Synthesis of Graphene and Graphene Films with Minimal Structural Defects.

Graphene is a carbon material with extraordinary properties that has been drawing a significant amount of attention in the recent decade. High-quality graphene can be produced by different methods, such as epitaxial growth, chemical vapor deposition, and micromechanical exfoliation. The reduced graphene oxide route is, however, the only current approach that leads to the large-scale production of graphene materials at a reasonable cost. Unfortunately, graphene oxide reduction normally yields graphene materials with a high defect density. Here, we introduce a new route for the large-scale synthesis of graphene that minimizes the creation of structural defects. The method involves high-quality hydrogen functionalization of graphite followed by thermal dehydrogenation. We also demonstrated that the hydrogenated graphene synthesis route can be used for the preparation of high-quality graphene films on glass substrates. A reliable method for the preparation of these types of films is essential for the widespread implementation of graphene devices. The structural evolution from the hydrogenated form to graphene, as well as the quality of the materials and films, was carefully evaluated by Raman spectroscopy.

The Impact of Treatment Delay on Hepatitis C Liver Transplant Outcomes.

Background: Direct-acting antivirals for the treatment of hepatitis C virus (HCV) have improved outcomes in liver transplant recipients (LTRs). However, the timing of HCV treatment and approach to treating rejection have not been well described. Additionally, pharmacists' roles in these comprehensive areas have not been investigated. Methods: This single-center, retrospective, cohort review compared 1-year graft and patient survival between HCV-positive and HCV-negative LTRs. Secondary endpoints included 1-year rejection rates, HCV sustained virologic response and time to HCV treatment. Results: Ninety-two HCV Nucleic Acid Amplification Test (NAT)-positive LTRs were matched 1:1 to HCV-seronegative LTRs. One-year graft and patient survival were similar between groups. HCV-positive LTRs were more likely to experience biopsy-proven acute rejection (BPAR), and despite treatment with pulse steroids, there was no impact on graft survival or occurrence of fibrosing cholestatic hepatitis (FCH). Time to HCV treatment was 5.4-6.4 months post-transplant, with no treatment failures or impact on graft or patient survival. Conclusions: No difference was seen in graft survival at 1 year between HCV-positive and HCV-seronegative LTRs. Delayed time to treatment of HCV and treatment of rejections in the HCV-positive cohort did not impact outcomes. However, pharmacist-driven protocols could ensure more efficient initiation of HCV treatment in the future.

An Economical and Scalable Method to Synthesize Graphitic-Like Films.

An economical and facile method to synthesize a precursor for carbon films and materials has been developed. This precursor can be easily coated onto substrates without binder reagents and then converted into a graphitic-like structure after mild thermal treatment. This approach potentially allows the coating of glass surfaces of different shapes and forms, such as the inside of a glass tube, for instance. The precursor consists of tetrahedral halocarbyne units which randomly combine through single electron transfer with organometallic compounds to create a poly(carbyne)-like polymeric material. Advanced characterization tools reveal that the synthesized product (poly(halocarbyne) or PXC, where X indicate the presence of halogens, is composed mostly of carbon, hydrogen, and a variable percentage of residual halocarbon groups. Therefore, it possesses good solubility in organic solvents and can be coated on any complex substrate. The coated PXC material produced here was annealed under mild conditions, leading to the production of a graphitic-like film on a glass substrate. The chemical homogeneity of the carbon material of the film was confirmed by Raman spectroscopy.

Interaction of the Mechanosensitive Channel, MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets.

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein-lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even "conservative" hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.

Towards measuring growth rates of pathogens during infections by D2 O-labeling lipidomics.

RATIONALE: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. METHODS: Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. RESULTS: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. CONCLUSIONS: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

The photochemical thiol-ene reaction as a versatile method for the synthesis of glutathione S-conjugates targeting the bacterial potassium efflux system Kef.

The thiol-ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione-S-conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K+ efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K+ homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs.

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea.

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis.

Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau ), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.

The evolution of bacterial mechanosensitive channels.

