Outcomes of kidney transplantation in patients with IgA nephropathy based on induction: A UNOS data analysis.

INTRODUCTION: IgA nephropathy (IgAN) can cause end-stage kidney disease (ESKD). This study assesses the impact of induction and maintenance immunosuppression on IgAN recurrence, graft survival, and mortality in living and deceased donor kidney transplants (LDKT and DDKT). METHODS: Retrospective analysis of the UNOS database in adults with ESKD secondary to IgAN who received kidney transplants between January 2000 and June 30, 2022. Patients with thymoglobulin (ATG), alemtuzumab, or basiliximab/daclizumab induction with calcineurin inhibitor (CNI) and mycophenolate mofetil (MMF) with or without prednisone maintenance were analyzed. Multivariate logistic regression was performed to identify factors correlated with IgA recurrence. Multivariable Cox regression analyses were performed for clinically suspected risk factors. Kaplan Meir Analysis was utilized for overall graft survival. RESULTS: Compared to ATG with steroid maintenance, alemtuzumab with steroid increased the odds of IgAN recurrence in DDKTs (OR 1.90, p < .010, 95% CI [1.169-3.101]). Alemtuzumab with and without steroid increased the odds of recurrence by 52% (p = .036) and 56% (p = .005), respectively, in LDKTs. ATG without steroids was associated with less risk of IgAN recurrence (HR .665, p = .044, 95% CI [.447-.989]), graft failure (HR .758, p = .002, 95% CI [.633-.907]), and death (HR .619, p < .001, 95% CI [.490-.783]) in DDKTs. Recurrence was strongly associated with risks of graft failure in DDKTs and LDKTs and death in LDKTs. CONCLUSION: In patients with IgAN requiring a kidney transplant, Alemtuzumab induction correlates with increased IgAN recurrence. Relapse significantly affects graft survival and mortality. ATG without steroids is associated with the least graft loss and mortality.

Systematic Review of Health-Related Quality of Life in Adolescents with Psoriasis.

BACKGROUND: Many cases of psoriasis begin in adolescence. The affected adolescents face the combined physical and psychosocial challenges of their disease-free peers with the added complexity of a visible disease. OBJECTIVE: To review the impact of psoriasis on the health-related quality of life (HRQL) in adolescents as compared to their peers with other chronic diseases and to determine the best tools to measure HRQL in this population. METHODS: A systematic literature review was completed using PubMed. RESULTS: 256 publications were screened for inclusion, 37 were relevant to objectives and included in the systematic review. Most studies are pediatric psoriasis studies with an adolescent subgroup, very few are dedicated to solely addressing HRQL in adolescents with psoriasis. Adolescents with psoriasis face both the challenges similar to an adult psoriasis population in addition to the complexities inherent to healthy adolescents. Studies often use a general pediatric HRQL measure, PedsQL 4.0, or a dermatology-specific measure adapted from an adult questionnaire. Only one psoriasis-specific measurement tool exists, and it is specifically for scalp psoriasis. CONCLUSION: Both dermatologists and primary care physicians should treat the visible cutaneous lesions and disease comorbidities and address the psychosocial impact of psoriasis in their adolescent patients. Use of both a general and dermatology-specific HRQL questionnaire may allow physicians to better identify the impact of the disease and recognize improvement or impairment over time.

Many-molecule encapsulation by an icosahedral shell.

We computationally study how an icosahedral shell assembles around hundreds of molecules. Such a process occurs during the formation of the carboxysome, a bacterial microcompartment that assembles around many copies of the enzymes ribulose 1,5-bisphosphate carboxylase/ oxygenase and carbonic anhydrase to facilitate carbon fixation in cyanobacteria. Our simulations identify two classes of assembly pathways leading to encapsulation of many-molecule cargoes. In one, shell assembly proceeds concomitantly with cargo condensation. In the other, the cargo first forms a dense globule; then, shell proteins assemble around and bud from the condensed cargo complex. Although the model is simplified, the simulations predict intermediates and closure mechanisms not accessible in experiments, and show how assembly can be tuned between these two pathways by modulating protein interactions. In addition to elucidating assembly pathways and critical control parameters for microcompartment assembly, our results may guide the reengineering of viruses as nanoreactors that self-assemble around their reactants.

The Role of Packaging Sites in Efficient and Specific Virus Assembly.

