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There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non-enzymatically generate natural or non-canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water-soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide-forming ligations between lipid precursors bearing hydroxylamines and α-ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non-canonical phospholipids at physiological pH conditions. The generated amide-linked phospholipids spontaneously self-assemble into cell-like micron-sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells.
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Amidas , Fosfolípidos , HidroxilaminasRESUMEN
In cells, a vast number of membrane lipids are formed by the enzymatic O-acylation of polar head groups with acylating agents such as fatty acyl-CoAs. Although such ester-containing lipids appear to be a requirement for life on earth, it is unclear if similar types of lipids could have spontaneously formed in the absence of enzymatic machinery at the origin of life. There are few examples of enzyme-free esterification of amphiphiles in water and none that can occur in water at physiological pH using biochemically relevant acylating agents. Here we report the unexpected chemoselective O-acylation of 1,2-amino alcohol amphiphiles in water directed by Cu(II) and several other transition metal ions. In buffers containing Cu(II) ions, mixing biological 1,2-amino alcohol amphiphiles such as sphingosylphosphorylcholine with biochemically relevant acylating agents, namely, acyl adenylates and acyl-CoAs, leads to the formation of the O-acylation product with high selectivity. The resulting O-acylated sphingolipids self-assemble into vesicles with markedly different biophysical properties than those formed from their N-acyl counterparts. We also demonstrate that Cu(II) can direct the O-acylation of alternative 1,2-amino alcohols, including prebiotically relevant 1,2-amino alcohol amphiphiles, suggesting that simple mechanisms for aqueous esterification may have been prevalent on earth before the evolution of enzymes.
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Prebióticos , Agua , Esterificación , Acilcoenzima A/metabolismo , Lípidos de la Membrana , Amino Alcoholes , AcilaciónRESUMEN
We have developed a three-dimensional coherent diffraction imaging algorithm to retrieve phases of diffraction patterns of samples in grazing incidence small angle x-ray scattering experiments. The algorithm interprets the diffraction patterns using the distorted-wave Born approximation instead of the Born approximation, as in this case, the existence of a reflected beam from the substrate causes the diffraction pattern to deviate significantly from the simple Fourier transform of the object. Detailed computer simulations show that the algorithm works. Verification with real experiments is planned.
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Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.
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Galactolípidos/química , Gotas Lipídicas , Orgánulos/química , Ingeniería Química , Detergentes , Membrana Dobles de Lípidos , Péptido Hidrolasas , Proteínas/química , Proteínas/metabolismoRESUMEN
All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.
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Gotas Lipídicas , O(6)-Metilguanina-ADN Metiltransferasa , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Fosfolípidos , ProteínasRESUMEN
Cell membranes define the boundaries of life and primarily consist of phospholipids. Living organisms assemble phospholipids by enzymatically coupling two hydrophobic tails to a soluble polar head group. Previous studies have taken advantage of micellar assembly to couple single-chain precursors, forming non-canonical phospholipids. However, biomimetic nonenzymatic coupling of two alkyl tails to a polar head-group remains challenging, likely due to the sluggish reaction kinetics of the initial coupling step. Here we demonstrate rapid de novo formation of biomimetic liposomes in water using dual oxime bond formation between two alkyl chains and a phosphocholine head group. Membranes can be generated from non-amphiphilic, water-soluble precursors at physiological conditions using micromolar concentrations of precursors. We demonstrate that functional membrane proteins can be reconstituted into synthetic oxime liposomes from bacterial extracts in the absence of detergent-like molecules.
