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The aerospace industry can benefit from conversational agents that provide efficient solutions for safety-of-life scenarios. This industry is characterized by products and systems that require years of engineering to achieve optimal performance within complex environments. With recent advances in retrieval and language models, conversational agents can be developed to enhance the system's question-answering capabilities. However, evaluating the added-value of such systems in the context of industrial applications, such as pilots in a cockpit, can be challenging. This article presents the design, implementation, and user evaluation of a conversational agent called Smart Librarian, which is tailored to the aerospace domain's specific requirements to support pilots in their tasks. Our results based on a controlled user experiment with flight school students indicate that the user's perception of the usefulness of the system in completing the search task is a good predictor of both task score and time spent. The system's responses to the relevance of the topic is also a good predictor of task score. The perceived difficulty of the search task and its interaction with the relevance of the system's responses to the topic also play a key role in search performance. The mixed-effects models constructed in this study had large effect sizes, demonstrating participants' ability to assess their performance accurately. Nevertheless, user satisfaction with the system's responses may not be a reliable predictor of user search performance. Implications for the design of conversational agents based on the domain's specific requirements are discussed. [ABSTRACT FROM AUTHOR]

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed 19F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the 19F chemical shift anisotropy, monitored through a C F bond order parameter (S CF), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning 19F ssNMR spectra with and without 1H decoupling, by studying alterations in the second spectral moment (M 2) as well as the 19F isotropic chemical shift, linewidth, T 1 , and T 2 relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T 1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using 19F ssNMR. [Display omitted] • Monofluorinated palmitic acid enables labeling of the red blood cell membrane. • 19F ssNMR reveals the interaction between the cationic antimicrobial peptide caerin 1.1 and erythrocyte membranes. • 19F chemical shift anisotropy provides information about lipid acyl chain order and phase. • MAS 19F ssNMR can differentiate between lamellar and non-lamellar phases. • Lipid bilayer dynamics determined by MAS 19F ssNMR informs on the formation of high curvature regions in the membrane. [ABSTRACT FROM AUTHOR]

Abstract Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo 2H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies. Graphical abstract Unlabelled Image Highlights • Marine bacteria are studied by in vivo solid-state NMR for the first time. • 2H isotopic labelling of marine bacteria membranes is optimized. • Membrane fluidity measured by NMR changes with bacterial growth time. • Fatty acid analysis shows bacterial adaptation to labelling conditions. • Labelled bacteria harvested in the late exponential phase are in a native state. [ABSTRACT FROM AUTHOR]

The hERG channel is a voltage-gated potassium channel found in cardiomyocytes that contributes to the repolarization of the cell membrane following the cardiac action potential, an important step in the regulation of the cardiac cycle. The lipids surrounding K channels have been shown to play a key role in their regulation, with anionic lipids shown to alter gating properties. In this study, we investigate how anionic lipids interact with the pore helix of hERG and compare the results with those from Kv1.5, which possesses a pore helix more typical of K channels. Circular dichroism studies of the pore helix secondary structure reveal that the presence of the anionic lipid DMPS within the bilayer results in a slight unfolding of the pore helices from both hERG and Kv1.5, albeit to a lesser extent for Kv1.5. In the presence of anionic lipids, the two pore helices exhibit significantly different interactions with the lipid bilayer. We demonstrate that the pore helix from hERG causes significant perturbation to the order in lipid bicelles, which contrasts with only small changes observed for Kv1.5. These observations suggest that the atypical sequence of the pore helix of hERG may play a key role in determining how anionic lipids influence its gating. [ABSTRACT FROM AUTHOR]