During the life cycle of many single-stranded RNA viruses, including many human pathogens, a protein shell called the capsid spontaneously assembles around the viral genome. Understanding the mechanisms by which capsid proteins selectively assemble around the viral RNA amidst diverse host RNAs is a key question in virology. In one proposed mechanism, short sequences (packaging sites) within the genomic RNA promote rapid and efficient assembly through specific interactions with the capsid proteins. In this work, we develop a coarse-grained particle-based computational model for capsid proteins and RNA that represents protein-RNA interactions arising both from nonspecific electrostatics and from specific packaging site interactions. Using Brownian dynamics simulations, we explore how the efficiency and specificity of assembly depend on solution conditions (which control protein-protein and nonspecific protein-RNA interactions) and the strength and number of packaging sites. We identify distinct regions in parameter space in which packaging sites lead to highly specific assembly via different mechanisms and others in which packaging sites lead to kinetic traps. We relate these computational predictions to in vitro assays for specificity in which cognate viral RNAs compete against non-cognate RNAs for assembly by capsid proteins.

Mechanisms of virus assembly.

Viruses are nanoscale entities containing a nucleic acid genome encased in a protein shell called a capsid and in some cases are surrounded by a lipid bilayer membrane. This review summarizes the physics that govern the processes by which capsids assemble within their host cells and in vitro. We describe the thermodynamics and kinetics for the assembly of protein subunits into icosahedral capsid shells and how these are modified in cases in which the capsid assembles around a nucleic acid or on a lipid bilayer. We present experimental and theoretical techniques used to characterize capsid assembly, and we highlight aspects of virus assembly that are likely to receive significant attention in the near future.

Thermal fluctuations in amphipol A8-35 particles: a neutron scattering and molecular dynamics study.

Amphipols are a class of polymeric surfactants that can stabilize membrane proteins in aqueous solutions as compared to detergents. A8-35, the best-characterized amphipol to date, is composed of a polyacrylate backbone with ~35% of the carboxylates free, ~25% grafted with octyl side-chains, and ~40% with isopropyl ones. In aqueous solutions, A8-35 self-organizes into globular particles with a molecular mass of ~40 kDa. The thermal dynamics of A8-35 particles was measured by neutron scattering in the 10-picosecond, 18-picosecond, and 1-nanosecond time-scales on natural abundance and deuterium-labeled molecules, which permitted to separate backbone and side-chain motions. A parallel analysis was performed on molecular dynamics trajectories (Perlmutter et al., Langmuir 27:10523-10537, 2011). Experimental results and simulations converge, from their respective time-scales, to show that A8-35 particles feature a more fluid hydrophobic core, predominantly containing the octyl chains, and a more rigid solvent-exposed surface, made up predominantly of the hydrophilic polymer backbone. The fluidity of the core is comparable to that of the lipid environment around proteins in the center of biological membranes, as also measured by neutron scattering. The biological activity of proteins depends sensitively on molecular dynamics, which itself is strongly dependent on the immediate macromolecular environment. In this context, the characterization of A8-35 particle dynamics constitutes a step toward understanding the effect of amphipols on membrane protein stability and function.

Pathways for virus assembly around nucleic acids.

Understanding the pathways by which viral capsid proteins assemble around their genomes could identify key intermediates as potential drug targets. In this work, we use computer simulations to characterize assembly over a wide range of capsid protein-protein interaction strengths and solution ionic strengths. We find that assembly pathways can be categorized into two classes, in which intermediates are either predominantly ordered or disordered. Our results suggest that estimating the protein-protein and the protein-genome binding affinities may be sufficient to predict which pathway occurs. Furthermore, the calculated phase diagrams suggest that knowledge of the dominant assembly pathway and its relationship to control parameters could identify optimal strategies to thwart or redirect assembly to block infection. Finally, analysis of simulation trajectories suggests that the two classes of assembly pathways can be distinguished in single-molecule fluorescence correlation spectroscopy or bulk time-resolved small-angle X-ray scattering experiments.

Molecular dynamics simulations of a membrane protein/amphipol complex.

Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

Viral genome structures are optimal for capsid assembly.

Understanding how virus capsids assemble around their nucleic acid (NA) genomes could promote efforts to block viral propagation or to reengineer capsids for gene therapy applications. We develop a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics. In contrast to recent theoretical models, we find that capsids spontaneously 'overcharge'; that is, the negative charge of the NA exceeds the positive charge on capsid. When applied to specific viruses, the optimal NA lengths closely correspond to the natural genome lengths. Calculations based on linear polyelectrolytes rather than base-paired NAs underpredict the optimal length, demonstrating the importance of NA structure to capsid assembly. These results suggest that electrostatics, excluded volume, and NA tertiary structure are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained, at least in part, on a thermodynamic basis. DOI:http://dx.doi.org/10.7554/eLife.00632.001.

The methionine-aromatic motif plays a unique role in stabilizing protein structure.

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1-1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized.

Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

NaCl interactions with phosphatidylcholine bilayers do not alter membrane structure but induce long-range ordering of ions and water.