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Liposomas , Oximas , Membrana Celular/metabolismo , Liposomas/química , Fosfolípidos/química , AguaRESUMEN
BACKGROUND: Pacemaker battery depletion triggers alert for replacement notification and results in automatic reprogramming, which has been shown to be associated with relevant cardiorespiratory symptoms and adverse clinical events. OBJECTIVE: Determine if electrocardiogram (ECG) pacing features may be predictive of pacemaker battery depletion and clinical risk. METHODS: This is an ECG substudy of a cohort analysis of 298 subjects referred for pacemaker generator replacement from 2006 to 2017. Electronic medical record review was performed; clinical, ECG, and pacemaker characteristics were abstracted. We applied two ECG prediction rules for pacemaker battery depletion that are relevant to all major pacemaker manufacturers except Boston Scientific and MicroPort: (1) atrial pacing not at a multiple of 10 and (2) nonsynchronous ventricular pacing not at a multiple of 10, to determine diagnostic sensitivity, specificity, and risk in applicable ECG subjects. RESULTS: We excluded 32 subjects not at replacement notification or duplicate surgeries. Overall, 176 of 266 subjects (66.2%) demonstrated atrial pacing or nonsynchronous ventricular pacing on preoperative ECG. When utilizing both rules, 139 of 176 preoperative ECGs and 12 of 163 postoperative ECGs met criteria for battery depletion yielding reasonable sensitivity (79.0%), high specificity (92.6%), and a positive likelihood ratio of 11.6:1. These rules were associated with significant increase in cardiorespiratory symptoms (P < .001) and adverse clinical events (P < .025). CONCLUSIONS: The "Rules of Ten" provided reasonable sensitivity and specificity for detecting replacement notification in pacemaker subjects with an applicable ECG. This ECG tool may help clinicians identify most patients with pacemaker battery depletion at significant clinical risk.
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Suministros de Energía Eléctrica , Electrocardiografía/métodos , Falla de Equipo , Marcapaso Artificial , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Ablation of atrial vagal ganglia has been associated with improved pulmonary vein isolation (PVI) outcomes. Disruption of vagal reflexes results in heart rate (HR) increase. We investigated the association between HR change after PVI and freedom from atrial fibrillation (AF) at 1 year. METHODS AND RESULTS: Patients who underwent PVI for paroxysmal AF were identified from the Johns Hopkins Hospital AF registry. Electrocardiograms taken pre-PVI and post-PVI were used to determine the change in HR. Patients followed-up at 3, 6, and 12 months. Of 257 patients (66% male, age 59+/-11 years), 134 (52%) remained free from AF at 1 year. The average HR increased from 60.6 ± 11.3 beats per minute (bpm) pre-PVI to 70.7 ± 12.0 bpm post-PVI. Patients with recurrence of AF had lower post-PVI HR than those who remained free from AF (67.8 ± 0.2 vs 73.3 ± 13.0 bpm; P <.001). The probability of AF recurrence at 1-year decreased as the change in HR increased (estimated odds ratio [OR], 0.83; 95% confidence interval [CI, 0.74-0.93]; P = .002). HR increase more than 15 bpm was associated with the lowest odds of AF recurrence (estimated OR, 0.39; 95% [0.17-0.85]; P = .018) compared to HR decrease. CONCLUSIONS: Resting HR was found to increase after PVI. Increase in HR more than 15 bpm has a positive association with remaining free from atrial fibrillation at 1 year.
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Fibrilación Atrial/cirugía , Ablación por Catéter , Criocirugía , Ganglios Parasimpáticos/cirugía , Frecuencia Cardíaca , Venas Pulmonares/cirugía , Nervio Vago/cirugía , Anciano , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/fisiopatología , Ablación por Catéter/efectos adversos , Criocirugía/efectos adversos , Supervivencia sin Enfermedad , Femenino , Ganglios Parasimpáticos/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Venas Pulmonares/inervación , Recurrencia , Reflejo , Sistema de Registros , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Nervio Vago/fisiopatologíaRESUMEN
We investigated the dynamics of polymer-grafted gold nanoparticles loaded into polymer melts using x-ray photon correlation spectroscopy. For low molecular weight host matrix polymer chains, normal isotropic diffusion of the gold nanoparticles is observed. For larger molecular weights, anomalous diffusion of the nanoparticles is observed that can be described by ballistic motion and generalized Lévy walks, similar to those often used to discuss the dynamics of jammed systems. Under certain annealing conditions, the diffusion is one-dimensional and related to the direction of heat flow during annealing and is associated with an dynamic alignment of the host polymer chains. Molecular dynamics simulations of a single gold nanoparticle diffusing in a partially aligned polymer network semiquantitatively reproduce the experimental results to a remarkable degree. The results help to showcase how nanoparticles can under certain circumstances move rapidly in polymer networks.