Bicelles (bilayered micelles) are model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- and short-chain phospholipids, usually dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because their composition and planar surface are similar to the native membrane environment. In this work, to improve the solubilization of membrane proteins and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family, of which dodecylphosphocholine (12PC) is known for its ability to solubilize membrane proteins. More specifically 12PC, tetradecyl- (14PC), and hexadecyl-PC (16PC) have been employed. To verify the possibility of making bicelles with different hydrophobic thicknesses to better accommodate membrane proteins, D14PC was also replaced by phospholipids with different alkyl chain lengths: dilauroyl-PC (D12PC), dipalmitoyl-PC (D16PC), distearoyl-PC (D18PC), and diarachidoyl-PC (D20PC). Results obtained by 31P solid-state nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) at several lipid-to-detergent molar ratios (q) and temperatures indicate that these new MAPCHO bicelles can be formed under a variety of conditions. The quality of their alignment is similar to that of classical bicelles, and the low critical micelle concentration (CMC) of the surfactants and their miscibility with phospholipids are likely to be advantageous for the reconstitution of membrane proteins. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes using model membrane systems. Ideally, considering the complexity of membranes, the interaction of molecules with membranes should be studied in vivo. The interactions of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis were investigated using deuterium solid-state NMR. Specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. The minimal inhibitory concentration value for E. coli and B. subtilis was about 100 μg/mL and 30 μg/mL, respectively, for both peptides. A protocol to deuterate the membrane phospholipids of non-mutated B. subtilis was established using deuterated palmitic acid. 2 H NMR spectra combined with spectral moment analysis support the interaction of the two AMPs with the hydrophobic core of the bacterial membranes. The presence of peptides decreased the order of the lipid acyl chains for both E. coli and B. subtilis , but at higher peptide concentrations for the Gram(+) bacteria. This may be explained by the presence of other cell wall components, such as the negatively-charged teichoic and lipoteichoic acids in the peptidoglycan, which would interact with the AMPs and decrease their actual concentration on the membrane surface. The mechanism of action of the AMPs thus depends on their local concentration as well as the membrane environment. The differences between the AMPs interaction with E. coli and B. subtilis reveal the importance of studying intact bacteria. [ABSTRACT FROM AUTHOR]

Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater ( Chlamydomonas reinhardtii ) and marine ( Pavlova lutheri and Nannochloropsis oculata ) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by 13 C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as 13 C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky. [ABSTRACT FROM AUTHOR]

Bicelles are modelmembranes generally made of long-chain dimyristoylphosphatidylcholine(DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensivelyused in the study of membrane interactions and structure determinationof membrane-associated peptides, since their composition and morphologymimic the widespread PC-rich natural eukaryotic membranes. At lowDMPC/DHPC (q) molar ratios, fast-tumbling bicellesare formed in which the DMPC bilayer is stabilized by DHPC moleculesin the high-curvature rim region. Experimental constraints imposedby techniques such as circular dichroism, dynamic light scattering,or microscopy may require the use of bicelles at high dilutions. Studieshave shown that such conditions induce the formation of small aggregatesand alter the lipid-to-detergent ratio of the bicelle assemblies.The objectives of this work were to determine the exact compositionof those DMPC/DHPC isotropic bicelles and study the lipid miscibility.This was done using 31P nuclear magnetic resonance (NMR)and exploring a wide range of lipid concentrations (2–400 mM)and qratios (0.15–2). Our data demonstratehow dilution modifies the actual DMPC/DHPC molar ratio in the bicelles.Care must be taken for samples with a total lipid concentration ≤250mM and especially at q∼ 1.5–2, sincemoderate dilutions could lead to the formation of large and slow-tumblinglipid structures that could hinder the use of solution NMR methods,circular dichroism or dynamic light scattering studies. Our results,supported by infrared spectroscopy and molecular dynamics simulations,also show that phospholipids in bicelles are largely segregated onlywhen q> 1. Boundaries are presented within whichcontrol of the bicelles’ qratio is possible.This work, thus, intends to guide the choice of qratio and total phospholipid concentration when using isotropicbicelles. [ABSTRACT FROM AUTHOR]

