A theoretical and experimental case study of the hydrogen bonding predilection of S-methylcysteine

S-containing amino acids can lead to two types of local NH···S interactions which bridge backbone NH sites to the side chain to form either intra- or inter-residue H-bonds. The present work reports on the conformational preferences of S-methyl-l-cysteine, Cys(Me), using a variety of investigating tools, ranging from quantum chemistry simulations, gas-phase UV and IR laser spectroscopy, and solution state IR and NMR spectroscopies, on model compounds comprising one or two Cys(Me) residues. We demonstrate that in gas phase and in low polarity solution, the C- and N-capped model compound for one Cys(Me) residue adopts a preferred C5–C6γ conformation which combines an intra-residue N–H···O=C backbone interaction (C5) and an inter-residue N–H···S interaction implicating the side-chain sulfur atom (C6γ). In contrast, the dominant conformation of the C- and N-capped model compound featuring two consecutive Cys(Me) residues is a regular type I β-turn. This structure is incompatible with concomitant C6γ interactions, which are no longer in evidence. Instead, C5γ interactions occur, that are fully consistent with the turn geometry and additionally stabilize the structure. Comparison with the thietane amino acid Attc, which exhibits a rigid cyclic side chain, pinpoints the significance of side chain flexibility for the specific conformational behavior of Cys(Me).

     

The contrasting genetic patterns of two sympatric flying fox species from the Comoros and the implications for conservation

Pteropus livingstonii and Pteropus seychellensis comorensis are endemic fruit bat species that are among the most threatened animals in the Comoros archipelago. Both species are pollinators and seed dispersers of native and cultivated plants and are thus of crucial importance for the regeneration of natural forests as well as for cultivated plantations. However, these species are subject to strong anthropogenic pressures and face one of the highest rates of natural habitat loss reported worldwide. Yet little is known about the population genetic structure of these two species, making it difficult to define relevant conservation strategies. In this study, we investigated for the two flying fox species (1) the level of genetic diversity within islands, as well as across the archipelago and (2) the genetic structure between the two islands (Anjouan and Mohéli) for P. livingstonii and between the four islands of the archipelago (Anjouan, Mohéli, Grande Comore and Mayotte) for P. s. comorensis using mitochondrial and microsatellite markers. The results revealed contrasting patterns of genetic structure, with P. s. comorensis showing low genetic structure between islands, whereas P. livingstonii exhibited high levels of inter-island genetic differentiation. Overall, the genetic analyses showed low genetic diversity for both species. These contrasting genetic patterns may be the result of different dispersal patterns and the populations’ evolutionary histories. Our findings lead us to suggest that in terms of conservation strategy, the two populations of P. livingstonii (on Anjouan and Mohéli islands) should be considered as two separate management units. We recommend focusing conservation efforts on the Anjouan population, which is the largest, exhibits the highest genetic diversity, and suffers the greatest anthropogenic pressure. As for P. s. comorensis, its four populations could be considered as a single unit for conservation management purposes. For this species, we recommend protecting roosting trees to reduce population disturbance.     

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.     

A computational approach for inferring the cell wall properties that govern guard cell dynamics

Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney‐shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin‐rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening.     

An automated quantitative image analysis tool for the identification of microtubule patterns in plants

High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule‐targeted herbicides and demonstrate that high throughput confocal imaging with integrated image analysis by CellArchitect can distinguish effects induced by the known herbicides indaziflam and trifluralin. The same platform was used to examine 6 other compounds with herbicidal activity, and at least 3 different effects induced by these compounds were profiled. We further show that CellArchitect can detect subcellular patterns tagged by actin and endoplasmic reticulum markers. Thus, the platform developed here can be used to automate image analysis of complex subcellular patterns for purposes such as herbicide discovery and mode of action characterisation. The capacity to use this tool to quantitatively characterize cellular responses lends itself to application across many areas of biology.      

Na v1.4 and Na v1.5 are modulated differently during muscle immobilization and contractile phenotype conversion

Muscle immobilization leads to modification in its fast/slow contractile phenotype. Since the properties of voltage-gated sodium channels (Na(v)) are different between "fast" and "slow" muscles, we studied the effects of immobilization on the contractile properties and the Na(v) of rat peroneus longus (PL). The distal tendon of PL was cut and fixed to the adjacent bone at neutral muscle length. After 4 or 8 wk of immobilization, the contractile and the Na(v) properties were studied and compared with muscles from control animals (Student's t-test). After 4 wk of immobilization, PL showed a faster phenotype with a rightward shift of the force-frequency curve and a decrease in both the Burke's index of fatigability and the tetanus-to-twitch ratio. These parameters showed opposite changes between 4 and 8 wk of immobilization. The maximal sodium current in 4-wk immobilized fibers was higher compared with that of control fibers (11.5 ± 1.2 vs. 7.8 ± 0.8 nA, P = 0.008), with partial recovery to the control values in 8-wk immobilized fibers (8.6 ± 0.7 nA, P = 0.48). In the presence of tetrodotoxin, the maximal residual sodium current decreased continuously throughout immobilization. Using the Western blot analysis, Na(v)1.4 expression showed a transient increase in 4-wk muscle, whereas Na(v)1.5 expression decreased during immobilization. Our results indicate that a muscle immobilized at optimal functional length with the preservation of neural inputs exhibits a transient fast phenotype conversion. Na(v)1.4 expression and current are related to the contractile phenotype variation.     

Lincomycin and clindamycin conformations. A fragment shared by macrolides, ketolides and lincosamides determined from TRNOE ribosome-bound conformations

Two important lincosamide antibiotics, lincomycin and clindamycin were studied in the complex state with the bacterial ribosome after a conformational analysis by 1H and 13C NMR spectroscopy and molecular modelling of the unbound molecules. Lincosamide-ribosome interactions were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), resulting in a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrates in solution indicate that specific conformations are preferred in the bound state. Clindamycin, the more bioactive antibiotic studied, displayed a stronger NMR response than lincomycin showing that in lincosamide-ribosome interactions, a low affinity binding level is associated to the tight binding one and is related to biological activity. This study shows that conformation plays an essential role for the low affinity binding site. Superimposition of lincosamide, macrolide and ketolide bound structures exhibited conformational similarities in a particular fragment which is in agreement with a hypothesis of partial overlapping lincosamide and macrolide binding sites.     

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.