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Single-chain amphiphiles (SCAs) that self-assemble into large vesicular structures are attractive components of synthetic cells because of the simplicity of bilayer formation and increased membrane permeability. However, SCAs commonly used for vesicle formation suffer from restricted working pH ranges, instability to divalent cations, and the inhibition of biocatalysts. Construction of more robust biocompatible membranes from SCAs would have significant benefits. We describe the formation of highly stable vesicles from alkyl galactopyranose thioesters. The compatibility of these uncharged SCAs with biomolecules makes possible the encapsulation of functional enzymes and nucleic acids during the vesicle generation process, enabling membrane protein reconstitution and compartmentalized nucleic acid amplification, even when charged precursors are supplied externally.
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Células Artificiales/química , Glucolípidos/química , Membrana Dobles de Lípidos/química , Tiogalactósidos/química , Animales , Bovinos , Permeabilidad de la Membrana Celular , ADN/genética , Replicación del ADN , Complejo IV de Transporte de Electrones/química , Glucolípidos/síntesis química , Membrana Dobles de Lípidos/síntesis química , Tensoactivos/síntesis química , Tensoactivos/química , Tiogalactósidos/síntesis químicaRESUMEN
Aims: Historical studies of ablation of atrioventricular nodal re-entrant tachycardia (AVNRT) have shown high long-term success rates and low complication rates. The potential impact of several recent practice trends has not been described. This study aims to characterize recent clinical practice trends in AVNRT ablation and their associated success rates and complications. Methods and results: Patients undergoing initial ablation of AVNRT between 1 July 2005 and 30 June 2015 were included in this study. Patient demographics and procedural data were abstracted from procedure reports. Follow-up data, including AVNRT recurrence and complications, was evaluated through electronic medical record review. In total, 877 patients underwent catheter ablation for AVNRT. By the last recorded year, three-dimension (3D) electroanatomical mapping (EAM) was used in 36.2%, 43.2% included anaesthesia, and 23.1% utilized irrigated catheters. Long-term procedural success was 95.5%. The use of anaesthesia, 3D EAM, and irrigated ablation catheters were not associated with differences in success. The presence of an atrial 'echo' or 'AH' jump at the end of an acutely successful procedure was not associated with long-term recurrence (P = 0.18, P = 0.15, respectively). Complications, including AV block requiring a pacemaker (0.4%), were uncommon. Conclusion: In a large, contemporary cohort, catheter ablation for AVNRT remains highly successful with low complications rates. The increased use of anaesthesia as well as modern mapping and ablation tools were not associated with changes in clinical outcomes. Further prospective evaluation of such contemporary practices is warranted given the lack of evidence to support their escalating use.
