Computational Design of Protein-Based Inhibitors of Plasmodium vivax Subtilisin-Like 1 Protease

Background

Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target.

Findings

Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range.

Conclusions

Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications.     

Comparison of Microbiological,Histological, and Immunomodulatory Parameters in Response to Treatment with Either Combination Therapy with Prednisone and Metronidazole or Probiotic VSL#3 Strains in Dogs with Idiopathic Inflammatory Bowel Disease

Background

Idiopathic inflammatory bowel disease (IBD) is a common chronic enteropathy in dogs. There are no published studies regarding the use of probiotics in the treatment of canine IBD. The objectives were to compare responses to treatment with either combination therapy (prednisone and metronidazole) or probiotic strains (VSL#3) in dogs with IBD.

Methodology and Principal Findings

Twenty pet dogs with a diagnosis of IBD, ten healthy pet dogs, and archived control intestinal tissues from three euthanized dogs were used in this open label study. Dogs with IBD were randomized to receive either probiotic (D-VSL#3, n = 10) or combination drug therapy (D-CT, n = 10). Dogs were monitored for 60 days (during treatment) and re-evaluated 30 days after completing treatment. The CIBDAI (P<0.001), duodenal histology scores (P<0.001), and CD3+ cells decreased post-treatment in both treatment groups. FoxP3+ cells (p<0.002) increased in the D-VSL#3 group after treatment but not in the D-CT group. TGF-β+ cells increased in both groups after treatment (P = 0.0043) with the magnitude of this increase being significantly greater for dogs in the D-VSL#3 group compared to the D-CT group. Changes in apical junction complex molecules occludin and claudin-2 differed depending on treatment. Faecalibacterium and Turicibacter were significantly decreased in dogs with IBD at T0, with a significant increase in Faecalibacterium abundance observed in the animals treated with VSL#3 strains.

Conclusions

A protective effect of VSL#3 strains was observed in dogs with IBD, with a significant decrease in clinical and histological scores and a decrease in CD3+ T-cell infiltration. Protection was associated with an enhancement of regulatory T-cell markers (FoxP3+ and TGF-β+), specifically observed in the probiotic-treated group and not in animals receiving combination therapy. A normalization of dysbiosis after long-term therapy was observed in the probiotic group. Larger scale studies are warranted to evaluate the clinical efficacy of VSL#3 in canine IBD.     

How Cheap Is Soaring Flight in Raptors? A Preliminary Investigation in Freely-Flying Vultures

Measuring the costs of soaring, gliding and flapping flight in raptors is challenging, but essential for understanding their ecology. Among raptors, vultures are scavengers that have evolved highly efficient soaring-gliding flight techniques to minimize energy costs to find unpredictable food resources. Using electrocardiogram, GPS and accelerometer bio-loggers, we report the heart rate (HR) of captive griffon vultures (Gyps fulvus and G. himalayensis) trained for freely-flying. HR increased three-fold at take-off (characterized by prolonged flapping flight) and landing (>300 beats-per-minute, (bpm)) compared to baseline levels (80–100 bpm). However, within 10 minutes after the initial flapping phase, HR in soaring/gliding flight dropped to values similar to baseline levels, i.e. slightly lower than theoretically expected. However, the extremely rapid decrease in HR was unexpected, when compared with other marine gliders, such as albatrosses. Weather conditions influenced flight performance and HR was noticeably higher during cloudy compared to sunny conditions when prolonged soaring flight is made easier by thermal ascending air currents. Soaring as a cheap locomotory mode is a crucial adaptation for vultures who spend so long on the wing for wide-ranging movements to find food.     

Jellyfish as Prey: Frequency of Predation and Selective Foraging of Boops boops (Vertebrata,Actinopterygii) on the Mauve Stinger Pelagia noctiluca (Cnidaria,Scyphozoa)

In recent years, jellyfish blooms have attracted considerable scientific interest for their potential impacts on human activities and ecosystem functioning, with much attention paid to jellyfish as predators and to gelatinous biomass as a carbon sink. Other than qualitative data and observations, few studies have quantified direct predation of fish on jellyfish to clarify whether they may represent a seasonally abundant food source. Here we estimate predation frequency by the commercially valuable Mediterranean bogue, Boops boops on the mauve stinger jellyfish, Pelagia noctiluca, in the Strait of Messina (NE Sicily). A total of 1054 jellyfish were sampled throughout one year to quantify predation by B. boops from bite marks on partially eaten jellyfish and energy density of the jellyfish. Predation by B. boops in summer was almost twice that in winter, and they selectively fed according to medusa gender and body part. Calorimetric analysis and biochemical composition showed that female jellyfish gonads had significantly higher energy content than male gonads due to more lipids and that gonads had six-fold higher energy content than the somatic tissues due to higher lipid and protein concentrations. Energetically, jellyfish gonads represent a highly rewarding food source, largely available to B. boops throughout spring and summer. During the remainder of the year, when gonads were not very evident, fish predation switched towards less-selective foraging on the somatic gelatinous biomass. P. noctiluca, the most abundant jellyfish species in the Mediterranean Sea and a key planktonic predator, may represent not only a nuisance for human leisure activities and a source of mortality for fish eggs and larvae, but also an important resource for fish species of commercial value, such as B. boops.     

