Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

Chronic Nitrogen Enrichment at the Watershed Scale Does Not Enhance Microbial Phosphorus Limitation

Increased N inputs through chronic atmospheric deposition has enriched temperate forest ecosystems, altering critical ecosystem functions such as decomposition and potentially resulting in a shift to P limitation. We used a combination of microbial biomass stoichiometry and enzymatic activity analyses to evaluate the potential for microbial nutrient limitation over the course of a growing season in response to multi-decadal, whole-watershed N enrichments and a one time, plot-scale P addition that occurred in the 22nd year of whole-watershed treatments. The one-time P addition increased microbial biomass threefold and reduced N-acetyl-glucosaminidase (NAG) and acid phosphatase (AP) activity 1 week after application, but there was no interaction with long-term experimental N enrichment to indicate a shift to P limitation. However, both N and P treatments increased C limitation independently of each other over the duration of the study based on measured increases in β-1,4-glucosidase (BG) activity relative to NAG and AP. Microbial biomass stoichiometry and enzyme activity indicated that BBWM is P limited regardless of N status. Our findings highlight the complex interactions between C, N, and P use and limitation in a forested ecosystem subjected to long-term N enrichment.     

Intra- and interspecific predation by adult female Metaseiulus occidentalis and Typhlodromus pyri (Acari: Phytoseiidae) when provisioned with varying densities and ratios of Tetranychus urticae (Acari: Tetranychidae) and phytoseiid larvae

The phytoseiid mites Metaseiulus occidentalis (Nesbitt) and Typhlodromus pyri Schueten are used together and alone as biological control agents against tetranychid pest mites of apple. Their effectiveness as control agents may be impacted by intraguild predation. The effects of prey species and prey density on the rates of inter- and intraspecific predation and oviposition by these two predators were investigated through a series of experiments. Adult female predators were given prey as mixed populations of phytoseiid larvae and larvae of a more preferred species, the spider mite, Tetranychus urticae Koch, at different densities and ratios. Typhlodromus pyri, more of a generalist predator, showed higher rates of predation and cannibalism on phytoseiid immatures at most prey densities and ratios. Manly preference indices indicated that T. pyri switched to feed on phytoseiid larvae at higher prey levels and ratios of T. urticae than M. occidentalis. This greater ability to use phytoseiid larvae as prey may help stabilize T. pyri populations when more preferred prey is unavailable. This may, in part, explain the observed persistence of T. pyri populations when M. occidentalis populations were decreasing in orchard test plots.     

Non-alkaloidal constituents of Virola elongata bark

Eleven of the major non-polar constituents of the dried bark of Virola elongata were isolated. A new neolignan, virolongin, two new lignans, dihydrosesartemin and β-dihydroyangambin, as well as the neolignan, eusiderin, the lignans, epi-sesartemin, epi-yangambin and yangambin, the cis and trans isomers of 3,5,4′-trimethoxystilbene and sitosterol were identified. The structures of virolongin, dihydrosesartemin and β-dihydroyangambin were determined.     

The Ulcerating Tuberculous Hilar Gland

In primary infection tuberculosis, the infected hilar gland(s) may cause involvement of peripheral lung tissue not only by pressure but also by rupture and discharge of caseous material into a bronchus. Atelectasis or lung infection or both may result and bronchiectasis may ensue.Early bronchoscopy is required when this form of tuberculosis fails to subside promptly under treatment.Bronchography is indicated to detect residual bronchiectasis which should be removed surgically.Three of six proved cases of Group A tuberculous tracheobronchitis caused by an ulcerating hilar gland required pulmonary resection for removal of residual bronchiectasis; two of these were complicated by atelectasis. All six patients are alive and well.