Links between global taxonomic diversity,ecological diversity and the expansion of vertebrates on land

Tetrapod biodiversity today is great; over the past 400 Myr since vertebrates moved onto land, global tetrapod diversity has risen exponentially, punctuated by losses during major extinctions. There are links between the total global diversity of tetrapods and the diversity of their ecological roles, yet no one fully understands the interplay of these two aspects of biodiversity and a numerical analysis of this relationship has not so far been undertaken. Here we show that the global taxonomic and ecological diversity of tetrapods are closely linked. Throughout geological time, patterns of global diversity of tetrapod families show 97 per cent correlation with ecological modes. Global taxonomic and ecological diversity of this group correlates closely with the dominant classes of tetrapods (amphibians in the Palaeozoic, reptiles in the Mesozoic, birds and mammals in the Cenozoic). These groups have driven ecological diversity by expansion and contraction of occupied ecospace, rather than by direct competition within existing ecospace and each group has used ecospace at a greater rate than their predecessors.     

Structure of the Recombinant Alphavirus Western Equine Encephalitis Virus Revealed by Cryoelectron Microscopy

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV''s structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.The alphaviruses comprise a genus of single-stranded, plus-sense, enveloped RNA viruses that, together with rubella virus, comprise the family Togaviridae. The current classification of the genus Alphavirus includes 29 different species, with multiple subtypes and/or varieties represented within some species (30). These species can be grouped into 8 different complexes based on antigenic and/or genetic similarities (20). Most viruses from the New World are found in the Eastern, Venezuelan, and Western equine encephalitis (EEE, VEE, and WEE, respectively) complexes and cause encephalitis in humans and a variety of domesticated animals. Old World alphaviruses, on the other hand, typically cause only an arthralgia and rash syndrome that is rarely life threatening (5, 24). Among the New World alphaviruses, EEE, VEE, and WEE viruses (EEEV, VEEV, and WEEV, respectively) are potential biological weapons as well as naturally emerging pathogens and are therefore included on the category B Priority Pathogens list of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (http://www.niaid.nih.gov/topics/biodefenserelated/biodefense/research/pages/cata.aspx).Alphaviruses replicate in the cytoplasm of infected cells after entry via receptor-mediated endocytosis (8). Following internalization, fusion of the viral envelope with the endocytic membrane is mediated by a low-pH-induced conformational change that exposes a fusion peptide found in the E1 envelope glycoprotein. The nucleocapsid then disassembles upon interactions with ribosomes, and an open reading frame (ORF) found in the 5′ two-thirds of the genome is translated. The resultant polyprotein is cleaved into 4 nonstructural proteins (nsP1 to -4) that mediate viral RNA replication, RNA capping, and polyprotein processing (Fig. ​(Fig.1).1). The structural proteins, including the two envelope glycoproteins E2 and E1 as well as the capsid protein, are encoded in a second ORF that is translated from a subgenomic message often referred to as 26S RNA. Following auto-cleavage of the capsid protein in the cytoplasm, the remaining polyprotein is inserted into the endoplasmic reticulum, where it is cleaved by host cell proteases and then processed through the secretory pathway, where the glycosylation of E2 and E1 occurs. Virion maturation occurs after E2/E1 heterodimers are inserted into the plasma membrane and 240 copies of the capsid protein interact with one copy of the genomic RNA to form nucleocapsids. These nucleocapsids then interact with a cytoplasmic domain of the E2 protein to initiate budding. The mature virion thus includes 240 copies of the capsid protein and 240 E2/E1 heterodimers arranged as trimeric spikes on the surface of the virus (8).Open in a separate windowFIG. 1.Diagram of the alphavirus genome, showing the 5′ cap, 5′ untranslated region, nonstructural polyprotein open reading frame, and major functions of the individual proteins, subgenomic promoter, structural polyprotein open reading frame, 3′ untranslated region, and poly(A) tail.The structures of several different alphaviruses, including Sindbis virus (SINV) (13), Ross River virus (RRV) (3, 35), Semliki Forest virus (SFV), (11), and VEEV (16), have been solved to subnanometer resolution using cryoelectron microscopy (cryoEM), and the X-ray crystallographic structure of the E1 protein from Semliki Forest virus has been determined to atomic resolution (9). The alphaviruses are ca. 700 Å in diameter, with 80 trimeric spikes on their surfaces. By fitting the E1 crystal structure into cryoEM reconstruction maps of whole viruses, the orientations of both envelope proteins within the spikes have been estimated (36). The E1 and E2 proteins are similar in shape, and the E2 proteins extend to the tips of the spikes, where most glycosylation and antibody-binding sites have been mapped (13). The underlying T=4 icosahedral capsid is constructed from regularly ordered capsomers arranged as hexons and pentons. These pentons and hexons consist of capsid protein monomers that apparently represent only the C-terminal half of the protein. Crystal structures of alphavirus capsid proteins also indicate that only the C terminus, including the protease domain, is ordered (25). cryoEM reconstructions of VEEV nucleocapsids isolated from virions have a less ordered structure, with density redistributed from the 3-fold to the 5-fold axis, suggesting that the envelope and/or the envelope glycoproteins constrain and stabilize the nucleocapsid in a compressed structure (15). Additionally, the VEEV nucleocapsids within viruses differ from those of Old World alphaviruses, with a counterclockwise rotation of the pentameric and hexameric capsomers in VEEV (16). Similar differences were observed in the capsid of Aura virus (AURAV), another New World alphavirus (34).In addition to being an important human and equine pathogen, WEEV is one of three alphaviruses that descended from a recombinant ancestor (6, 31). This ancestor derived its nonstructural and capsid protein genes from an ancestral EEEV strain, whereas its envelope glycoprotein genes were provided from an ancestral SINV. The recombination event was apparently followed by compensatory mutations in the cytoplasmic domain of the E2 protein that restored efficient interactions with the EEEV-like capsid protein (6). If this interpretation of the WEEV ancestral recombination event is correct, its nucleocapsids, constructed from capsid proteins derived from the New World EEEV ancestor, would be expected be more similar to those of the New World VEEV than to those of the Old World SINV, RRV, and SFV. To test this hypothesis and to investigate other structural features of interest related to its recombinant history and pathogenicity, we determined the structure of WEEV to a 13-Å resolution using cryoEM image reconstruction.     