Abstract: The human ether-a-go-go-related gene (hERG) voltage-gated K+ channels are located in heart cell membranes and hold a unique selectivity filter (SF) amino acid sequence (SVGFG) as compared to other K+ channels (TVGYG). The hERG provokes the acquired long QT syndrome (ALQTS) when blocked, as a side effect of drugs, leading to arrhythmia or heart failure. Its pore domain – including the SF – is believed to be a cardiotoxic drug target. In this study combining solution and solid-state NMR experiments we examine the structure and function of hERG's L622-K638 segment which comprises the SF, as well as its role in the ALQTS using reported active drugs. We first show that the SF segment is unstructured in solution with and without K+ ions in its surroundings, consistent with the expected flexibility required for the change between the different channel conductive states predicted by computational studies. We also show that the SF segment has the potential to perturb the membrane, but that the presence of K+ ions cancels this interaction. The SF moiety appears to be a possible target for promethazine in the ALQTS mechanism, but not as much for bepridil, cetirizine, diphenhydramine and fluvoxamine. The membrane affinity of the SF is also affected by the presence of drugs which also perturb model DMPC-based membranes. These results thus suggest that the membrane could play a role in the ALQTS by promoting the access to transmembrane or intracellular targets on the hERG channel, or perturbing the lipid–protein synergy. [Copyright &y& Elsevier]

Abstract: Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate 2H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. 2H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences. [Copyright &y& Elsevier]

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using P- and H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics. [ABSTRACT FROM AUTHOR]

Abstract: The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS. [Copyright &y& Elsevier]

Excipients in the pharmaceutical formulation of oral drugs are notably employed to improve drug stability. However, they can affect drug absorption and bioavailability. Passive transport through intestinal cell walls is the main absorption mechanism of drugs and, thus, involves an interaction with the membrane lipids. Therefore in this work, the effect of the excipient NaHCO on the interaction of the anticholesterolemic drug fluvastatin sodium (FS) with membrane phospholipids was investigated by H NMR and FTIR spectroscopy. Sodium bicarbonate is often combined with fluvastatin for oral delivery to prevent its degradation. We have used model DMPC/DMPS membranes to mimic the phospholipid content of gut cell membranes. The results presented in this work show a 100% affinity of FS for the membrane phospholipids that is not modified by the presence of the excipient. However, NaHCO is shown to change the interaction mechanism of the drug. According to our data, FS enters the DMPC/DMPS bilayer interface by interacting with the lipids' polar headgroups and burying its aromatic moieties into the apolar core. Moreover, lipid segregation takes place between the anionic and zwitterionic lipids in the membranes due to a preferential interaction of FS with phosphatidylserines. The excipient counteracts this favored interaction without affecting the drug affinity and location in the bilayer. This work illustrates that preferential interactions with lipids can be involved in passive drug permeation mechanisms and gives evidence of a possible nonpassive role of certain excipients in the interaction of drugs with membrane lipids. [ABSTRACT FROM AUTHOR]

The design of stable redox active liposomes where the organometallic electroactive pendent was covalently bound to the phospholipid headgroup through a phospholipase D (PLD)-catalyzed transphosphatidylation reaction between a choline-bearing phospholipid and a primary alcohol containing a ferrocene derivative is reported. The functionalization of a liposome surface with this organometallic redox phospholipid allowed the study of membrane-bound electrochemical reactions, which are important in the design of redox-sensitive liposome delivery systems. [ABSTRACT FROM AUTHOR]

