Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.     

Activity stabilization of Aspergillus niger and Escherichia coli phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays

The aim of this work was to study the stabilization of the activity of two commercial microbial phytases (Aspergillus niger and Escherichia coli) after immobilization on nanoclays and to establish optimal conditions for their immobilization. Synthetic allophane, synthetic iron-coated allophanes and natural montmorillonite were chosen as solid supports for phytase immobilization. Phytase immobilization patterns at different pH values were strongly dependent on both enzyme and support characteristics. After immobilization, the residual activity of both phytases was higher under acidic conditions. Immobilization of phytases increased their thermal stability and improved resistance to proteolysis, particularly on iron-coated allophane (6% iron oxide), which showed activation energy (E(a)) and activation enthalpy (ΔH(#)) similar to free enzymes. Montmorillonite as well as allophanic synthetic compounds resulted in a good support for immobilization of E. coli phytase, but caused a severe reduction of A. niger phytase activity.     

Development and validation of a high-throughput screening assay for the hepatitis C virus p7 viroporin

The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.     

Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design

Carbazole 1,9a-dioxygenase (CarA), the first enzyme in the carbazole degradation pathway used by Pseudomonas sp., was expressed in E. coli under different conditions defined by experimental design. This enzyme depends on the coexistence of three components containing [2Fe-2S] clusters: CarAa, CarAc, and CarAd. The catalytic site is present in CarAa. The genes corresponding to components of carbazole 1,9a-dioxygenase from P. stutzeri were cloned and expressed by salt induction in E. coli BL21-SI (a host that allows the enhancement of overexpressed proteins in the soluble fraction), using the vector pDEST?14. The expression of these proteins was performed under different induction conditions (cell concentration, temperature, and time), with the help of two-level factorial design. Cell concentration at induction (measured by absorbance at 600 nm) was tested at 0.5 and 0.8. After salt induction, expression was performed at 30 and 37°C, for 4 h and 24 h. Protein expression was evaluated by densitometry analysis. Expression of CarAa was enhanced by induction at a lower cell concentration and temperature and over a longer time, according to the analysis of the experimental design results. The results were validated at Abs (ind) = 0.3, 25°C, and 24 h, at which CarAa expression was three times higher than under the standard condition. The behavior of CarAc and CarAd was the inverse, with the best co-expression condition tested being the standard one (Abs (ind) = 0.5, T = 37°C, and t = 4 h). The functionality of the proteins expressed in E. coli was confirmed by the degradation of 20 ppm carbazole.     

Global human footprint on the linkage between biodiversity and ecosystem functioning in reef fishes

Difficulties in scaling up theoretical and experimental results have raised controversy over the consequences of biodiversity loss for the functioning of natural ecosystems. Using a global survey of reef fish assemblages, we show that in contrast to previous theoretical and experimental studies, ecosystem functioning (as measured by standing biomass) scales in a non-saturating manner with biodiversity (as measured by species and functional richness) in this ecosystem. Our field study also shows a significant and negative interaction between human population density and biodiversity on ecosystem functioning (i.e., for the same human density there were larger reductions in standing biomass at more diverse reefs). Human effects were found to be related to fishing, coastal development, and land use stressors, and currently affect over 75% of the world's coral reefs. Our results indicate that the consequences of biodiversity loss in coral reefs have been considerably underestimated based on existing knowledge and that reef fish assemblages, particularly the most diverse, are greatly vulnerable to the expansion and intensity of anthropogenic stressors in coastal areas.     

Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells

Background

Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.

Methodology/Principal Findings

We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.

Conclusions/Significance

Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.