ESWT may be a good option for the management of proximal fifth metatarsal stress fractures in soccer players. If this approach proves successful in larger trials, the shockwave approach might help avoid known complications of the surgical treatment like wound problems, nerve injury, and hardware intolerance. Further investigations with larger sample size should be conducted in order to confirm the present conclusions. LEVEL OF EVIDENCE: Level II, therapeutic, pilot randomized controlled trial.

Decarboxylation of Ang-(1-7) to Ala1-Ang-(1-7) leads to significant changes in pharmacodynamics.

The heptapeptide angiotensin (Ang)-(1-7) is part of the beneficial arm of the renin-angiotensin system. Ang-(1-7) has cardiovascular protective effects, stimulates regeneration, and opposes the often detrimental effects of AngII. We recently identified the G protein-coupled receptors Mas and MrgD as receptors for the heptapeptide. Ala1-Ang-(1-7) (Alamandine), a decarboxylated form of Ang-(1-7), has similar vasorelaxant effects, but has been described as only stimulating MrgD. Therefore, this study aimed to characterise the consequences of the lack of the carboxyl group in amino acid 1 on intracellular signalling and to identify the receptor fingerprint for Ala1-Ang-(1-7). In primary endothelial and mesangial cells, Ala1-Ang-(1-7) elevated cAMP concentration. Dose response curves generated with Ang-(1-7) and Ala1-Ang-(1-7) significantly differed from each other, with a much lower EC50 and a bell-shape curve for Ala1-Ang-(1-7). We provided pharmacological proof that both, Mas and MrgD, are functional receptors for Ala1-Ang-(1-7). Consequently, in primary mesangial cells with genetic deficiency in both receptors, the heptapeptide failed to increase cAMP concentration. As we previously described for Ang-(1-7), the Ala1-Ang-(1-7)-mediated cAMP increase in Mas/MrgD-transfected HEK293 cells and primary cells was blocked by the AT2 receptor blocker, PD123319. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, electrostatic potential calculations, and an involvement of Galpha i for higher concentrations of Ala1-Ang-(1-7). Our results identify Ala1-Ang-(1-7) as a peptide with specific pharmacodynamic properties and builds the basis for the design of more potent and efficient Ang-(1-7) analogues for therapeutic intervention in a rapidly growing number of diseases.

Identification of PPARgamma partial agonists of natural origin (II): in silico prediction in natural extracts with known antidiabetic activity.

BACKGROUND: Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action. METHODOLOGY/PRINCIPAL FINDINGS: From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (i.e., they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature. CONCLUSIONS/SIGNIFICANCE: Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the prevention/treatment of type 2 Diabetes Mellitus.

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.

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.

Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (part I): virtual screening and activity assays.

BACKGROUND: There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. METHODOLOGY/PRINCIPAL FINDINGS: We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.

Development of docking-based 3D-QSAR models for PPARgamma full agonists.

Peroxisome proliferator-activated receptor gamma (PPARγ) has become an attractive molecular target for drugs that aim to treat diabetes mellitus type II, and its therapeutic potency against skin cancer and other skin diseases is also currently being explored. To study the relationship between the structure of several PPARγ full agonists and the trans-activation activity of PPARγ, we have performed a three-dimensional quantitative structure-activity relationship (3D-QSAR) study of tyrosine-based derivatives, based on the 3D alignment of conformations obtained by docking. Highly predictive 3D-QSAR models, with Pearson-R values of 0.86 and 0.90, were obtained for the transactivation activity and binding affinity of PPARγ, respectively. These models are in good agreement with the structural characteristics of the binding pocket of PPARγ and provide some structural insights for the improvement of PPARγ full agonist bioactivities.

Identification of human IKK-2 inhibitors of natural origin (Part II): in Silico prediction of IKK-2 inhibitors in natural extracts with known anti-inflammatory activity.

Human inhibitor NF-κB kinase 2 (hIKK-2) is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Thus, synthetic ATP-competitive inhibitors for hIKK-2 have been developed as anti-inflammatory compounds. We recently reported a virtual screening protocol (doi:10.1371/journal.pone.0016903) that is able to identify hIKK-2 inhibitors that are not structurally related to any known molecule that inhibits hIKK-2 and that have never been reported to have anti-inflammatory activity. In this study, a stricter version of this protocol was applied to an in-house database of 29,779 natural products annotated with their natural source. The search identified 274 molecules (isolated from 453 different natural extracts) predicted to inhibit hIKK-2. An exhaustive bibliographic search revealed that anti-inflammatory activity has been previously described for: (a) 36 out of these 453 extracts; and (b) 17 out of 30 virtual screening hits present in these 36 extracts. Only one of the remaining 13 hit molecules in these extracts shows chemical similarity with known synthetic hIKK-2 inhibitors. Therefore, it is plausible that a significant portion of the remaining 12 hit molecules are lead-hopping candidates for the development of new hIKK-2 inhibitors.