Abstract: The human ether-à-go-go related gene (hERG) potassium channels are located in the myocardium cell membrane where they ensure normal cardiac activity. The binding of drugs to this channel, a side effect known as drug-induced (acquired) long QT syndrome (ALQTS), can lead to arrhythmia or sudden cardiac death. The hERG channel is a unique member of the family of voltage-gated K+ channels because of the long extracellular loop connecting its transmembrane S5 helix to the pore helix in the pore domain. Considering the proximal position of the S5-P linker to the membrane surface, we have investigated the interaction of its central segment I583–Y597 with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I583–Y597 segment for the membrane where it would sit on the surface with no defined secondary structure. A structural dependence of this segment on model membrane composition was observed. A helical conformation is favoured in detergent micelles and in the presence of negative charges. Our results suggest that the interaction of the S5-P linker with the membrane could participate in the stabilization of transient channel conformations, but helix formation would be triggered by interactions with other hERG domains. Because potential drug binding sites on the S5-P linker have been identified, we have explored the role of this segment in ALQTS. Four LQTS-liable drugs were studied which showed more affinity for the membrane than this hERG segment. Our results, therefore, identify two possible roles for the membrane in channel functioning and ALQTS. [ABSTRACT FROM AUTHOR]

We report the observation of undetected (until now) residues of the prion protein fragment HET-s(218-289) which give rise to well-resolved 13C, 15N, and ¹H NMR resonances under high-resolution magic-angle spinning (HAMAS) conditions. The observed signals belong to large polymeric units as shown by measuring the lateral diffusion constants. The amino acids identified in the spectra are compatible with their localization in the segments of the protein which could not be detected in earlier solid-state NMR experiments. The observed chemical shifts indicate that these residues are in a random-coil conformation. Complementary experiments which detect only dynamic or static residues, respectively, strongly suggest that they belong to different parts of the same molecule. [ABSTRACT FROM AUTHOR]

Abstract: In this work, the temperature and pressure dependent growth of domains in DMPC/DSPC monolayers at various molar ratios was studied by Brewster angle microscopy. Upon compression, roughly discoidal domains with some branching are formed. Further compression leads to an increase in both the number and the average size of the domains, which range between ca. 5 and 20μm. The isobaric heating of the monolayers results in a gradual decrease of the domain size until their disappearance. The size and morphology of the domains depend not only on equilibrium parameters such as temperature, pressure and composition, but appear to be also strongly dependent on non-equilibrium parameters such as the rate of perturbation. The comparison between our results and those previously published for bilayers allows us to infer that the growth behaviour in monolayers can be qualitatively but not quantitatively extrapolated to bilayers. [Copyright &y& Elsevier]

Abstract: 31P and 1H solid-state nuclear magnetic resonance (NMR) experiments have been designed with the aim of studying directly the formation of supported bilayers tethered inside nanoporous aluminum oxide supports as a model of biomimetic membranes. The static and magic angle spinning 31P NMR spectra of the supported bilayers have been compared with the experimental and simulated spectra of a simpler model with cylindrical geometry, namely a phospholipid bilayer adsorbed on an oriented polymer sheet. The broadening observed for the nanoporous model is most likely due to the presence of paramagnetic ions in the aluminum oxide. A phospholipid lateral diffusion coefficient of (2.8±0.4)×10−8cm2/s has been measured for the tethered bilayer on a spherical support, indicating a good fluidity as compared with adsorbed membrane models. [Copyright &y& Elsevier]

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo 2H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(−) bacteria membranes. We also investigated the effect of marennine using 31P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis. [Display omitted] • The action mechanism of marennine on Vibrio splendidus is studied in vivo by 2H solid-state NMR. • The specific interaction with phospholipids is studied by 31P solid-state NMR with model membranes. • Marennine stiffens phospholipid acyl chains, even at low concentration. • The lipid ordering effect is greater for bacteria sampled in the stationary phase. • Marennine does not disrupt bacterial membranes but would act via alteration of the membrane fluidity. [ABSTRACT FROM AUTHOR]

