Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.     

Intima-Media Thickness Does Not Differ between Two Common Carotid Artery Segments in Children

Carotid intima-media thickness (cIMT) is a surrogate marker of early atherosclerotic changes in children. cIMT-studies are hard to compare, due to variations in ultrasound protocols, especially regarding the common carotid artery (CCA) segment measured in relation to the bulb. This study’s purpose was therefore to compare two distinct CCA segments in children, to see if cIMT values differ substantially according to the site of measurement. cIMT was assessed after power calculation in 30 children (15 girls) aged 8–17, using B-Mode ultrasound (5–13 MHz) at two CCA locations. The first measurement was performed over a distance of 1 cm immediately after the bulb (A), the second 1cm proximal the bulb (B) over the same distance of 1cm length. Means of end-diastolic far wall cIMT were compared between measurement A and B. cIMT in 30 participants was 0.51±0.06 mm for measurement A and 0.51±0.05 mm for measurement B. Results did not differ significantly (p = .947) over a distance of 2 cm after the bulb. According to our results, studies measuring CCA IMT within the first 2 cm, either close to the bulb or further proximal, can be compared. This will improve interpretation of data and application of reference values.     

Early vigour in wheat: Could it lead to more severe terminal drought stress under elevated atmospheric [CO2] and semi‐arid conditions?

Early vigour in wheat is a trait that has received attention for its benefits reducing evaporation from the soil surface early in the season. However, with the growth enhancement common to crops grown under elevated atmospheric CO₂ concentrations (e[CO₂]), there is a risk that too much early growth might deplete soil water and lead to more severe terminal drought stress in environments where production relies on stored soil water content. If this is the case, the incorporation of such a trait in wheat breeding programmes might have unintended negative consequences in the future, especially in dry years. We used selected data from cultivars with proven expression of high and low early vigour from the Australian Grains Free Air CO₂ Enrichment (AGFACE) facility, and complemented this analysis with simulation results from two crop growth models which differ in the modelling of leaf area development and crop water use. Grain yield responses to e[CO₂] were lower in the high early vigour group compared to the low early vigour group, and although these differences were not significant, they were corroborated by simulation model results. However, the simulated lower response with high early vigour lines was not caused by an earlier or greater depletion of soil water under e[CO₂] and the mechanisms responsible appear to be related to an earlier saturation of the radiation intercepted. Whether this is the case in the field needs to be further investigated. In addition, there was some evidence that the timing of the drought stress during crop growth influenced the effect of e[CO₂] regardless of the early vigour trait. There is a need for FACE investigations of the value of traits for drought adaptation to be conducted under more severe drought conditions and variable timing of drought stress, a risky but necessary endeavour.     

Na+-translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-translocating pyrophosphatases

Membrane pyrophosphatases (PPases), divided into K(+)-dependent and K(+)-independent subfamilies, were believed to pump H(+) across cell membranes until a recent demonstration that some K(+)-dependent PPases function as Na(+) pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na(+) transport, and H(+) transport activities as well as their K(+) and Na(+) requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K(+)-dependent Na(+) transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na(+)-transporting PPases (Na(+)-PPases) within the K(+)-dependent subfamily. H(+)-transporting PPases (H(+)-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na(+)-PPases predominantly constitute the K(+)-dependent subfamily. Furthermore, Na(+)-PPases possibly preceded H(+)-PPases in evolution, and transition from Na(+) to H(+) transport may have occurred in several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na(+)-PPases but shifted to within the membrane or helix 5 in H(+)-PPases. These results contribute to the prediction of the transport specificity and K(+) dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K(+) dependence signature, and position of the membrane-located Glu residue.     

Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are the two ligands of the Tie-2 receptor, a receptor tyrosine kinase that is expressed on the endothelium. A balanced angiopoietin/Tie-2 system is critical for the maintenance of vascular integrity. We investigated the potential role of a disrupted angiopoietin/Tie-2 system on hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Using streptozotocin (STZ) mice subjected to myocardial ischemia, we examined the effects of shifting the Ang-2-to-Ang-1 ratio on myocardial infarction size, apoptosis, bone marrow (BM) cell-endothelial progenitor cell (EPC) differentiation, and angiogenesis. In control mice, myocardial ischemia increased expression of both Ang-2 and Tie-2. In STZ mice, Ang-2 expression was elevated, whereas Tie-2 expression was reduced, and neither was significantly altered by ischemia. Myocardial infarct size and apoptosis were increased in STZ compared with control mice. Using in vivo administration of an adenovirus containing Ang-1 or Ang-2, we found that shifting the Ang-2-to-Ang-1 ratio to favor Ang-1 reduced myocardial apoptosis and infarct size in STZ mice, while shifting the Ang-2-to-Ang-1 ratio to favor Ang-2 resulted in a significant increase in myocardial infarct size and apoptosis in control mice. Myocardial ischemia-stimulated BM cell-EPC differentiation was inhibited and myocardial angiogenesis was reduced in STZ mice. Systemic administration of Ad-Ang-1 restored BM cell-EPC differentiation and increased myocardial VEGF expression and angiogenesis in STZ mice. Our data demonstrate that disturbed angiopoietin/Tie-2 signaling contributes to the hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Restoration of the Ang-2-to-Ang-1 ratio may be a novel therapeutic strategy for the treatment of diabetic myocardial ischemic diseases.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Slow Orthograde Axonal Transport of Radiolabelled Protein in Sciatic Motoneurones of Rats with Short-Term Experimental Diabetes: Effects of Treatment with an Aldose Reductase Inhibitor or myo-Inositol

Abstract: This study examined the effect of streptozotocin diabetes of 5 weeks duration on the profile of slow orthogradely transported radiolabelled protein in rat sciatic motoneurones. The diabetic rats showed a retardation of the tail of the slow-component profile. This selective retardation was unaffected by treatment with an aldose reductase inhibitor, although this treatment reduced the accumulation of sorbitol and prevented the depletion of myo -inositol in the sciatic nerves of the treated diabetic rats. Other groups, treated with myo -inositol, had normal or elevated sciatic nerve myo -inositol levels in the presence of accumulated sorbitol. The axonal transport profiles from both control and diabetic myo-inositol-treated groups gave normal tail velocities but an altered shape such that retardation of the tail of the profile may have been present in both. The study concludes that rats with 5 weeks streptozotocin diabetes show retardation of the velocity of the most slowly transported proteins in sciatic motoneurones, and that this defect is not linked to the polyol pathway.     

Versican expression during synovial joint morphogenesis

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.     

Modeling Interventions in the Owned Cat Population to Decrease Numbers,Knox County,TN

To find management strategies for controlling the owned cat population in Knox County, TN, the authors formulated a mathematical model using biological properties of such nonhuman animals and spay actions on certain age classes. They constructed this discrete-time model to predict the future owned cat population in this county and to evaluate intervention strategies to surgically sterilize some proportion of the population. Using the predicted population size and the number of surgeries for specific scenarios, they showed that focusing on specific age classes can be an effective feature in spay programs.     

Phylogeography and population differentiation in terrestrial island populations of Banksia arborea (Proteaceae)

The Banded Iron Formations (BIFs) of south‐western Australia are terrestrial islands characterized by high species richness and endemism. Regional endemics occur across multiple formations without inhabiting the intervening landscape matrix. We investigated whether the occurrence on BIF terrestrial islands has led to genetic differentiation among the eight known populations of the regional endemic, Banksia arborea. Genetic structure was assessed using three chloroplast DNA sequence markers and 11 nuclear microsatellite loci. Phylogenetic relationships were assessed with statistical parsimony and Bayesian methods. Dates of haplotype divergence were estimated using the time to most recent common ancestor of B. arborea and Banksia purdieana, as well as a conservative angiosperm chloroplast (cp)DNA mutation rate. Population genetic diversity and structure was assessed amongst and within populations by genotyping 24 geographically clustered individuals from each BIF and three subpopulations within the Die Hardy Range BIF. Indirect gene flow estimates were determined using a method based on the frequency of private alleles. Banksia arborea showed low genetic diversity in (cp)DNA and a complex structural pattern, with genetic differentiation of some BIF populations and an absence of differentiation amongst others, reflecting either retention of ancestral polymorphism across northern BIF populations or more recent connectivity of these populations. There was little evidence of pollen dispersal both between BIFs and within the large BIF known as Die Hardy Range. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 860–872.