Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.     

Evaluation of the Performance of Information Theory-Based Methods and Cross-Correlation to Estimate the Functional Connectivity in Cortical Networks

Functional connectivity of in vitro neuronal networks was estimated by applying different statistical algorithms on data collected by Micro-Electrode Arrays (MEAs). First we tested these “connectivity methods” on neuronal network models at an increasing level of complexity and evaluated the performance in terms of ROC (Receiver Operating Characteristic) and PPC (Positive Precision Curve), a new defined complementary method specifically developed for functional links identification. Then, the algorithms better estimated the actual connectivity of the network models, were used to extract functional connectivity from cultured cortical networks coupled to MEAs. Among the proposed approaches, Transfer Entropy and Joint-Entropy showed the best results suggesting those methods as good candidates to extract functional links in actual neuronal networks from multi-site recordings.     

Amplification of trial-to-trial response variability by neurons in visual cortex

The visual cortex responds to repeated presentations of the same stimulus with high variability. Because the firing mechanism is remarkably noiseless, the source of this variability is thought to lie in the membrane potential fluctuations that result from summated synaptic input. Here this hypothesis is tested through measurements of membrane potential during visual stimulation. Surprisingly, trial-to-trial variability of membrane potential is found to be low. The ratio of variance to mean is much lower for membrane potential than for firing rate. The high variability of firing rate is explained by the threshold present in the function that converts inputs into firing rates. Given an input with small, constant noise, this function produces a firing rate with a large variance that grows with the mean. This model is validated on responses recorded both intracellularly and extracellularly. In neurons of visual cortex, thus, a simple deterministic mechanism amplifies the low variability of summated synaptic inputs into the large variability of firing rate. The computational advantages provided by this amplification are not known.     

Inferring protein interactions from phylogenetic distance matrices

Finding the interacting pairs of proteins between two different protein families whose members are known to interact is an important problem in molecular biology. We developed and tested an algorithm that finds optimal matches between two families of proteins by comparing their distance matrices. A distance matrix provides a measure of the sequence similarity of proteins within a family. Since the protein sets of interest may have dozens of proteins each, the use of an efficient approximate solution is necessary. Therefore the approach we have developed consists of a Metropolis Monte Carlo optimization algorithm which explores the search space of possible matches between two distance matrices. We demonstrate that by using this algorithm we are able to accurately match chemokines and chemokine-receptors as well as the tgfbeta family of ligands and their receptors.     

Dual Utility of a Melanin‐Based Ornament in Bearded Tits

A general mechanism to account for the maintenance of sexually selected traits is the armament–ornament model, which predicts that females exploit signals used in male–male aggressive contests. Melanin‐based ornaments are good candidates to be dual utility traits because they are usually involved in male–male competition and function as signals of male dominance or fighting ability. Despite this, very few studies have investigated the dual utility of melanin‐based plumage traits and results remain generally ambiguous. In this study, we investigated the dual function of a melanin‐based trait (the black beard) in the male bearded tit, Panurus biarmicus. In a controlled aviary experiment, beard length was the best predictor of dominance in male–male competition. In a choice experiment, females showed a proximity preference for males whose beard length was experimentally elongated. Our results indicate that beard length plays an important role in both male–male competition and female choice in bearded tits, as proposed by the dual utility model.     

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.     

Interaction between Calpain-1 and HSP90: New Insights into the Regulation of Localization and Activity of the Protease

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca²⁺. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca²⁺, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca²⁺]_i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca²⁺-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.     

Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.