The cytoskeletal regulator Genghis khan is required for columnar target specificity in the Drosophila visual system

A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity.     

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

Global conservation priorities for marine turtles

Where conservation resources are limited and conservation targets are diverse, robust yet flexible priority-setting frameworks are vital. Priority-setting is especially important for geographically widespread species with distinct populations subject to multiple threats that operate on different spatial and temporal scales. Marine turtles are widely distributed and exhibit intra-specific variations in population sizes and trends, as well as reproduction and morphology. However, current global extinction risk assessment frameworks do not assess conservation status of spatially and biologically distinct marine turtle Regional Management Units (RMUs), and thus do not capture variations in population trends, impacts of threats, or necessary conservation actions across individual populations. To address this issue, we developed a new assessment framework that allowed us to evaluate, compare and organize marine turtle RMUs according to status and threats criteria. Because conservation priorities can vary widely (i.e. from avoiding imminent extinction to maintaining long-term monitoring efforts) we developed a “conservation priorities portfolio” system using categories of paired risk and threats scores for all RMUs (n = 58). We performed these assessments and rankings globally, by species, by ocean basin, and by recognized geopolitical bodies to identify patterns in risk, threats, and data gaps at different scales. This process resulted in characterization of risk and threats to all marine turtle RMUs, including identification of the world''s 11 most endangered marine turtle RMUs based on highest risk and threats scores. This system also highlighted important gaps in available information that is crucial for accurate conservation assessments. Overall, this priority-setting framework can provide guidance for research and conservation priorities at multiple relevant scales, and should serve as a model for conservation status assessments and priority-setting for widespread, long-lived taxa.     

Phylogenetic relationships and biogeography of Fuchsia (Onagraceae) based on noncoding nuclear and chloroplast DNA data

To examine relationships and test previous sectional delimitations within Fuchsia, this study used parsimony and maximum likelihood analyses with nuclear ITS and chloroplast trnL-F and rpl16 sequence data for 37 taxa representing all sections of Fuchsia and four outgroup taxa. Results support previous sectional delimitations, except for F. verrucosa, which is related to a Central American clade rather than to section Fuchsia and is described here as a new section Verrucosa. The basal relationships within Fuchsia are poorly resolved, suggesting an initial rapid diversification of the genus. Among the species sampled, there is strong support for a single South Pacific lineage, a southern South American/southern Brazilian lineage, a tropical Andean lineage, and one or two Central American and Mexican lineages. There is no clear support for an austral origin of the genus, as previously proposed, which is more consistent with Fuchsia's sister group relationship with the boreal Circaea. An ultrametric molecular clock analysis (all minimal dates) places the split between Fuchsia and Circaea at 41 million years ago (mya), with the diversification of the modern-day lineages of Fuchsia beginning at 31 mya. The South Pacific Fuchsia lineage branches off around 30 mya, consistent with fossil records from Australia and New Zealand. The large Andean section Fuchsia began to diversify around 22 mya, preceded by the divergence of the Caribbean F. triphylla at 25 mya. The Brazilian members of section Quelusia separated from the southern Andean F. magellanica around 13 mya, and the ancestor of the Tahitian F. cyrtandroides split off from the New Zealand species of section Skinnera approximately 8 mya.     

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.     

Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.     

Aging attenuates the coronary blood flow response to cold air breathing and isometric handgrip in healthy humans

The purpose of this echocardiography study was to measure peak coronary blood flow velocity (CBV(peak)) and left ventricular function (via tissue Doppler imaging) during separate and combined bouts of cold air inhalation (-14 ± 3°C) and isometric handgrip (30% maximum voluntary contraction). Thirteen young adults and thirteen older adults volunteered to participate in this study and underwent echocardiographic examination in the left lateral position. Cold air inhalation was 5 min in duration, and isometric handgrip (grip protocol) was 2 min in duration; a combined stimulus (cold + grip protocol) and a cold pressor test (hand in 1°C water) were also performed. Heart rate, blood pressure, O(2) saturation, and inspired air temperature were monitored on a beat-by-beat basis. The rate-pressure product (RPP) was used as an index of myocardial O(2) demand, and CBV(peak) was used as an index of myocardial O(2) supply. The RPP response to the grip protocol was significantly blunted in older subjects (Δ1,964 ± 396 beats·min(-1)·mmHg) compared with young subjects (Δ3,898 ± 452 beats·min(-1)·mmHg), and the change in CBV(peak) was also blunted (Δ6.3 ± 1.2 vs. 11.2 ± 2.0 cm/s). Paired t-tests showed that older subjects had a greater change in the RPP during the cold + grip protocol [Δ2,697 ± 391 beats·min(-1)·mmHg compared with the grip protocol alone (Δ2,115 ± 375 beats·min(-1)·mmHg)]. An accentuated RPP response to the cold + grip protocol (compared with the grip protocol alone) without a concomitant increase in CBV(peak) may suggest a dissociation between the O(2) supply and demand in the coronary circulation. In conclusion, older adults have blunted coronary blood flow responses to isometric exercise.     

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host''s antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi''s sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.     

Time-calibrated phylogeny of the woody Australian genus Hakea (Proteaceae) supports multiple origins of insect-pollination among bird-pollinated ancestors

? Premise of the study: A past study based on morphological data alone showed that the means by which plants of the Australian genus Hakea reduce florivory is related to the evolution of bird pollination. For example, bird pollination was shown to have arisen only in insect-pollinated lineages that already produced greater amounts of floral cyanide, a feature that reduces florivory. We examine a central conclusion of that study, and a common assumption in the literature, that bird pollination arose in insect-pollinated lineages, rather than the reverse. ? Methods: We combined morphological and DNA data to infer the phylogeny and age of the Australian genus Hakea, using 9.2 kilobases of plastid and nuclear DNA and 46 morphological characters from a taxonomically even sampling of 55 of the 149 species. ? Key results: Hakea is rooted confidently in a position that has not been suggested before. The phylogeny implies that bird pollination is primitive in Hakea and that multiple shifts to insect pollination have occurred. The unexpectedly young age of Hakea (a crown age of ca. 10 Ma) makes it coincident with its primary bird pollinators (honeyeaters) throughout its history. ? Conclusions: Our study demonstrates that Hakea is an exception to the more commonly described shift from insect to bird pollination. However, we note that only one previous phylogenetic study involved Australian plants and their honeyeater pollinators and that our finding might prove to be more common on that continent.