Membrane-perturbing properties of three peptides corresponding to the ectodomain of hepatitis C virus E2 envelope protein

Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2(430-449) and E2(603-624) were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2(543-560) did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a beta-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.     

Intake rates and the functional response in shorebirds (Charadriiformes) eating macro-invertebrates

As field determinations take much effort, it would be useful to be able to predict easily the coefficients describing the functional response of free-living predators, the function relating food intake rate to the abundance of food organisms in the environment. As a means easily to parameterise an individual-based model of shorebird Charadriiformes populations, we attempted this for shorebirds eating macro-invertebrates. Intake rate is measured as the ash-free dry mass (AFDM) per second of active foraging; i.e. excluding time spent on digestive pauses and other activities, such as preening. The present and previous studies show that the general shape of the functional response in shorebirds eating approximately the same size of prey across the full range of prey density is a decelerating rise to a plateau, thus approximating the Holling type II ('disc equation') formulation. But field studies confirmed that the asymptote was not set by handling time, as assumed by the disc equation, because only about half the foraging time was spent in successfully or unsuccessfully attacking and handling prey, the rest being devoted to searching.A review of 30 functional responses showed that intake rate in free-living shorebirds varied independently of prey density over a wide range, with the asymptote being reached at very low prey densities (<150/m-2). Accordingly, most of the many studies of shorebird intake rate have probably been conducted at or near the asymptote of the functional response, suggesting that equations that predict intake rate should also predict the asymptote.A multivariate analysis of 468 'spot' estimates of intake rates from 26 shorebirds identified ten variables, representing prey and shorebird characteristics, that accounted for 81% of the variance in logarithm-transformed intake rate. But four-variables accounted for almost as much (77.3%), these being bird size, prey size, whether the bird was an oystercatcher Haematopus ostralegus eating mussels Mytilus edulis, or breeding. The four variable equation under-predicted, on average, the observed 30 estimates of the asymptote by 11.6%, but this discrepancy was reduced to 0.2% when two suspect estimates from one early study in the 1960s were removed. The equation therefore predicted the observed asymptote very successfully in 93% of cases. We conclude that the asymptote can be reliably predicted from just four easily measured variables. Indeed, if the birds are not breeding and are not oystercatchers eating mussels, reliable predictions can be obtained using just two variables, bird and prey sizes. A multivariate analysis of 23 estimates of the half-asymptote constant suggested they were smaller when prey were small but greater when the birds were large, especially in oystercatchers. The resulting equation could be used to predict the half-asymptote constant, but its predictive power has yet to be tested. As well as predicting the asymptote of the functional response, the equations will enable research workers engaged in many areas of shorebird ecology and behaviour to estimate intake rate without the need for conventional time-consuming field studies, including species for which it has not yet proved possible to measure intake rate in the field.     

Desulfovibrio vulgaris CbiKP cobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues

The sulfate‐reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiK^P and CbiK^C, that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C‐terminus of CbiK^P confirmed that the protein is transported to the periplasm. The structure‐function relationship of CbiK^P was studied by constructing eleven site‐directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal‐chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiK^P is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiK^P, which are absent from all other known bacterial cobaltochelatases, has evolved CbiK^P a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter.     

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43^def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43^def cell death. Cx43^def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43^def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21^fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43^def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43^def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43^def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.     

The Effect of Nanostructured Chitosan and Chitosan‐thyme Essential Oil Coatings on Colletotrichum gloeosporioides Growth in vitro and on cv Hass Avocado and Fruit Quality

In this study, nanostructured edible coatings based on chitosan nanoparticles (CSNPs) and chitosan‐thyme essential oil nanoparticles (CSTEO‐NPs) were characterized and evaluated on in vitro growth of Colletotrichum gloeosporioides and on inoculated avocado fruit cv. Hass, to evaluate their fungicidal activity and the effect on fruit quality. From TEM and particle size distribution characterization, the size of nanoparticles increased after thyme essential oil incorporation. Overall a synergistic effect between the chitosan and thyme essential oil (TEO) was observed. For in vitro evaluation, incorporation of this essential oil to CSNPs improved the control of C. gloeosporioides as there was a complete growth inhibition. CSNPs with concentrations of TEO at 3 and 5% had a fungicidal effect. The coating formulation with 55% CSTEO‐NPs notably reduced the incidence of C. gloeosporioides on avocado cv. Hass by up to 60%. Also, at the end of the 8‐day storage period, CSTEO‐NPs incorporation into the coating did not affect the quality of avocado; moreover, fruit firmness was better maintained than untreated fruit.     

Development and Validation of a Diagrammatic Scale to Assess the Severity of Black Rot of Crucifers in Kale

The aim of this study was to develop a diagrammatic scale to evaluate black rot (Xanthomonas campestris pv. compestris) severity on kale (Brassica oleraceae var. acephala) leaves. The diagrammatic scale was developed and validated with eight levels of severity, ranging from 0.19 to 48.8%. More than 95% of the leaves collected from the field showed severity levels ranging from 0.1 to 21%, and 5% of the leaves showed severities higher than 22%. The validation of the scale was performed by 10 inexperienced evaluators, and the data were analysed with two methods: linear regression and Lin's statistics. Without the scale, most evaluators overestimated disease severity, whereas the use of the scale resulted in increased precision, accuracy, repeatability, and reproducibility of the estimates according to both validation methods. In conclusion, the proposed diagrammatic scale proved to be useful for assessments of black rot severity in kale leaves. The scale may be of interest to researches performing studies on epidemiology or breeding for resistance.     