Structural insights for the design of new PPARgamma partial agonists with high binding affinity and low transactivation activity.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) full agonists are molecules with powerful insulin-sensitizing action that are used as antidiabetic drugs. Unfortunately, these compounds also present various side effects. Recent results suggest that effective PPARγ agonists should show a low transactivation activity but a high binding affinity to inhibit phosphorylation at Ser273. We use several structure activity relationship studies of synthetic PPARγ agonists to explore the different binding features of full and partial PPARγ agonists with the aim of differentiating the features needed for binding and those needed for the transactivation activity of PPARγ. Our results suggest that effective partial agonists should have a hydrophobic moiety and an acceptor site with an appropriate conformation to interact with arm II and establish a hydrogen bond with Ser342 or an equivalent residue at arm III. Despite the fact that interactions with arm I increase the binding affinity, this region should be avoided in order to not increase the transactivation activity of potential PPARγ partial agonists.

Identification of human IKK-2 inhibitors of natural origin (part I): modeling of the IKK-2 kinase domain, virtual screening and activity assays.

BACKGROUND: Their large scaffold diversity and properties, such as structural complexity and drug similarity, form the basis of claims that natural products are ideal starting points for drug design and development. Consequently, there has been great interest in determining whether such molecules show biological activity toward protein targets of pharmacological relevance. One target of particular interest is hIKK-2, a serine-threonine protein kinase belonging to the IKK complex that is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Indeed, this has led to the development of synthetic ATP-competitive inhibitors for hIKK-2. Therefore, the main goals of this study were (a) to use virtual screening to identify potential hIKK-2 inhibitors of natural origin that compete with ATP and (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits. METHODOLOGY/PRINCIPAL FINDINGS: We thus predicted that 1,061 out of the 89,425 natural products present in the studied database would inhibit hIKK-2 with good ADMET properties. Notably, when these 1,061 molecules were merged with the 98 synthetic hIKK-2 inhibitors used in this study and the resulting set was classified into ten clusters according to chemical similarity, there were three clusters that contained only natural products. Five molecules from these three clusters (for which no anti-inflammatory activity has been previously described) were then selected for in vitro activity testing, in which three out of the five molecules were shown to inhibit hIKK-2. CONCLUSIONS/SIGNIFICANCE: We demonstrated that our virtual-screening protocol was successful in identifying lead compounds for developing new inhibitors for hIKK-2, a target of great interest in medicinal chemistry. Additionally, all the tools developed during the current study (i.e., the homology model for the hIKK-2 kinase domain and the pharmacophore) will be made available to interested readers upon request.

Dietary procyanidins enhance transcriptional activity of bile acid-activated FXR in vitro and reduce triglyceridemia in vivo in a FXR-dependent manner.

Consumption of dietary flavonoids has been associated with reduced mortality and risk of cardiovascular disease, partially by reducing triglyceridemia. We have previously reported that a grape seed procyanidin extract (GSPE) reduces postprandial triglyceridemia in normolipidemic animals signaling through the orphan nuclear receptor small heterodimer partner (SHP) a target of the bile acid receptor farnesoid X receptor (FXR). Our aim was to elucidate whether FXR mediates the hypotriglyceridemic effect of procyanidins. In FXR-driven luciferase expression assays GSPE dose-dependently enhanced FXR activity in the presence of chenodeoxycholic acid. GSPE gavage reduced triglyceridemia in wild type mice but not in FXR-null mice, revealing FXR as an essential mediator of the hypotriglyceridemic actions of procyanidins in vivo. In the liver, GSPE downregulated, in an FXR-dependent manner, the expression of the transcription factor steroid response element binding protein 1 (SREBP1) and several SREBP1 target genes involved in lipogenesis, and upregulated ApoA5 expression. Altogether, our results indicate that procyanidins lower triglyceridemia following the same pathway as bile acids: activation of FXR, transient upregulation of SHP expression and subsequent downregulation of SREBP1 expression. This study adds dietary procyanidins to the arsenal of FXR ligands with potential therapeutic use to combat hypertriglyceridemia, type 2 diabetes and metabolic syndrome.

In vivo, in vitro, and in silico studies of Cu/Zn-superoxide dismutase regulation by molecules in grape seed procyanidin extract.

The potential beneficial effects of flavonoids on human health have aroused considerable interest and were initially attributed to their antioxidant activities. Recent studies have speculated that as well as their antioxidant role, flavonoids can act by modulating cell signaling pathways and/or gene expression. In this respect, we have used streptozotocin-induced diabetic rats as an oxidative stress model to study whether grape seed procyanidin extract (GSPE) regulates copper/zinc-superoxide dismutase (Cu/Zn-SOD), an enzyme that defends against oxidative stress. The results indicate that the expression profile of Cu/Zn-SOD in diabetic rats was similar to the profile in nondiabetic rats. Nevertheless, the administration of GSPE increased Cu/Zn-SOD activity in both diabetic and nondiabetic rats. Therefore, to evaluate whether this increase in activity was dose-dependent, we also studied the effect of GSPE on Cu/Zn-SOD expression by using an in vitro model (Fao cell line hepatocytes). The cells were exposed to GSPE doses between 0 and 150 mg/L for 24 h, and the results showed that enzyme activity was enhanced only with 15 mg/L of GSPE. Therefore, we decided to explore whether this increase in Cu/Zn-SOD activity was due to direct interaction between some of the molecules in GSPE and the enzyme (in vitro experiments) and, if so, to analyze how this interaction occurs (in silico experiments). The results of these studies showed that direct interaction between some small- or medium-sized GSPE components and the enzyme is responsible for the increase in Cu/Zn-SOD activity.

Hernia obturadora recidivada en paciente de 85 años / Recurrent obturator hernia in an 85-year-old patient

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