Biological and biochemical characterization of HIV‐1 Gag/dgp41 virus‐like particles expressed in Nicotiana benthamiana

The transmembrane HIV‐1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus‐like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant‐optimized HIV‐1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus‐based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV‐1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV‐1.     

Quantifying variation in forest disturbance,and its effects on aboveground biomass dynamics,across the eastern United States

The role of tree mortality in the global carbon balance is complicated by strong spatial and temporal heterogeneity that arises from the stochastic nature of carbon loss through disturbance. Characterizing spatio‐temporal variation in mortality (including disturbance) and its effects on forest and carbon dynamics is thus essential to understanding the current global forest carbon sink, and to predicting how it will change in future. We analyzed forest inventory data from the eastern United States to estimate plot‐level variation in mortality (relative to a long‐term background rate for individual trees) for nine distinct forest regions. Disturbances that produced at least a fourfold increase in tree mortality over an approximately 5 year interval were observed in 1–5% of plots in each forest region. The frequency of disturbance was lowest in the northeast, and increased southwards along the Atlantic and Gulf coasts as fire and hurricane disturbances became progressively more common. Across the central and northern parts of the region, natural disturbances appeared to reflect a diffuse combination of wind, insects, disease, and ice storms. By linking estimated covariation in tree growth and mortality over time with a data‐constrained forest dynamics model, we simulated the implications of stochastic variation in mortality for long‐term aboveground biomass changes across the eastern United States. A geographic gradient in disturbance frequency induced notable differences in biomass dynamics between the least‐ and most‐disturbed regions, with variation in mortality causing the latter to undergo considerably stronger fluctuations in aboveground stand biomass over time. Moreover, regional simulations showed that a given long‐term increase in mean mortality rates would support greater aboveground biomass when expressed through disturbance effects compared with background mortality, particularly for early‐successional species. The effects of increased tree mortality on carbon stocks and forest composition may thus depend partly on whether future mortality increases are chronic or episodic in nature.     

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae,Macrobdellidae)

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant‐orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well‐characterized anticoagulants from a variety of leeches with different feeding habits, including non‐sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx‐orthology with five and three anticoagulant‐families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.     

Intestinal Methanobrevibacter smithii but not total bacteria is related to diet‐induced weight gain in rats

It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism.

Objective:

Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions.

Design and Methods:

Sprague‐Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high‐fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high‐fat and normal chow until euthanasia.

Results:

Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high‐fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high‐fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high‐fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high‐fat chow which had minimal M. smithii colonization.

Conclusions:

We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model.     

Niche Separation of Methanotrophic Archaea (ANME-1 and -2) in Methane-Seep Sediments of the Eastern Japan Sea Offshore Joetsu

In this study, we investigated the diversity and spatial distribution of anaerobic methanotrophic archaea (ANMEs) in sediments of a gas hydrate field off Joetsu in the Japan Sea. Distribution of ANMEs in sediments was identified by targeting the gene for methyl coenzyme M reductase alpha subunit (mcrA), a phylogenetically conserved gene that occurs uniquely in methanotrophic and methanogenic archaea, in addition to 16S rRNA genes. Quantitative PCR analyses of mcrA genes in 14 piston core samples suggested that members of ANME-1 group would dominate AOM communities in sulfate-depleted sediments, even below the sulfate-methane interface, while ANME-2 archaea would prefer to populate in shallower sediments containing comparatively higher sulfate concentrations. These results suggest that, although the potential electron acceptors in sulfate-depleted habitats remain elusive, the niche separation of ANME-1 and -2 may be controlled by in situ concentration of sulfate and the availability in sediments.     

Evidence for Recycling of Invariant Surface Transmembrane Domain Proteins in African Trypanosomes

Intracellular trafficking is a vital component of both virulence mechanisms and drug interactions in Trypanosoma brucei, the causative agent of human African trypanosomiasis and n''agana of cattle. Both maintaining the surface proteome composition within a life stage and remodeling the composition when progressing between life stages are important features of immune evasion and development for trypanosomes. Our recent work implicates the abundant transmembrane invariant surface glycoproteins (ISGs) in the uptake of first-line therapeutic suramin, suggesting a potential therapeutic route into the cell. RME-8 is a mediator of recycling pathways in higher eukaryotes and is one of a small cohort of intracellular transport gene products upregulated in mammal-infective trypanosomes, suggesting a role in controlling the copy number of surface proteins in trypanosomes. Here we investigate RME-8 function and its contribution to intracellular trafficking and stability of ISGs. RME-8 is a highly conserved protein and is broadly distributed across multiple endocytic compartments. By knockdown we find that RME-8 is essential and mediates delivery of endocytic probes to late endosomal compartments. Further, we find ISG accumulation within endosomes, but that RME-8 knockdown also increases ISG turnover; combined with previous data, this suggests that it is most probable that ISGs are recycled, and that RME-8 is required to support recycling.