Migratory preferences of humpback whales between feeding and breeding grounds in the eastern South Pacific

Latitudinal preferences within the breeding range have been suggested for Breeding Stock G humpback whales that summer in different feeding areas of the eastern South Pacific. To address this hypothesis, humpback whales photo‐identified from the Antarctic Peninsula and the Fueguian Archipelago (southern Chile) were compared with whales photo‐identified from lower latitudes extending from northern Peru to Costa Rica. This comparison was performed over a time span that includes 18 austral seasons. A total of 238 whales identified from the Antarctic Peninsula and 25 whales from the Fueguian Archipelago were among those photo‐identified at the breeding grounds. Our findings showed that humpback whales from each feeding area were resighted unevenly across the breeding grounds, which suggests a degree of spatial structuring in the migratory pathway. Humpback whales that feed at the Antarctic Peninsula were more likely to migrate to the southern breeding range between northern Peru and Colombia, whereas whales that feed at the Fueguian Archipelago were more likely to be found in the northern range of the breeding ground off Panama. Further photo‐identification efforts and genetic sampling from poorly sampled or unsampled areas are recommended to confirm these reported connectivity patterns.     

Shadow conservation and the persistence of sacred church forests in northern Ethiopia

Land‐use change threatens biodiversity and ecosystem function worldwide. These changes have impacts on weather patterns, carbon storage, biodiversity, and other ecosystem services from regional to local scales. Only 8 percent of tropical forests are formally recognized as conservation areas, however globally, there is a network of sites that are protected because they are sacred and as a result act as ‘shadow’ conservation for biodiversity. Unlike other types of protected sites (e.g., national parks), these sites are seats of religious ritual that anchor a community's cultural identity, while also conserving biological diversity and other ecosystem services. We studied the extent and status of sacred forests in northern Ethiopia, which are threatened because of their small size (~5 ha) and isolation, increasing their exposure to edge effects and human pressures. Using historical and modern imagery, we found that over the last 50 yr, sacred forests have increased in area, but decreased in crown closure. We also found that forest ecological status, via ground‐level investigation, had high mean human disturbance (e.g., trails, plantations, exotic planting; 37%); and that forests close to markets (e.g., cities) increased in area due to planting of Eucalyptus (exotic), indicating a potential threat to their persistence and value as shelters of the church.     

Is the maximum reproductive rate of <Emphasis Type="Italic">Centris analis</Emphasis> (Hymenoptera,Apidae, Centridini) associated with floral resource availability?

Spatiotemporal variation in the availability of food resources may be a determining factor for reproductive success and maintenance of bees, but the extent of these variations is poorly understood. For management and conservation of bees, the first step is to know the behavior and the food resources used. Currently, urban areas are considered refuge zones for bees, and understanding the availability of floral resources and the influence on reproductive processes is very important for management of bees. We used the protocols applied in phenological studies with bees and plant species to evaluate both throughout the year in an urbanized area. At the same time, we used palynology protocols to analyze the pollen material collected from brood cells (food and feces) of immature Centris analis. These protocols allowed to evaluate the availability of floral resources in the studied area and the plant species effectively used by C. analis females to feed immature larvae during the reproductive period. The maximum reproductive period of C. analis was not associated with the highest floral resources availability. However, there was a strong selectivity of pollen in flowers of Malpighia emarginata (Malpighiaceae), which represented more than 59% of all the pollen grains provisioned throughout the year. This means that in the case of more specialized bees like C. analis, the availability of the preferred plants is more important than the overall floral resource availability in the area. Thus, to keep C. analis in the city, it is necessary to maintain or introduce Malpighiaceae species in the urban planning. On the other hand, at least 27% of the plant species found in the study area are pollinated by C. analis, emphasizing the importance of preserving this bee.     

Riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral RNA

Initiation of translation driven by internal ribosome entry site (IRES) elements depends upon the structural organization of this mRNA region. Besides translation initiation factors (eIFs), auxiliary proteins can also affect IRES activity. With the aim to identify proteins interacting with two unrelated IRESs present in the genome of foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) we have used a proteomic approach. This procedure allowed the identification of 21 RNA-binding proteins interacting with discrete regions of the FMDV IRES, domains 3 and 5, and 16 interacting with domain III of the HCV IRES. In support of the binding specificity, the factors interacting with domain 3 differed from those interacting with domain 5, and included three poly(rC)-binding protein (PCBP) members, besides proliferation-associated 2G4 (PA2G4) and deleted-azoospermia 1 (DAZ1) protein. Around 71% of the identified factors associated with the FMDV IRES differ from those interacting with the HCV IRES. The group of proteins interacting with the FMDV or the HCV IRES includes eIF4B and 5 subunits of eIF3, respectively, known to interact with each of these RNAs, validating the results of this approach. According to the function of the identified proteins, 55% are involved in translation control, whereas 35% play a role in different aspects of RNA lifespan. Compilation of factors preferentially associated with FMDV or HCV IRES provides a basis for examining the strategies used by IRESs to recruit the translation machinery.