We present a new membrane mimetic system using a membrane softening detergent commonly known as Tween 80 (TW80), to form oriented systems for solid-state NMR applications. TW80 is a fatty acid ester (oleate) of sorbitan polyethoxylate and a mild non-ionic surfactant. Phosphatidylcholine (PC)/TW80 model membrane systems were characterized by solid-state NMR and FTIR spectroscopy. 31P and 2H NMR spectra showed that DMPC (14:0) and DPPC (16:0) self-assemble with TW80 to form oriented structures, and maintain alignment over a wide range of molar ratios and temperatures. The addition of lanthanide ions revealed that the membrane alignment can be flipped from parallel to perpendicular with respect to the magnetic field direction. Using 15N solid-state NMR and a labeled model transmembrane peptide, we showed that TW80-based membranes can be employed to determine the peptide orientation in the magnetic field, which is useful for structural determination. Altogether, our work showed that TW80 could be exploited for direct and efficient membrane protein extraction and to enhance membrane and membrane protein orientation without using a detergent removal step. This approach could be extended to a wide range of membranes including native ones. Unlabelled Image • Phosphatidylcholine model membranes with improved magnetic orientation can be formed by adding the mild surfactant Tween-80 • The magnetic orientation is sufficient to yield highly resolved 31P and 2H solid-state NMR spectra • A transmembrane peptide incorporated in such membranes is shown to be well oriented • The membrane systems, with and without proteins, can be flipped to align the bilayer normal with the magnetic field using paramagnetic ions [ABSTRACT FROM AUTHOR]

Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its bioavailability for an organism, as well as its value in the food industry. Using two-dimensional (2D) high resolution solid-state nuclear magnetic resonance (SS-NMR) on 13C-labelled starches that were obtained from Chlamydomonas reinhardtii microalgae, we established a complete and unambiguous assignment for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We also assigned so far unreported non-reducing end groups and assessed starch chain length, crystallinity and amylose content. Starch was then characterized in situ, i.e., by 13C solid-state NMR of intact microalgal cells. Our in-cell methodology also enabled the identification of the effect of nitrogen starvation on starch metabolism. This work shows how solid-state NMR can enable the identification of starch structure, chemical modifications and biosynthesis in situ in intact microorganisms, eliminating time consuming and potentially altering purification steps. [ABSTRACT FROM AUTHOR]

• Mussel tissues accumulated a mean of 26 µg STX eq. 100 g–1 after a 120 h exposure to cultured A. catenella. • Regardless of saxitoxin concentration or immune cell type, most cells remained alive and non-apoptotic. • The neutral lipid content of mussel immune cells decreased by a factor of two with A. catenella exposure. • A. catenella exposure affected the energy metabolism and osmolyte composition of mussel immune cells. The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes – the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella. [ABSTRACT FROM AUTHOR]

Owing to the presence of abundant interchain interactions such as hydrogen bonds, polyureas (PU) are only swellable or soluble in a limited number of highly protonic solvents, and the viscosity of the solutions obtained is very high, making their chemical structure characterization hard or even impossible by standard NMR. Accurate structure analysis is also hard by solid-state NMR due to low spectral resolution. The presence of a side reaction in their synthesis generating biuret cross-links is often invoked to explain their insolubility. Here we demonstrate that High Resolution Magic Angle Spinning (HR-MAS) NMR is an efficient tool for the chemical structure analysis even for cross-linked PU. With ¹H, 13C, and ¹H-15N HSQC combined, a variety of linear and cross-linked PU is analyzed by HR-MAS NMR, and conclusive information on their chemical structure is obtained, which reveals for the first time that the biuret group is absent in all PU. [ABSTRACT FROM AUTHOR]

We develop an approach for quantifying cure that applies thermodynamic modelling to interpret DSC data that is consistent with solid state NMR. The effect of elements of monomer geometry, such as the angle between functional groups and the distance between them, on curing of epoxy-amine thermosets is explored experimentally using this new technique. The effect of the angle between functional groups on the glass transition temperature of the final cured network is significant. The effect of spacing between functional groups is masked by the rigidity of the spacer. Our approach to interpreting DSC data allows us to detect processes that would have been missed otherwise. This is exemplified by the observation of slow exothermic deterioration reaction. Our NMR results indicate that significant etherification occurs at 140 °C, unlike the commonly held belief that it is insignificant at such low temperatures. [ABSTRACT FROM AUTHOR]