Mechanosensitive channels are ubiquitous and highly studied. However, the evolution of the bacterial channels remains enigmatic. It can be argued that mechanosensitivity might be a feature of all membrane proteins with some becoming progressively less sensitive to membrane tension over the course of evolution. Bacteria and archaea exhibit two main classes of channels, MscS and MscL. Present day channels suggest that the evolution of MscL may be highly constrained, whereas MscS has undergone elaboration via gene fusion (and potentially gene fission) events to generate a diversity of channel structures. Some of these channel variants are constrained to a small number of genera or species. Some are only found in higher organisms. Only exceptionally have these diverse channels been investigated in any detail. In this review we consider both the processes that might have led to the evolved complexity but also some of the methods exploiting the explosion of genome sequences to understand (and/or track) their distribution. The role of MscS-related channels in calcium-mediated cell biology events is considered.

Bacterial mechanosensitive channels: progress towards an understanding of their roles in cell physiology.

Bacterial mechanosensitive channels sense the changes in lateral tension in the bilayer of the cytoplasmic membrane generated by rapid water flow into the cell. Two major structural families are found widely distributed across bacteria and archaea: MscL and MscS. Our understanding of the mechanisms of gating has advanced rapidly through genetic analysis, structural biology and electrophysiology. It is only recently that the analysis of the physiological roles of the channels has kept pace with mechanistic studies. Recent advances have increased our understanding of the role of the channels in preventing structural perturbation during osmotic transitions and its relationship to water flow across the membrane. It is to these recent developments that this review is dedicated.

Understanding the structural requirements for activators of the Kef bacterial potassium efflux system.

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.

Probing the structure of the mechanosensitive channel of small conductance in lipid bilayers with pulsed electron-electron double resonance.

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.

Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper?

Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.

Integrated stress response of Escherichia coli to methylglyoxal: transcriptional readthrough from the nemRA operon enhances protection through increased expression of glyoxalase I.

Methylglyoxal (MG) elicits activation of K(+) efflux systems to protect cells against the toxicity of the electrophile. ChIP-chip targeting RNA polymerase, supported by a range of other biochemical measurements and mutant creation, was used to identify genes transcribed in response to MG and which complement this rapid response. The SOS DNA repair regulon is induced at cytotoxic levels of MG, even when exposure to MG is transient. Glyoxalase I alone among the core MG protective systems is induced in response to MG exposure. Increased expression is an indirect consequence of induction of the upstream nemRA operon, encoding an enzyme system that itself does not contribute to MG detoxification. Moreover, this induction, via nemRA only occurs when cells are exposed to growth inhibitory concentrations of MG. We show that the kdpFABCDE genes are induced and that this expression occurs as a result of depletion of cytoplasmic K(+) consequent upon activation of the KefGB K(+) efflux system. Finally, our analysis suggests that the transcriptional changes in response to MG are a culmination of the damage to DNA and proteins, but that some integrate specific functions, such as DNA repair, to augment the allosteric activation of the main protective system, KefGB.

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.

Characterization of three novel mechanosensitive channel activities in Escherichia coli.

Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves.

Single-celled organisms must survive exposure to environmental extremes. Perhaps one of the most variable and potentially life-threatening changes that can occur is that of a rapid and acute decrease in external osmolarity. This easily translates into several atmospheres of additional pressure that can build up within the cell. Without a protective mechanism against such pressures, the cell will lyse. Hence, most microbes appear to possess members of one or both families of bacterial mechanosensitive channels, MscS and MscL, which can act as biological emergency release valves that allow cytoplasmic solutes to be jettisoned rapidly from the cell. While this is undoubtedly a function of these proteins, the discovery of the presence of MscS homologues in plant organelles and MscL in fungus and mycoplasma genomes may complicate this simplistic interpretation of the physiology underlying these proteins. Here we compare and contrast these two mechanosensitive channel families, discuss their potential physiological roles, and review some of the most relevant data that underlie the current models for their structure and function.

Bacterial mechanosensitive channels--MscS: evolution's solution to creating sensitivity in function.

The discovery of mechanosensing channels has changed our understanding of bacterial physiology. The mechanosensitive channel of small conductance (MscS) is perhaps the most intensively studied of these channels. MscS has at least two states: closed, which does not allow solutes to exit the cytoplasm, and open, which allows rapid efflux of solvent and solutes. The ability to appropriately open or close the channel (gating) is critical to bacterial survival. We briefly review the science that led to the isolation and identification of MscS. We concentrate on the structure-function relationship of the channel, in particular the structural and biochemical approaches to understanding channel gating. We highlight the troubling discrepancies between the various models developed to understand MscS gating.