Vector Choice Determines Immunogenicity and Potency of Genetic Vaccines against Angola Marburg Virus in Nonhuman Primates

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4⁺ and CD8⁺ cellular immune responses, with skewing toward CD4⁺ T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8⁺ T-cell dominance. Across vaccine groups, CD8⁺ T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8⁺ T cells may determine vaccine efficacy against infection by MARV Angola.The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmid-based vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA vectors exhibited clinical symptoms associated with MARV hemorrhagic fever (MHF) that were absent in NHP receiving a single inoculation with recombinant adenovirus (rAd) vectors, suggesting qualitative differences in the immune responses elicited by the different modalities.     

Mathematical aspects of protein structure determination with NMR orientational restraints

The field of structural biology is becoming increasingly important as new technological developments facilitate the collection of data on the atomic structures of proteins and nucleic acids. The solid-state NMR method is a relatively new biophysical technique that holds particular promise for determining the structures of peptides and proteins that are located within the cell membrane. This method provides information on the orientation of the peptide planes relative to an external magnetic field. In this article, we discuss some of the mathematical methods and tools that are useful in deriving the atomic structure from these orientational data. We first discuss how the data are viewed as tensors, and how these tensors can be used to construct an initial atomic model, assuming ideal stereochemistry. We then discuss methods for refining the models using global optimization, with stereochemistry constraints treated as penalty functions. These two processes, initial model building followed by refinement, are the two crucial steps between data collection and the final atomic model.     

A unique set of 11,008 onion expressed sequence tags reveals expressed sequence and genomic differences between the monocot orders Asparagales and Poales

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.     

