Patterns of Vertebrate Diversity and Protection in Brazil

Most conservation decisions take place at national or finer spatial scales. Providing useful information at such decision-making scales is essential for guiding the practice of conservation. Brazil is one of the world’s megadiverse countries, and consequently decisions about conservation in the country have a disproportionate impact on the survival of global biodiversity. For three groups of terrestrial vertebrates (birds, mammals, and amphibians), we examined geographic patterns of diversity and protection in Brazil, including that of endemic, small-ranged, and threatened species. To understand potential limitations of the data, we also explored how spatial bias in collection localities may influence the perceived patterns of diversity. The highest overall species richness is in the Amazon and Atlantic Forests, while the Atlantic Forest dominates in terms of country endemics and small-ranged species. Globally threatened species do not present a consistent pattern. Patterns for birds were similar to overall species richness, with higher concentrations of threatened species in the Atlantic Forest, while mammals show a more generalized pattern across the country and a high concentration in the Amazon. Few amphibians are listed as threatened, mostly in the Atlantic Forest. Data deficient mammals occur across the country, concentrating in the Amazon and southeast Atlantic Forest, and there are no data deficient birds in Brazil. In contrast, nearly a third of amphibians are data deficient, widespread across the country, but with a high concentration in the far southeast. Spatial biases in species locality data, however, possibly influence the perceived patterns of biodiversity. Regions with low sampling density need more biological studies, as do the many data deficient species. All biomes except the Amazon have less than 3% of their area under full protection. Reassuringly though, rates of protection do correlate with higher biodiversity, including higher levels of threatened and small-ranged species. Our results indicate a need for expanded formal protection in Brazil, especially in the Atlantic forest, and with an emphasis on fully protected areas.     

Effects of Rhododendron removal and prescribed fire on bees and plants in the southern Appalachians

Rhododendron maximum is an evergreen shrub native to the Appalachian Mountains of North America that has expanded in recent decades due to past disturbances and land management. The purpose of this study was to explore how bees and plants were affected by the experimental removal of R. maximum followed by a prescribed fire in one watershed compared to a neighboring reference watershed. Bees and plants were sampled for three years in both watersheds. Comparisons were based on the rarefaction and extrapolation sampling curves of Hill numbers as well as multivariate methods to assess effects on community composition. Bee richness, Shannon''s diversity, and Simpson''s diversity did not differ between watersheds in the year after removal but were all significantly higher in the removal watershed in year two, following the prescribed fire. Bee Shannon''s diversity and Simpson''s diversity, but not richness, remained significantly higher in the removal watershed in the third year. Similar but weaker patterns were observed for plants. Comparisons of community composition found significant differences for bees in the second and third year and significant differences for plants in all three years. For both groups, significant indicator taxa were mostly associated with the removal watershed. Because bees appeared to respond more strongly to the prescribed fire than to the removal of R. maximum and these benefits weakened considerably one year after the fire, clearing R. maximum does not appear to dramatically improve pollinator habitat in the southern Appalachians. This conclusion is underscored by the fact that about one quarter of the bee species in our study area were observed visiting R. maximum flowers. The creation of open areas with wildflowers may be a better way to benefit bees in this region judging from the high diversity of bees captured in the small roadside clearings in this study.     

The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.     

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ~ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ~ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.     

Noise pollution alters ecological services: enhanced pollination and disrupted seed dispersal

Noise pollution is a novel, widespread environmental force that has recently been shown to alter the behaviour and distribution of birds and other vertebrates, yet whether noise has cumulative, community-level consequences by changing critical ecological services is unknown. Herein, we examined the effects of noise pollution on pollination and seed dispersal and seedling establishment within a study system that isolated the effects of noise from confounding stimuli common to human-altered landscapes. Using observations, vegetation surveys and pollen transfer and seed removal experiments, we found that effects of noise pollution can reverberate through communities by disrupting or enhancing these ecological services. Specifically, noise pollution indirectly increased artificial flower pollination by hummingbirds, but altered the community of animals that prey upon and disperse Pinus edulis seeds, potentially explaining reduced P. edulis seedling recruitment in noisy areas. Despite evidence that some ecological services, such as pollination, may benefit indirectly owing to noise, declines in seedling recruitment for key-dominant species such as P. edulis may have dramatic long-term effects on ecosystem structure and diversity. Because the extent of noise pollution is growing, this study emphasizes that investigators should evaluate the ecological consequences of noise alongside other human-induced environmental changes that are reshaping human-altered landscapes worldwide.     

Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Modeling the glucose-insulin regulatory system and ultradian insulin secretory oscillations with two explicit time delays

In the glucose-insulin regulatory system, ultradian insulin secretory oscillations are observed to have a period of 50-150 min. After pioneering work traced back to the 1960s, several mathematical models have been proposed during the last decade to model these ultradian oscillations as well as the metabolic system producing them. These currently existing models still lack some of the key physiological aspects of the glucose-insulin system. Applying the mass conservation law, we introduce two explicit time delays and propose a more robust alternative model for better understanding the glucose-insulin endocrine metabolic regulatory system and the ultradian insulin secretory oscillations for the cases of continuous enteral nutrition and constant glucose infusion. We compare the simulation profiles obtained from this two time delay model with those from the other existing models. As a result, we notice many unique features of this two delay model. Based on our intensive simulations, we suspect that one of the possibly many causes of ultradian insulin secretion oscillations is the time delay of the insulin secretion stimulated by the elevated glucose concentration.     

Continuous delivery of IFN-beta promotes sustained maturation of intratumoral vasculature

IFNs have pleiotropic antitumor mechanisms of action. The purpose of this study was to further investigate the effects of IFN-beta on the vasculature of human xenografts in immunodeficient mice. We found that continuous, systemic IFN-beta delivery, established with liver-targeted adeno-associated virus vectors, led to sustained morphologic and functional changes of the tumor vasculature that were consistent with vessel maturation. These changes included increased smooth muscle cell coverage of tumor vessels, improved intratumoral blood flow, and decreased vessel permeability, tumor interstitial pressure, and intratumoral hypoxia. Although these changes in the tumor vasculature resulted in more efficient tumor perfusion, further tumor growth was restricted, as the mature vasculature seemed to be unable to expand to support further tumor growth. In addition, maturation of the intratumoral vasculature resulted in increased intratumoral penetration of systemically administered chemotherapy. Finally, molecular analysis revealed increased expression by treated tumors of angiopoietin-1, a cytokine known to promote vessel stabilization. Induction of angiopoietin-1 expression in response to IFN-beta was broadly observed in different tumor lines but not in those with defects in IFN signaling. In addition, IFN-beta-mediated vascular changes were prevented when angiopoietin signaling was blocked with a decoy receptor. Thus, we have identified an alternative approach for achieving sustained vascular remodeling-continuous delivery of IFN-beta. In addition to restricting tumor growth by inhibiting further angiogenesis, maturation of the tumor vasculature also improved the efficiency of delivery of adjuvant therapy. These results have significant implications for the planning of combination anticancer therapy.     

Automatic particle selection: results of a comparative study

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.