This work proposes a new approach to characterize cell membranes in intact cells by 2 H solid-state nuclear magnetic resonance (NMR) in only a few hours using magic-angle spinning (MAS) and spectral moment analysis. The method was first validated on model dipalmitoylphosphatidylcholine (DPPC) membranes, allowing the detection of lipid fluctuations below the main transition temperature. Then the lipid dynamics in Escherichia coli membranes was compared in bacteria grown under different diets. More specifically, deuterated palmitic acid was used to isotopically label the phospholipid acyl chains in bacteria membranes, with or without the presence of protonated oleic acid. Our results showed improved lipid fluidity when bacteria were grown in the presence of oleic acid, which helps preserving the natural fatty acid profile in E. coli membranes. The MAS 2 H solid-state NMR study of membranes combined with spectral moment analysis showed to be a fast method compatible with in vivo bacterial studies, and should also be applicable to other micro-organisms to obtain molecular information on living cells by solid-state NMR. [ABSTRACT FROM AUTHOR]

Protein misfolding and amyloid deposition are associated with numerous diseases. The detailed characterization of the proteospecies mediating cell death remains elusive owing to the (supra)structural polymorphism and transient nature of the assemblies populating the amyloid pathway. Here we describe the identification of toxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). While N21Q filaments share structural properties with cytocompatible fibrils, including the 4.7 Å inter-strand distance and β-sheet-rich conformation, they concurrently display characteristics of oligomers, such as low thioflavin-T binding, high surface hydrophobicity and recognition by the A11 antibody, leading to high potency to disrupt membranes and cause cellular dysfunction. The toxic oligomer-like conformation of N21Q fibrils, which is preserved upon elongation, is transmissible to naïve IAPP. These stable fibrils expanding the conformational diversity of amyloid assemblies represent an opportunity to elucidate the structural basis of amyloid disorders. Nguyen et al identified cytotoxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). They presented evidence to show that these stable fibrils expand the conformational diversity of amyloid assemblies, which represents an opportunity to elucidate the structural basis of amyloid disorders. [ABSTRACT FROM AUTHOR]

Diabetes can elicit direct deleterious effects on the myocardium, independent of coronary artery disease or hypertension. These cardiac disturbances are termed diabetic cardiomyopathy showing increased risk of heart failure with or without reduced ejection fraction. Presently, there is no specific treatment for this type of cardiomyopathy and in the case of type I diabetes, it may start in early childhood independent of glycemic control. We hypothesized that alterations in isolated myocyte contractility and cardiac function are present in the early stages of experimental diabetes in rats before overt changes in myocardium structure occur. Diabetes was induced by single-dose injection of streptozotocin (STZ) in rats with data collected from control and diabetic animals 3 weeks after injection. Left ventricle myocyte contractility was measured by single-cell length variation under electrical stimulation. Cardiac function and morphology were studied by high-resolution echocardiography with pulsed-wave tissue Doppler imaging (TDI) measurements and three-lead surface electrocardiogram. Triglycerides, cholesterol and liver enzyme levels were measured from plasma samples obtained from both groups. Myocardial collagen content and perivascular fibrosis of atria and ventricle were studied by histological analysis after picrosirius red staining. Diabetes resulted in altered contractility of isolated cardiac myocytes with increased contraction and relaxation time intervals. Echocardiography showed left atrium dilation, increased end-diastolic LV and posterior wall thickness, with reduced longitudinal systolic peak velocity (S') of the septum mitral annulus at the apical four-chamber view obtained by TDI. Triglycerides, aspartate aminotransferase and alkaline phosphatase were elevated in diabetic animals. Intertitial collagen content was higher in atria of both groups and did not differ among control and diabetic animals. Perivascular intramyocardial arterioles collagen did not differ between groups. These results suggest that alterations in cardiac function are present in the early phase in this model of diabetes type 1 and occur before overt changes in myocardium structure appear as evaluated by intersticial collagen deposition and perivascular fibrosis of intramyocardial arterioles. [ABSTRACT FROM AUTHOR]