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Ablación por Catéter , Taquicardia por Reentrada en el Nodo Atrioventricular/cirugía , Anestesia/métodos , Ablación por Catéter/efectos adversos , Ablación por Catéter/tendencias , Técnicas Electrofisiológicas Cardíacas , Humanos , Complicaciones Posoperatorias/etiología , Pautas de la Práctica en Medicina/tendencias , Recurrencia , Estudios Retrospectivos , Factores de Riesgo , Taquicardia por Reentrada en el Nodo Atrioventricular/diagnóstico , Taquicardia por Reentrada en el Nodo Atrioventricular/fisiopatología , Factores de Tiempo , Resultado del Tratamiento , Flujo de TrabajoRESUMEN
BACKGROUND: Pacemaker patients experience battery depletion that activates pacemaker's alert for replacement notification. Automatic reprogramming at replacement notification can result in loss of rate response and atrioventricular (AV) synchrony. OBJECTIVE: To determine if relevant symptoms or clinical events may be associated with automatic reprogramming at replacement notification. METHODS: Electronic medical record review was undertaken for 298 patients referred for pacemaker generator replacement. Primary endpoints were symptoms or clinical events during replacement notification period. RESULTS: Following elimination of duplicate pacemaker replacements (n = 12), "near-replacement notification" or "recalled" (n = 15) and pacemakers at "end of life" (n = 5), 266 subjects were included. Three distinct reprogramming cohorts were identified; those with no change (control) in pacing mode (n = 46), those with loss of rate response (n = 154), and those with loss of AV synchrony ± rate response (n = 66). In total, 83 subjects (31.2%) had symptoms with significant differences seen between groups (control = 4.3%, loss of rate response = 26.0%, loss of AV synchrony ± rate response = 62.1%, P < 0.001). Overall, 28 subjects (10.5%) experienced clinical events with significant differences seen between groups (control = 0.0%, loss of rate response = 6.5%, loss of AV synchrony ± rate response = 27.3%, P < 0.001). CONCLUSIONS: Automatic reprogramming at replacement notification was associated with significant symptoms in 26% of those who lost rate response and in 62% of those who lost AV synchrony ± rate response. Additionally, 27% of the latter cohort required nonelective clinical care.
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Suministros de Energía Eléctrica , Marcapaso Artificial , Anciano , Anciano de 80 o más Años , Remoción de Dispositivos , Electrodos Implantados , Falla de Equipo , Análisis de Falla de Equipo , Femenino , Humanos , Masculino , Estudios Retrospectivos , Factores de TiempoRESUMEN
BACKGROUND: Patients go without pacemaker, defibrillator, and cardiac resynchronization therapies (devices) each year due to the prohibitive costs of devices. OBJECTIVE: We sought to examine data available from studies regarding contemporary risks of reused devices in comparison with new devices. METHODS: We searched online indexing sites to identify recent studies. Peer-reviewed manuscripts reporting infection, malfunction, premature battery depletion, and device-related death with reused devices were included. The primary study outcome was the composite risk of infection, malfunction, premature battery depletion, and death. Secondary outcomes were the individual risks. RESULTS: Nine observational studies (published 2009-2017) were identified totaling 2,302 devices (2,017 pacemakers, 285 defibrillators). Five controlled trials were included in meta-analysis (2,114 devices; 1,258 new vs 856 reused). All device reuse protocols employed interrogation to confirm longevity and functionality, disinfectant therapy, and, usually, additional biocidal agents, packaging, and ethylene oxide gas sterilization. Demographic characteristics, indications for pacing, and median follow-up were similar. There were no device-related deaths reported and no statistically significant difference in risk between new versus reused devices for the primary outcome (2.23% vs 3.86% respectively, P = 0.807, odds ratio = 0.76). There were no significant differences seen in the secondary outcomes for the individual risks of infection, malfunction, and premature battery depletion. CONCLUSIONS: Device reuse utilizing modern protocols did not significantly increase risk of infection, malfunction, premature battery depletion, or device-related death in observational studies. These data provide rationale for proceeding with a prospective multicenter noninferiority randomized control trial.
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Desfibriladores Implantables , Equipo Reutilizado , Marcapaso Artificial , Remoción de Dispositivos , Falla de Equipo , Humanos , Factores de RiesgoAsunto(s)
Organizaciones de Beneficencia , Marcapaso Artificial , Autopsia , Bradicardia/terapia , HumanosRESUMEN
All contemporary pacemakers undergo automatic reprogramming upon reaching elective replacement indication due to battery depletion. The majority of such reprogramming will result in changes to both pacing mode and pacing rate. The exact software reprogramming varies considerably among pacemaker manufacturers and may even vary among models of the same manufacturer. Accordingly, it is essential for healthcare providers managing pacemaker patients to have a detailed understanding of the automatic reprogramming seen at elective replacement indication as well as their potential physiological and clinical consequences.
