Structural Optimization and De Novo Design of Dengue Virus Entry Inhibitory Peptides

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding “pseudoenergies”, we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 µM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery.     

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi’s virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.     

Endogenous cytokinin dynamics in micropropagated tulips during bulb formation process influenced by TDZ and iP pretreatment

There are substantial variations in bulbing (bulb formation) efficiency among micropropagated tulip cultivars. The mechanisms involved are poorly understood, but presumably involve cytokinins (CKs) for several reasons. Therefore, we explored CK profiles and dynamics in ‘Blue Parrot’ and ‘Prominence’ cultivars (which have low and high bulbing efficiency, respectively) during the in vitro propagation stages: the last shoot multiplication subculture extended to 14 weeks (S1–S2), the shoot cooling at 5 °C for induction of bulb formation (S3–S4) and the bulb growth initiation after the end of cooling (S5–S6). The CK thidiazuron (TDZ) is routinely used in tulip micropropagation at the shoot multiplication stage, but replacing it with isopentenyladenine (iP) during the last multiplication subculture substantially changed CK dynamics in later stages, and significantly increased bulb formation rates in both cultivars. Generally, the most abundant CKs in both cultivars were the isoprenoid CK types, trans-zeatin (tZ), iP, cis-zeatin and dihydrozeatin. However, ‘Prominence’ shoots had much lower cis- to trans-Z-type CK ratios than ‘Blue Parrot’ shoots, and generally higher levels of physiologically active CKs (free bases tZ, iP and their ribosides) until the last phase of bulb formation, S6 (bulb growth initiation, i.e. swelling of shoot bases), 6 weeks after the end of cold treatment. In this phase total active CK and O-glucoside contents sharply declined in ‘Prominence’ shoots, but not in ‘Blue Parrot’ shoots pretreated with iP. In contrast, the low bulbing ability observed in ‘Prominence’ shoots pretreated with TDZ and ‘Blue Parrot’ shoots pretreated with either TDZ or iP was associated with a gradual rise in active CK and O-glucoside contents after the end of cooling. The results suggest that low bulbing efficiency may be related to down-regulation of tZ biosynthesis, and high bulbing efficiency to a transient increase in active CK forms (mainly tZs) in response to cold treatment during the bulb induction phase, S4 (at the end of cold treatment), followed by a rapid decrease during bulb formation, S6 (6 weeks after the end of cooling).     

Genetic mapping of DArT markers in the Festuca�CLolium complex and their use in freezing tolerance association analysis

Species belonging to the Festuca-Lolium complex are important forage and turf species and as such, have been studied intensively. However, their out-crossing nature and limited availability of molecular markers make genetic studies difficult. Here, we report on saturation of F. pratensis and L. multiflorum genetic maps using Diversity Array Technology (DArT) markers and the DArTFest array.The 530 and 149 DArT markers were placed on genetic maps of L. multiflorum and F. pratensis, respectively, with overlap of 20 markers, which mapped in both species. The markers were sequenced and comparative sequence analysis was performed between L. multiflorum, rice and Brachypodium. The utility of the DArTFest array was then tested on a Festulolium population FuRs0357 in an integrated analysis using the DArT marker map positions to study associations between markers and freezing tolerance. Ninety six markers were significantly associated with freezing tolerance and five of these markers were genetically mapped to chromosomes 2, 4 and 7. Three genomic loci associated with freezing tolerance in the FuRs0357 population co-localized with chromosome segments and QTLs previously identified to be associated with freezing tolerance. The present work clearly confirms the potential of the DArTFest array in genetic studies of the Festuca-Lolium complex. The annotated DArTFest array resources could accelerate further studies and improvement of desired traits in Festuca-Lolium species.     

Cushions of Thylacospermum caespitosum (Caryophyllaceae) do not facilitate other plants under extreme altitude and dry conditions in the north-west Himalayas

Background

Cushion plants are commonly considered as keystone nurse species that ameliorate the harsh conditions they inhabit in alpine ecosystems, thus facilitating other species and increasing alpine plant biodiversity. A literature search resulted in 25 key studies showing overwhelming facilitative effects of different cushion plants and hypothesizing greater facilitation with increased environmental severity (i.e. higher altitude and/or lower rainfall). At the same time, emerging ecological theory alongside the cushion-specific literature suggests that facilitation might not always occur under extreme environmental conditions, and especially under high altitude and dryness.

Methods

To assess these hypotheses, possible nursing effects of Thylacospermum caespitosum (Caryophyllaceae) were examined at extremely high altitude (5900 m a.s.l.) and in dry conditions (precipitation <100 mm year⁻¹) in Eastern Ladakh, Trans-Himalaya. This is, by far, the highest site, and the second driest, at which the effects of cushions have been studied so far.

Key Results

In accordance with the theoretical predictions, no nursing effects of T. caespitosum on other alpine plants were detected. The number and abundance of species were greater outside cushions than within and on the edge of cushions. None of the 13 species detected was positively associated with cushions, while nine of them were negatively associated. Plant diversity increased with the size of the area sampled outside cushions, but no species–area relationship was found within cushions.

Conclusions

The results support the emerging theoretical prediction of restricted facilitative effects under extreme combinations of cold and dryness, integrating these ideas in the context of the ecology of cushion plants. This evidence suggests that cases of missing strong facilitation are likely to be found in other extreme alpine conditions.     