It is generally accepted that ions interact directly with lipids in biological membranes. Decades of biophysical studies on pure lipid bilayer systems have shown that only certain types of ions, most significantly large anions and multivalent cations, can fundamentally alter the structure and dynamics of lipid bilayers. It has long been accepted that at physiological concentrations NaCl ions do not alter the physical behavior or structure of bilayers composed solely of zwitterionic phosphatidylcholine (PC) lipids. Recent X-ray scattering experiments have reaffirmed this dogma, showing that below 1 M concentration, NaCl does not significantly alter bilayer structure. However, despite this history, there is an ongoing controversy within the molecular dynamics (MD) simulation community regarding NaCl/PC interactions. In particular, the CHARMM and GROMOS force fields show dramatically different behavior, including the effect on bilayer structure, surface potential, and the ability to form stable, coordinated ion-lipid complexes. Here, using long-timescale, constant-pressure simulations under the newest version of the CHARMM force field, we find that Na⁺ and Cl⁻ associate with PC head groups in a POPC bilayer with approximately equal, though weak, affinity, and that the salt has a negligible effect on bilayer structure, consistent with earlier CHARMM results and more recent X-ray data. The results suggest that interpretation of simulations where lipids interact with charged groups of any sort, including charged proteins, must be carefully scrutinized.

All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer.

Amphipathic polymers called amphipols (APols) have been developed as an alternative to detergents for stabilizing membrane proteins (MPs) in aqueous solutions. APols provide MPs with a particularly mild environment and, as a rule, keep them in a native functional state for longer periods than do detergents. Amphipol A8-35, a derivative of polyacrylate, is widely used and has been particularly well studied experimentally. In aqueous solutions, A8-35 molecules self-assemble into well-defined globular particles with a mass of ∼40 kDa and a R(g) of ∼2.4 nm. As a first step towards describing MP/A8-35 complexes by molecular dynamics (MD), we present three sets of simulations of the pure APol particle. First, we performed a series of all-atom MD (AAMD) simulations of the particle in solution, starting from an arbitrary initial configuration. Although AAMD simulations result in stable cohesive particles over a 45 ns simulation, the equilibration of the particle organization is limited. This motivated the use of coarse-grained MD (CGMD), allowing us to investigate processes on the microsecond time scale, including de novo particle assembly. We present a detailed description of the parametrization of the CGMD model from the AAMD simulations and a characterization of the resulting CGMD particles. Our third set of simulations utilizes reverse coarse-graining (rCG), through which we obtain all-atom coordinates from a CGMD simulation. This allows a higher-resolution characterization of a configuration determined by a long-timescale simulation. Excellent agreement is observed between MD models and experimental, small-angle neutron scattering data. The MD data provides new insight into the structure and dynamics of A8-35 particles, which is possibly relevant to the stabilizing effects of APols on MPs, as well as a starting point for modeling MP/A8-35 complexes.

Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations.

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics simulations of phase separated lipid bilayers. The simulations are motivated by differences in lipid and cholesterol composition in the inner and outer leaflets of biological membranes. Over the past several years, this phenomenon has inspired a series of experiments in model membrane systems which have explored the effects of lipid compositional asymmetry in the two leaflets. The simulations are directed at understanding one potential consequence of compositional asymmetry, that being regions of bilayers where liquid-ordered (L(o)) domains in one leaflet are opposite liquid-disordered (L(d)) domains in the other leaflet (phase asymmetry). The simulated bilayers are of two sorts: 1) Compositionally symmetric leaflets where each of the two leaflets contains an identical, phase separated (L(o)/L(d)) mixture of cholesterol, saturated and unsaturated phospholipid; and 2) Compositionally asymmetric leaflets, where one leaflet contains a phase separated (L(o)/L(d)) mixture while the other contains only unsaturated lipid, which on its own would be in the L(d) phase. In addition, we have run simulations where the lengths of the saturated lipid chains as well as the mole ratios of the three lipid components are varied. Collectively, we report on three types of interleaflet coupling within a bilayer. First, we show the effects of compositional asymmetry on acyl chain tilt and order, lipid rotational dynamics, and lateral diffusion in regions of leaflets that are opposite L(o) domains. Second, we show substantial effects of compositional asymmetry on local bilayer curvature, with the conclusion that phase separated leaflets resist curvature, while inducing large degrees of curvature in an opposing L(d) leaflet. Finally, in compositionally symmetric, phase separated bilayers, we find phase asymmetry (domain antiregistration) between the two leaflets occurs as a consequence of mismatched acyl chain-lengths in the saturated and unsaturated lipids.

Inhibiting lateral domain formation in lipid bilayers: simulations of alternative steroid headgroup chemistries.