Allocation of energy use in petroleum refineries to petroleum products

Aim, Scope, and Background  Studies to evaluate the energy and emission impacts of vehicle/fuel systems have to address allocation of the energy use and emissions associated with petroleum refineries to various petroleum products because refineries produce multiple products. The allocation is needed in evaluating energy and emission effects of individual transportation fuels. Allocation methods used so far for petroleum-based fuels (e.g., gasoline, diesel, and liquefied petroleum gas [LPG]) are based primarily on mass, energy content, or market value shares of individual fuels from a given refinery. The aggregate approach at the refinery level is unable to account for the energy use and emission differences associated with producing individual fuels at the next sub-level: individual refining processes within a refinery. The approach ignores the fact that different refinery products go through different processes within a refinery. Allocation at the subprocess level (i.e., the refining process level) instead of at the aggregate process level (i.e., the refinery level) is advocated by the International Standard Organization. In this study, we seek a means of allocating total refinery energy use among various refinery products at the level of individual refinery processes. Main Features  We present a petroleum refinery-process-based approach to allocating energy use in a petroleum refinery to petroleum refinery products according to mass, energy content, and market value share of final and intermediate petroleum products as they flow through refining processes within a refinery. The approach is based on energy and mass balance among refining processes within a petroleum refinery. By using published energy and mass balance data for a simplified U.S. refinery, we developed a methodology and used it to allocate total energy use within a refinery to various petroleum products. The approach accounts for energy use during individual refining processes by tracking product stream mass and energy use within a refinery. The energy use associated with an individual refining process is then distributed to product streams by using the mass, energy content, or market value share of each product stream as the weighting factors. Results  The results from this study reveal that product-specific energy use based on the refinery process-level allocation differs considerably from that based on the refinery-level allocation. We calculated well-to-pump total energy use and greenhouse gas (GHG) emissions for gasoline, diesel, LPG, and naphtha with the refinery process-based allocation approach. For gasoline, the efficiency estimated from the refinery-level allocation underestimates gasoline energy use, relative to the process-level based gasoline efficiency. For diesel fuel, the well-to-pump energy use for the process-level allocations with the mass- and energy-content-based weighting factors is smaller than that predicted with the refinery-level allocations. However, the process-level allocation with the market-value-based weighting factors has results very close to those obtained by using the refinery-level allocations. For LPG, the refinery-level allocation significantly overestimates LPG energy use. For naphtha, the refinery-level allocation overestimates naphtha energy use. The GHG emission patterns for each of the fuels are similar to those of energy use. Conclusions  We presented a refining-process-level-based method that can be used to allocate energy use of individual refining processes to refinery products. The process-level-based method captures process-dependent characteristics of fuel production within a petroleum refinery. The method starts with the mass and energy flow chart of a refinery, tracks energy use by individual refining processes, and distributes energy use of a given refining process to products from the process. In allocating energy use to refinery products, the allocation method could rely on product mass, product energy contents, or product market values as weighting factors. While the mass- and energy-content-based allocation methods provide an engineering perspective of energy allocation within a refinery, the market-value-based allocation method provides an economic perspective. The results from this study show that energy allocations at the aggregate refinery level and at the refining process level could make a difference in evaluating the energy use and emissions associated with individual petroleum products. Furthermore, for the refining-process-level allocation method, use of mass — energy content- or market value share-based weighting factors could lead to different results for diesel fuels, LPG, and naphtha. We suggest that, when possible, energy use allocations should be made at the lowest subprocess level — a confirmation of the recommendation by the International Standard Organization for life cycle analyses. Outlook  The allocation of energy use in petroleum refineries at the refining process level in this study follows the recommendation of ISO 14041 that allocations should be accomplished at the subprocess level when possible. We developed a method in this study that can be readily adapted for refineries in which process-level energy and mass balance data are available. The process-level allocation helps reveal some additional energy and emission burdens associated with certain refinery products that are otherwise overlooked with the refinery-level allocation. When possible, process-level allocation should be used in life-cycle analyses.     

Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943

To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na(+)-K(+)-ATPase inhibition), and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff-perfused rat heart for external [Ca(2+)] of 0.5 and 1.5 mM under control conditions and in the presence of the reverse-mode Na(+)/Ca(2+) exchange inhibitor KB-R7943 (0.1 microM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 microg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high-affinity/low-capacity and a low-affinity/high- capacity binding site, response generation was >80% mediated by binding to the high-affinity receptor. Digoxin sensitivity increased at lower external [Ca(2+)] due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 microg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na(+)-K(+)-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca(2+) influx rate via the reverse mode Na(+)/Ca(2+) exchanger might become the limiting factor in digoxin response generation.     

Tyrosine phosphorylation of caveolin-2 at residue 27: differences in the spatial and temporal behavior of phospho-Cav-2 (pY19 and pY27)

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct properties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functional analysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, but clearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstream mediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that in A431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However, the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporal response to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylation at tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to the common characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) may each possess a set of unique functional roles.     

Recruitment of histone modifications by USF proteins at a vertebrate barrier element

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.