Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V_max of 3.9 ± 0.2 μmol/min/mg and K _m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.     

MolMod – an open access database of force fields for molecular simulations of fluids

The MolMod database is presented, which is openly accessible at http://molmod.boltzmann-zuse.de and contains intermolecular force fields for over 150 pure fluids at present. It was developed and is maintained by the Boltzmann-Zuse Society for Computational Molecular Engineering (BZS). The set of molecular models in the MolMod database provides a coherent framework for molecular simulations of fluids. The molecular models in the MolMod database consist of Lennard-Jones interaction sites, point charges, and point dipoles and quadrupoles, which can be equivalently represented by multiple point charges. The force fields can be exported as input files for the simulation programmes ms2 and ls1 mardyn, GROMACS, and LAMMPS. To characterise the semantics associated with the numerical database content, a force field nomenclature is introduced that can also be used in other contexts in materials modelling at the atomistic and mesoscopic levels. The models of the pure substances that are included in the database were generally optimised such as to yield good representations of experimental data of the vapour–liquid equilibrium with a focus on the vapour pressure and the saturated liquid density. In many cases, the models also yield good predictions of caloric, transport, and interfacial properties of the pure fluids. For all models, references to the original works in which they were developed are provided. The models can be used straightforwardly for predictions of properties of fluid mixtures using established combination rules. Input errors are a major source of errors in simulations. The MolMod database contributes to reducing such errors.     

The use of electromyography for the assessment of sense of muscular effort: a test–retest reliability study

This study sought to examine the test–retest reliability to measure sense of muscular effort with electromyography (EMG). The EMG activity of the tibialis anterior muscle from 23 participants was recorded. Targets of EMG amplitudes produced at 10 and 20% of the maximum voluntary contraction (MVC) were calculated. Participants matched the target EMG level with and without visual feedback (FB). With NFB, the reliability was good to excellent when errors were represented as the average standard deviation (SD) of the error from the target (ICC_1,2 = 0.75 and 0.69 for 10 and 20% targets, respectively). Also, reliability was good when errors were presented as the average SD as a percentage of the MVC EMG (intraclass correlation coefficient (ICC_1,2) = 0.67 and 0.66, respectively, for 10 and 20% targets). Standard deviation around the target was the most reliable method to represent the error. This approach could be used as a simple cost-effective method to assess the sense of muscular effort.     

Effect of electrical stimulation of antagonist muscles for voluntary motor drive

Voluntary motor drive is an important central command that descends via the corticospinal tract to initiate muscle contraction. When electrical stimulation (ES) is applied to an antagonist or agonist muscle, it changes the agonist muscle’s representative motor cortex and thus its voluntary motor drive. In this study, we used a reaction time task to compare the effects of weak and strong ES of the antagonist or agonist muscle during the premotor period of a wrist extension. We recorded motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS) that was applied to the extensor carpi radialis (ECR; agonist) and flexor carpi radialis (FCR; antagonist). When stronger ES intensities were applied to the antagonist, the MEP control ratio in the ECR significantly increased during the premotor time. Furthermore, the MEP control ratio with stronger antagonist ES intensity was significantly larger than that in the agonist for the same ES intensity. In the FCR, the MEP control ratio was also significantly greater at the strong ES intensity than at the weak ES intensity. Furthermore, the MEP control ratio in the antagonist with a strong ES intensity was significantly larger than that in the agonist with the same ES intensity. These results suggest that agonist corticomotor excitability might be enhanced by ES of the antagonist, which in turn strongly activates the descending motor system in the preparation of agonist contraction.     

Colour‐based foraging diverges after multiple generations under different light environments

When the environment changes, sensory systems can adapt plastically or evolve genetically to the new surroundings, and traits and behaviours reliant on these sensory systems may also change, leading to altered evolutionary trajectories. We tested for differences in colour‐based foraging preferences of guppies (Poecilia reticulata) that lived for 6–10 generations under each of three light environments (green, lilac or control) to determine whether evolution under different light environments alters visually based behaviour. When tested in a common light environment, we found differences in pecking behaviour between treatments that were likely due to changes in the visual system. Pecking behaviour towards green stimuli was consistent across light treatments, possibly reflecting the importance of detecting green algae in the wild. The blue stimulus was only pecked at by fish from the control environments. Behaviour towards long wavelength stimuli varied, possibly due to the polymorphic nature of the long wavelength opsins. These results are consistent with one component of sensory drive but do not allow us to conclude whether these differences are due to plastic or evolved responses.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Local and long range potentials for heparin-protein systems for coarse-grained simulations

Heparin belongs to glycosaminoglycans (GAGs), a class of periodic linear anionic polysaccharides, which are functionally important components of the extracellular matrix owing to their interactions with various protein targets. Heparin is known to be involved in many cell signaling processes, while the experimental data available for heparin are significantly more abundant than for other GAGs. At the same time, the length and conformational flexibility of the heparin represent major challenges for its theoretical analysis. Coarse-grained (CG) approaches, which enable us to extend the size- and time-scale by orders of magnitude owing to reduction of system representation, appear, therefore, to be useful in simulating these systems. In this work, by using umbrella-sampling molecular dynamics simulations, we derived and parameterized the CG backbone-local potentials of heparin chains and the orientational potentials for the interactions of heparin with amino acid side chains to be further included in the physics-based Unified Coarse-Grained Model of biological macromolecules. With these potentials, simulations of extracellular matrix processes where both heparin and multiple proteins participate will be possible.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.     

Lower-limb muscle function during sidestep cutting

To investigate lower-limb muscle function during sidestep cutting, prior studies have analysed electromyography (EMG) data together with three dimensional motion analysis. Such an approach does not directly quantify the biomechanical role of individual lower-limb muscles during a sidestep cut. This study recorded three dimensional motion analysis, ground reaction force (GRF) and EMG data for eight healthy males executing an unanticipated sidestep cut. Using a musculoskeletal modelling approach, muscle function was determined by computing the muscle contributions to the GRFs and lower-limb joint moments. We found that bodyweight support (vertical GRF) was primarily provided by the vasti, gluteus maximus, soleus and gastrocnemius. These same muscles, along with the hamstrings, were also primarily responsible for modulating braking and propulsion (anteroposterior GRF). The vasti, gluteus maximus and gluteus medius were the key muscles for accelerating the centre-of-mass towards the desired cutting direction by generating a medially-directed GRF. Our findings have implications for designing retraining programs to improve sidestep cutting technique.