Abstract We report a novel and versatile method of grafting polymers onto lithium titanium oxide (LTO) by dispersion-mediated interfacial polymerization. A myriad of polymers can be grafted by this method, and the hybrid-polymer particles are compatible with all kinds of binders used in lithium-ion batteries. This method is capable of producing thin and homogenous polymer films that are useful for battery applications. A coating of poly(styrene) effectively prevents degradation of the cell after float test without affecting performance at high C rates. Graphical abstract Image 1 Highlights • Versatile method of grafting polymers onto lithium titanium oxide (LTO) is reported. • Hybrid-polymer particles are compatible with all kinds of binders. • Effect of different polymer coatings on cell performances is investigated. • Poly(styrene) coating mitigates degradation of cell after float test. [ABSTRACT FROM AUTHOR]

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and β-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells. [Display omitted] • IAPP induces the clustering of lipid vesicles derived from mammalian cell plasma membranes. • Membrane clusters exhibit colocalization between lipid bilayer vesicles. • Perturbation of cell-derived vesicles induced by IAPP can be detected by FRET. • FRET signal obtained from damaged cell-derived lipid vesicles can be kinetically followed using a microplate format. • A new assay is reported to investigate cell plasma membrane perturbation under biologically relevant conditions. [ABSTRACT FROM AUTHOR]

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membranebound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr- 545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels. [ABSTRACT FROM AUTHOR]

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase. [Display omitted] • A fast and facile lipid profile characterization pipeline is proposed. • Complete bacterial lipid profiles are determined at different growth stages. • Membrane fluidity is measured for bacteria under various labeling conditions. • Optimized lipid labeling protocol is established for whole-cell solid-state NMR. • Bacteria should be harvested at the early stationary phase. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) are incorporated in many consumer products and have a high-releasing potential in aquatic environments. In these environments, AgNPs could interact with colloidal natural organic matter (NOM) affecting their behavior and fate. Aggregation kinetics of AgNPs were conducted at 10 NaCl concentrations during 2 h with 2.5 or 25 mgNOM L−1. Four (4) colloidal NOM fractions were extracted from 50 m3 of St.-Lawrence estuarine waters: fulvic acids (SLFA), humic acids (SLHA), hydrophilic (SL4ILE), and hydrophobic (SL4OBE) fractions. The addition of NOM to AgNP dispersion in nanopure water induced a small increase in AgNP size depending on NOM concentration and fraction used. The presence of both salt and NOM significantly altered the size of silver aggregates. With added 2.5 mgNOM L−1, more than 90% of particles have a size smaller than 200 nm, independently of the NOM fraction used. At 25 mgNOM L−1, the particle size distribution was strongly affected by the nature of NOM fractions. Our results show that NOM with highly aromatic carbon content such as SLFA and SLHA promoted the formation of larger aggregates with a hydrodynamic diameter larger than 200 nm. A high content in non-polar aliphatic carbon as observed in SL4ILE and SL4OBE stabilized aggregates at a size below 200 nm. This study highlights the importance of the chemical composition of NOM fractions and their content in non-polar aliphatic and aromatic carbons as they are involved in hydrophobic effects and strongly influence the aggregate size distribution. Sulfur, also present at low concentration NOM fractions, appears to be involved in the aggregation of AgNPs, but its action mechanism was not determined. [Display omitted] • Effects of concentration of natural organic matter on the size of aggregates. • At 2.5 mgNOM L−1, more than 90% of particles have a size smaller than 200 nm. • At 25 mgNOM L−1, the particle size distribution was affected by the nature of NOM. • NOM with high content of aromatic carbon promote aggregates > 200 nm. • NOM with high content of non-polar aliphatic carbon promote aggregates < 200 nm. [ABSTRACT FROM AUTHOR]