Pairwise binding competition experiments for sorting hub-protein/effector interaction hierarchy and simultaneous equilibria

NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide a tool to understand complex biological interaction networks. Competition among proteins for binding to signaling hubs is often at the basis of the information transmission across signaling networks in every organism. Changes in affinity towards one or more partners, as well as changes of the relative concentration of the competing partners, can determine pathways alterations that lead to pathological consequences. Overall, the knowledge of the interaction hierarchy of the multiple partners to a single signaling hub can lead to new therapeutic strategies. Smith and Ikura (Nat Chem Biol 10:223–230, 2014) have recently proposed pairwise competition NMR experiments to determine the binding hierarchy in network interactions. We have taken the moves from their approach to show how from pairwise competition NMR experiments the ratios between the equilibrium constants for multiple binding partners can be determined, and thus, given their concentration in solution, the concentrations of all the possible complexes can be obtained.     

Transglutaminase activity in primary and subcultured rat astroglial cells

Transglutaminases, calcium-dependent thiol enzymes, may be involved in cellular growth control and differentiation, having an intracellular regulatory role in some post-traslational modifications found in various classes of proteins. In order to elucidate the involvement of this class of enzymes in cellular differentiation processes, we have assayed transglutaminase activity in primary and subcultured rat glial cells. Reduced activity was found from 3rd to 5th passage. In the 5th passage the activity was some 50% of that found in the primary cultures and was not restored by addition of 10 M retinoic acid. The decrease of TGase activity, observed during serial passages, could represent an early metabolic alteration related to cell dedifferentiation and loss of growth control. In fact, the subcultured cells may have undergone a disarranged state, as confirmed by a decrease in GFAP-stained cells and glutamine synthetase activity, respectively, immunocytochemical and biochemical markers of astroglial cells.     

Herbivory, connectivity, and ecosystem resilience: response of a coral reef to a large-scale perturbation

Coral reefs world-wide are threatened by escalating local and global impacts, and some impacted reefs have shifted from coral dominance to a state dominated by macroalgae. Therefore, there is a growing need to understand the processes that affect the capacity of these ecosystems to return to coral dominance following disturbances, including those that prevent the establishment of persistent stands of macroalgae. Unlike many reefs in the Caribbean, over the last several decades, reefs around the Indo-Pacific island of Moorea, French Polynesia have consistently returned to coral dominance following major perturbations without shifting to a macroalgae-dominated state. Here, we present evidence of a rapid increase in populations of herbivorous fishes following the most recent perturbation, and show that grazing by these herbivores has prevented the establishment of macroalgae following near complete loss of coral on offshore reefs. Importantly, we found the positive response of herbivorous fishes to increased benthic primary productivity associated with coral loss was driven largely by parrotfishes that initially recruit to stable nursery habitat within the lagoons before moving to offshore reefs later in life. These results underscore the importance of connectivity between the lagoon and offshore reefs for preventing the establishment of macroalgae following disturbances, and indicate that protecting nearshore nursery habitat of herbivorous fishes is critical for maintaining reef resilience.     

Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from dehydrocholic acid. The specific reduction of dehydrocholic with NADH was catalysed by 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)⁺ oxidoreductase, EC 1.1.1.50) and 7α-hydroxysteroid dehydrogenase (7α-hydroxysteroid:NAD⁺ 7-oxidoreductase, EC 1.1.1.159). Cofactor regeneration was obtained through the formate dehydrogenase (formate:NAD⁺ oxidoreductase, EC 1.2.1.2) catalysed oxidation of formate. Complete transformation of dehydrocholic acid to the 12-keto derivative was achieved with a coenzyme turnover number up to 1200. No steroid by-products were detected by high performance liquid chromatography and thin layer chromatography. The process yielded 9 g product l^?1 in 66–84 h. The high purity of the enzymatically prepared 12-ketochenodeoxycholic acid should drastically reduce the formation of the toxic by-product lithocholic acid, which occurs in the synthesis of chenodeoxycholic acid when using chemical methods alone.