Phylogenetic diversity of fungal communities in areas accessible and not accessible to tourists in Naracoorte Caves

The fungal diversity in areas accessible and not accessible to tourists at UNESCO World Heritage-listed Naracoorte Caves was investigated with culture-dependent and culture-independent techniques for assistance in cave management protocol development. The caves were selected based on tourist numbers and configurations: Stick Tomato (open, high numbers), Alexandra (lockable openings, high numbers) and Strawhaven (control; no access). Culture-based survey revealed Ascomycota dominance irrespective of sampling area with Microascales (Trichurus sp.) being most frequently isolated. Some Hypocreales-like sequences belonging to Fusarium sp., Trichoderma sp. and Neonectria sp. (Stick Tomato) were cultured only from areas not accessible to tourists. These orders also were detected by DGGE assay irrespective of sampling area. The predominance of Ascomycota (especially Microascales) suggested their important ecological roles in these caves. Culture-independent analysis showed higher Shannon fungal diversity values (from ITS-based DGGE profiles) in tourist-accessible areas of these caves than in inaccessible areas with the fungal community banding patterns being substantially different in Stick Tomato Cave. Further investigations are needed to determine the cause of the differences in the fungal communities of Stick Tomato Cave, although cave-related factors such as use, configuration and sediment heterogeneity might have contributed to these differences.     

Multiple,concentration-dependent effects of sucrose,auxins and cytokinins in explant cultures of kale and tobacco

We describe complex multiple concentration dependencies for the response of isolated pith tissues to plant biologically active substances. Kale and tobacco stem pith explants were cultured on agar media containing combinations of sucrose, cytokinin [kinetin or benzyladenine (BA)] and auxin [indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA)]. Absorption of these components by explants and their effects on explant mass, contents of soluble proteins, starch and sugars, and activity of ADP-glucose pyrophosphorylase (AGPase) were studied in relation to their concentration. Up to ten pronounced statistically significant maxima (peaks or waves) were repeatedly detected in the dose–response curves over a concentration range of several logarithmic orders. Slight maxima were observed in the corresponding absorption curves. Pronounced maxima of sucrose absorption were induced by IAA and BA, and those of NAA absorption were induced by sucrose. Both types of multiple maxima (in dose–response and absorption curves) may be due to changes in concentration of intracellular solutes (sugars, auxins and cytokinins), thereby affecting metabolic processes that act as sinks for external solutes and elicit feedback appearance of maxima in absorption curves. Good correspondence between external concentrations at which maxima of different compared curves occur in addition to statistical significance of individual maxima and repeatability of experimental results supports the conclusion that the multiple maxima exhibited are genuine. We consider it possibile that the multiple maxima are associated with endopolyploidy or mixoploidy and/or epigenomic diversity of pith cells that show different sensitivities to biologically active solutes.     

Physiological effects of a novel aromatic cytokinin analogue in micropropagated Aloe arborescens and Harpagophytum procumbens

Globally, there is a continuous effort geared toward improving micropropagation protocols with much emphasis on the type of cytokinins (CKs). We investigated the effect of meta-topolin tetrahydropyran-2-yl [mTTHP—a novel derivative of the aromatic cytokinin meta-topolin (mT)] on shoot proliferation, photosynthetic pigment content, phytochemicals and antioxidant activity of two widely used medicinal plants, Aloe arborescens and Harpagophytum procumbens. In terms of shoot proliferation, besides mTTHP and mT (at equimolar level) showing comparable effects, both topolins were significantly better than the control and benzyladenine riboside (BAR) in micropropagated A. arborescens. In H. procumbens, mT-treated cultures were the most responsive treatment at 2.5 μM when compared to the control. At 5.0 μM concentration, mTTHP and meta-topolin riboside (mTR) demonstrated a comparable effect on shoot proliferation. To a certain extent (particularly at low concentrations), mTTHP had a better rooting stimulatory effect when compared to the other CKs in both investigated species. In micropropagated A. arborescens, 2.5 μM mTTHP showed the best antioxidant activity (ferric reducing power) which was significantly higher than the control and all BAR-treated plants. Although 2.5 μM mTTHP exhibited a significant antioxidant activity (in β-carotene based assay) when compared to mTR at equimolar level in H. procumbens, it was not significantly different from the treatment with 2.5 μM mT and the control. As evidenced in the current study, the efficacy of the novel cytokinin may be species-specific and beneficial for few physiological parameters. It is conceivable that mTTHP is another viable alternative topolin with the extra advantage of inducing rooting at a low concentration.     

Sexual Dimorphism in Bite Performance Drives Morphological Variation in Chameleons

Phenotypic performance in different environments is central to understanding the evolutionary and ecological processes that drive adaptive divergence and, ultimately, speciation. Because habitat structure can affect an animal’s foraging behaviour, anti-predator defences, and communication behaviour, it can influence both natural and sexual selection pressures. These selective pressures, in turn, act upon morphological traits to maximize an animal’s performance. For performance traits involved in both social and ecological activities, such as bite force, natural and sexual selection often interact in complex ways, providing an opportunity to understand the adaptive significance of morphological variation with respect to habitat. Dwarf chameleons within the Bradypodion melanocephalum-Bradypodion thamnobates species complex have multiple phenotypic forms, each with a specific head morphology that could reflect its use of either open- or closed-canopy habitats. To determine whether these morphological differences represent adaptations to their habitats, we tested for differences in both absolute and relative bite performance. Only absolute differences were found between forms, with the closed-canopy forms biting harder than their open-canopy counterparts. In contrast, sexual dimorphism was found for both absolute and relative bite force, but the relative differences were limited to the closed-canopy forms. These results indicate that both natural and sexual selection are acting within both habitat types, but to varying degrees. Sexual selection seems to be the predominant force within the closed-canopy habitats, which are more protected from aerial predators, enabling chameleons to invest more in ornamentation for communication. In contrast, natural selection is likely to be the predominant force in the open-canopy habitats, inhibiting the development of conspicuous secondary sexual characteristics and, ultimately, enforcing their overall diminutive body size and constraining performance.