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Suministros de Energía Eléctrica , Marcapaso Artificial , HumanosRESUMEN
Ludwig's angina is potentially lethal, rapidly spreading cellulitis of the floor of mouth and neck. The anticipated difficult airway becomes even more challenging when it occurs in children. In children, the larynx is positioned relatively higher in the neck, and one does not have the option for blind nasal intubation or awake fiberoptic, which otherwise is the technique of choice in adult patients. We present the clinical course of 16 children and highlight various problems encountered during the anesthetic management of six children who required emergency surgical drainage under general anesthesia.
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The cholesterol partitioning and condensing effect in the liquid-ordered (Lo) and liquid-disordered (Ld) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the Lo phase was observed for total cholesterol concentration of 20â¼30%.
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Colesterol/metabolismo , Membrana Dobles de Lípidos/química , Transición de Fase , 1,2-Dipalmitoilfosfatidilcolina/química , Humedad , Fosfatos/química , Electricidad Estática , Difracción de Rayos XRESUMEN
Ternary lipid mixtures incorporating cholesterol are well-known to phase separate into liquid-ordered (L(o)) and liquid-disordered (L(d)) phases. In multilayers of these systems, the laterally phase separated domains register in columnar structures with different bilayer periodicities, resulting in hydrophobic mismatch energies at the domain boundaries. In this paper, we demonstrate via synchrotron-based X-ray diffraction measurements that the system relieves the hydrophobic mismatch at the domain boundaries by absorbing larger amounts of inter-bilayer water into the L(d) phase with lower d-spacing as the relative humidity approaches 100%. The lamellar repeat distance of the L(d) phase swells by an extra 4 Å, well beyond the equilibrium spacing predicted by the inter-bilayer forces. This anomalous swelling is caused by the hydrophobic mismatch energy at the domain boundaries, which produces a surprisingly long-range effect. We also demonstrate that the d-spacings of the lipid multilayers at 100% relative humidity do not change when bulk water begins to condense on the sample.
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Colesterol/química , Humedad , Lípidos/química , Agua/química , Interacciones Hidrofóbicas e Hidrofílicas , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
In this study, we have designed a compact sample chamber that can achieve accurate and continuous control of the relative humidity (RH) in the vicinity of 100%. A 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilayer can be used as a humidity sensor by measuring its inter-layer repeat distance (d-spacing) via X-ray diffraction. We convert from DOPC d-spacing to RH according to a theory given in the literature and previously measured data of DOPC multilamellar vesicles in polyvinylpyrrolidone (PVP) solutions. This curve can be used for calibration of RH close to 100%, a regime where conventional sensors do not have sufficient accuracy. We demonstrate that this control method can provide RH accuracies of 0.1 to 0.01%, which is a factor of 10-100 improvement compared to existing methods of humidity control. Our method provides fine tuning capability of RH continuously for a single sample, whereas the PVP solution method requires new samples to be made for each PVP concentration. The use of this cell also potentially removes the need for an X-ray or neutron beam to pass through bulk water if one wishes to work close to biologically relevant conditions of nearly 100% RH.
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Liquid-crystalline phases of stacked lipid bilayers represent a pervasive motif in biomolecular assemblies. Here we report that, in addition to the usual smectic order, multicomponent multilayer membranes can exhibit columnar order arising from the coupling of two-dimensional intralayer phase separation and interlayer smectic ordering. This coupling propagates across hundreds of membrane lamellae, producing long-range alignment of phase-separated domains. Quantitative analysis of real-time dynamical experiments reveals that there is an interplay between intralayer domain growth and interlayer coupling, suggesting the existence of cooperative multilayer epitaxy. We postulate that such long-range epitaxy is solvent-assisted, and that it originates from the surface tension associated with differences in the network of hydrogen-bonded water molecules at the hydrated interfaces between the domains and the surrounding phase. Our findings might inspire the development of self-assembly-based strategies for the long-range alignment of functional lipid domains.