Folding Pathways of a Knotted Protein with a Realistic Atomistic Force Field

We report on atomistic simulation of the folding of a natively-knotted protein, MJ0366, based on a realistic force field. To the best of our knowledge this is the first reported effort where a realistic force field is used to investigate the folding pathways of a protein with complex native topology. By using the dominant-reaction pathway scheme we collected about 30 successful folding trajectories for the 82-amino acid long trefoil-knotted protein. Despite the dissimilarity of their initial unfolded configuration, these trajectories reach the natively-knotted state through a remarkably similar succession of steps. In particular it is found that knotting occurs essentially through a threading mechanism, involving the passage of the C-terminal through an open region created by the formation of the native -sheet at an earlier stage. The dominance of the knotting by threading mechanism is not observed in MJ0366 folding simulations using simplified, native-centric models. This points to a previously underappreciated role of concerted amino acid interactions, including non-native ones, in aiding the appropriate order of contact formation to achieve knotting.     

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Diversity of honey stores and their impact on pathogenic bacteria of the honeybee,Apis mellifera

Honeybee colonies offer an excellent environment for microbial pathogen development. The highest virulent, colony killing, bacterial agents are Paenibacillus larvae causing American foulbrood (AFB), and European foulbrood (EFB) associated bacteria. Besides the innate immune defense, honeybees evolved behavioral defenses to combat infections. Foraging of antimicrobial plant compounds plays a key role for this “social immunity” behavior. Secondary plant metabolites in floral nectar are known for their antimicrobial effects. Yet, these compounds are highly plant specific, and the effects on bee health will depend on the floral origin of the honey produced. As worker bees not only feed themselves, but also the larvae and other colony members, honey is a prime candidate acting as self‐medication agent in honeybee colonies to prevent or decrease infections. Here, we test eight AFB and EFB bacterial strains and the growth inhibitory activity of three honey types. Using a high‐throughput cell growth assay, we show that all honeys have high growth inhibitory activity and the two monofloral honeys appeared to be strain specific. The specificity of the monofloral honeys and the strong antimicrobial potential of the polyfloral honey suggest that the diversity of honeys in the honey stores of a colony may be highly adaptive for its “social immunity” against the highly diverse suite of pathogens encountered in nature. This ecological diversity may therefore operate similar to the well‐known effects of host genetic variance in the arms race between host and parasite.     

Mechanical Predictors of Discomfort during Load Carriage

Discomfort during load carriage is a major issue for activities using backpacks (e.g. infantry maneuvers, children carrying school supplies, or outdoor sports). It is currently unclear which mechanical parameters are responsible for subjectively perceived discomfort. The aim of this study was to identify objectively measured mechanical predictors of discomfort during load carriage. We compared twelve different configurations of a typical load carriage system, a commercially available backpack with a hip belt. The pressure distribution under the hip belt and the shoulder strap, as well as the tensile force in the strap and the relative motion of the backpack were measured. Multiple linear regression analyses were conducted to investigate possible predictors of discomfort. The results demonstrate that static peak pressure, or alternatively, static strap force is a significant (p<0.001) predictor of discomfort during load carriage in the shoulder and hip region, accounting for 85% or more of the variation in discomfort. As an additional finding, we discovered that the regression coefficients of these predictors are significantly smaller for the hip than for the shoulder region. As static peak pressure is measured directly on the body, it is less dependent on the type of load carriage system than static strap force. Therefore, static peak pressure is well suited as a generally applicable, objective mechanical parameter for the optimization of load carriage system design. Alternatively, when limited to load carriage systems of the type backpack with hip belt, static strap force is the most valuable predictor of discomfort. The regionally differing regression coefficients of both predictors imply that the hip region is significantly more tolerant than the shoulder region. In order to minimize discomfort, users should be encouraged to shift load from the shoulders to the hip region wherever possible, at the same time likely decreasing the risk of low back pain or injury.     

Heavy metals in sewage sludges contribute to their adverse effects on the arbuscular mycorrhizal fungus<Emphasis Type="Italic">Glomus mosseae</Emphasis>

Applying sewage sludges to agricultural land is a widespread practice because of the sludges’ agronomic value as a source of plant nutrients and organic matter. Nevertheless, sludges often contain micropollutants that can constitute a menace for health and the environment. Arbuscular mycorrhizal fungi are sensitive to sewage sludges that have been spiked, or not, with metallic trace elements (MTE). Here we have investigated if MTE in sewage sludges could be responsible for effects on mycorrhizal development betweenGlomus mosseae andMedicago truncatula. The impact of a dehydrated or composted urban sewage sludge spiked or not with MTE, was tested on spore germination and root colonization byG. mosseae. The sewage sludges depressed both the presymbiotic andin planta stages of development of the mycorrhizal fungus. This negative effect was more related to the metallic pollutant contents of the sludges than to the presence of antagonistic microorganisms or phosphorus.