High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.     

Mode of Death on Chagas Heart Disease: Comparison with Other Etiologies. A Subanalysis of the REMADHE Prospective Trial

Background

Sudden death has been considered the main cause of death in patients with Chagas heart disease. Nevertheless, this information comes from a period before the introduction of drugs that changed the natural history of heart failure. We sought to study the mode of death of patients with heart failure caused by Chagas heart disease, comparing with non-Chagas cardiomyopathy.

Methods and results

We examined the REMADHE trial and grouped patients according to etiology (Chagas vs non-Chagas) and mode of death. The primary end-point was all-cause, heart failure and sudden death mortality; 342 patients were analyzed and 185 (54.1%) died. Death occurred in 56.4% Chagas patients and 53.7% non-Chagas patients. The cumulative incidence of all-cause mortality and heart failure mortality was significantly higher in Chagas patients compared to non-Chagas. There was no difference in the cumulative incidence of sudden death mortality between the two groups. In the Cox regression model, Chagas etiology (HR 2.76; CI 1.34–5.69; p = 0.006), LVEDD (left ventricular end diastolic diameter) (HR 1.07; CI 1.04–1.10; p<0.001), creatinine clearance (HR 0.98; CI 0.97–0.99; p = 0.006) and use of amiodarone (HR 3.05; CI 1.47–6.34; p = 0.003) were independently associated with heart failure mortality. LVEDD (HR 1.04; CI 1.01–1.07; p = 0.005) and use of beta-blocker (HR 0.52; CI 0.34–0.94; p = 0.014) were independently associated with sudden death mortality.

Conclusions

In severe Chagas heart disease, progressive heart failure is the most important mode of death. These data challenge the current understanding of Chagas heart disease and may have implications in the selection of treatment choices, considering the mode of death.

Trial Registration

ClinicalTrails.gov {"type":"clinical-trial","attrs":{"text":"NCT00505050","term_id":"NCT00505050"}}NCT00505050 (REMADHE)     

Evolution of swarm robotics systems with novelty search

Novelty search is a recent artificial evolution technique that challenges traditional evolutionary approaches. In novelty search, solutions are rewarded based on their novelty, rather than their quality with respect to a predefined objective. The lack of a predefined objective precludes premature convergence caused by a deceptive fitness function. In this paper, we apply novelty search combined with NEAT to the evolution of neural controllers for homogeneous swarms of robots. Our empirical study is conducted in simulation, and we use a common swarm robotics task—aggregation, and a more challenging task—sharing of an energy recharging station. Our results show that novelty search is unaffected by deception, is notably effective in bootstrapping evolution, can find solutions with lower complexity than fitness-based evolution, and can find a broad diversity of solutions for the same task. Even in non-deceptive setups, novelty search achieves solution qualities similar to those obtained in traditional fitness-based evolution. Our study also encompasses variants of novelty search that work in concert with fitness-based evolution to combine the exploratory character of novelty search with the exploitatory character of objective-based evolution. We show that these variants can further improve the performance of novelty search. Overall, our study shows that novelty search is a promising alternative for the evolution of controllers for robotic swarms.     

Potential Application of Turnip Oil (Raphanus sativus L.) for Biodiesel Production: Physical–Chemical Properties of Neat Oil, Biofuels and their Blends with Ultra-Low Sulphur Diesel (ULSD)

Turnip oil (TO; Raphanus sativus L.) produces seeds that contain around 26 wt% of inedible base stock that are suitable as a potential feedstock for biodiesel production. A turnip oil methyl ester (TME) was prepared from acid-catalyzed pretreated TO in an effort to evaluate important fuel properties of turnip oil-based biodiesel, such as kinematic viscosity, cloud point, pour point (PP), cold filter plugging point, acid value, oxidative stability and lubricity. A comparison was made with soybean oil methyl esters (SME) as per biodiesel fuel standards such as ASTM D6751 and EN 14214. TME was characterized using FTIR, HPLC and ¹H NMR. Except PP property, SME displays superior fuel properties compared to TME. Blends (B5 and B20) of TME in ultra-low sulphur diesel fuel (ULSD) were also assessed for the aforesaid fuel properties and compared to an analogous set of blends of soybean oil methyl ester in ULSD as per petro diesel fuel standards such as ASTM D975 and D7467. TME B5 blends in ULSD displayed improved PP property in comparison to neat ULSD and blends of SME in ULSD. It was demonstrated that the B5 and B20 blends of TME in ULSD had acceptable fuel properties as per ASTM D975 (for B5 blend) and ASTM D7467 (for B20 blend). In summary, turnip oil has potential as an alternative, non-food feedstock for biodiesel production.     

Systemic Down-Regulation of Delta-9 Desaturase Promotes Muscle Oxidative Metabolism and Accelerates Muscle Function Recovery following Nerve Injury

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles.     

Courtship Sounds Advertise Species Identity and Male Quality in Sympatric Pomatoschistus spp. Gobies

Acoustic signals can encode crucial information about species identity and individual quality. We recorded and compared male courtship drum sounds of the sand goby Pomatoschistus minutus and the painted goby P. pictus and examined if they can function in species recognition within sympatric populations. We also examined which acoustic features are related to male quality and the factors that affect female courtship in the sand goby, to determine whether vocalisations potentially play a role in mate assessment. Drums produced by the painted goby showed significantly higher dominant frequencies, higher sound pulse repetition rates and longer intervals between sounds than those of the sand goby. In the sand goby, male quality was predicted by visual and acoustic courtship signals. Regression analyses showed that sound amplitude was a good predictor of male length, whereas the duration of nest behaviour and active calling rate (i.e. excluding silent periods) were good predictors of male condition factor and fat reserves respectively. In addition, the level of female courtship was predicted by male nest behaviour. The results suggest that the frequency and temporal patterns of sounds can encode species identity, whereas sound amplitude and calling activity reflects male size and fat reserves. Visual courtship duration (nest-related behaviour) also seems relevant to mate choice, since it reflects male condition and is related to female courtship. Our work suggests that acoustic communication can contribute to mate choice in the sand goby group, and invites further study.     

The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.