Cholesterol is an essential component of lateral domain formation in lipid bilayers. Experiments using structural analogues of cholesterol have revealed that slight changes to the steroid headgroup chemistry can lead to significantly different degrees of domain formation. For example, the seemingly slight modification to 4-cholesten-3beta-one changes the molecule from domain enhancing to domain inhibiting. To investigate the relationship between steroid headgroup structure and domain formation, we have performed a series of coarse-grain molecular dynamics simulations in which the hydrophobicity of the steroid headgroup is altered. We show that bilayers containing steroids with polar headgroups undergo lateral phase separation, with the steroids adopting the canonical, upright orientation. However, increasing the hydrophobicity of the steroid's headgroup dramatically changes the orientation of the molecule such that it lies at the center of the bilayer, perpendicular to the bilayer normal. As a fundamental consequence, we show that these steroids inhibit the formation of phase-separated domains in membranes.

Experimental verification of lipid bilayer structure through multi-scale modeling.

Great progress has been made in applying coarse-grain molecular dynamics (CGMD) simulations to the investigation of membrane biophysics. In order to validate the accuracy of CGMD simulations of membranes, atomistic scale detail is necessary for direct comparison to structural experiments. Here, we present our strategy for verifying CGMD lipid bilayer simulations. Through reverse coarse graining and subsequent calculation of the bilayer electron density profile, we are able to compare the simulations to our experimental low angle X-ray scattering (LAXS) data. In order to determine the best match to the experimental data, atomistic simulations are run at a range of areas (in the NP(N)AT ensemble), starting from distinct configurations extracted from the CGMD simulation (run in the NPT ensemble). We demonstrate the effectiveness of this procedure with two small, single-component bilayers, and suggest that the greater utility of our algorithm will be for CGMD simulations of more complex structures.

Curvature dynamics of alpha-synuclein familial Parkinson disease mutants: molecular simulations of the micelle- and bilayer-bound forms.

Alpha-synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. Herein, we present a series of atomistic molecular dynamics simulations of wild-type alpha-synuclein and three Parkinson disease familial mutants (A30P, A53T, and E46K) in two distinct environments. First, in order to match recent NMR experiments, we have simulated each protein bound to an SDS detergent micelle. Second, in order to connect more closely to the true biological environment, we have simulated the proteins bound to a 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid bilayer. In the micelle-bound case, we find that the wild type and all of the variants of alpha-synuclein flatten the underlying micelle, decreasing its surface area. A30P is known to lessen alpha-synuclein/membrane affinity and, consistent with experiment, destabilizes the simulated secondary structure. In the case of A53T, our simulations reveal a range of stabilizing hydrogen bonds that form with the threonine. In both environments, the E46K mutation, which is known to increase bilayer affinity, leads to an additional hydrogen bond between the protein and either the detergent or lipid. Simulations indicate that alphaS and its variants are less dynamic in the bilayer than in the micelle. Furthermore, the simulations of the mutants suggest how changes in the structure and dynamics of alpha-synuclein may affect its biological role.

Validation of a Method for the Determination of Total Chromium in Rat Feces by Inductively Coupled Plasma Optical Emission Spectrometry.

The validation of a method for the determination of total chromium in Fischer-344 rat feces by inductively coupled plasma optical emission spectrometry following a rapid, atmospheric-pressure microwave digestion is described. The performance of the method was evaluated over the concentration range of 5.00 to 200 µg Cr/g feces. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limit of detection and limit of quantitation data. Data from a cross-validation method for B6C3F1 mouse feces are also presented. Following validation, the method was applied to analyze samples collected in support of two chronic toxicological investigations.

The effect of cholesterol on short- and long-chain monounsaturated lipid bilayers as determined by molecular dynamics simulations and X-ray scattering.

We investigate the structure of cholesterol-containing membranes composed of either short-chain (diC14:1PC) or long-chain (diC22:1PC) monounsaturated phospholipids. Bilayer structural information is derived from all-atom molecular dynamics simulations, which are validated via direct comparison to x-ray scattering experiments. We show that the addition of 40 mol % cholesterol results in a nearly identical increase in the thickness of the two different bilayers. In both cases, the chain ordering dominates over the hydrophobic matching between the length of the cholesterol molecule and the hydrocarbon thickness of the bilayer, which one would expect to cause a thinning of the diC22:1PC bilayer. For both bilayers there is substantial headgroup rearrangement for lipids directly in contact with cholesterol, supporting the so-called umbrella model. Importantly, in diC14:1PC bilayers, a dynamic network of hydrogen bonds stabilizes long-lived reorientations of some cholesterol molecules, during which they are found to lie perpendicular to the bilayer normal, deep within the bilayer's hydrophobic core. Additionally, the simulations show that the diC14:1PC bilayer is significantly more permeable to water. These differences may be correlated with faster cholesterol flip-flop between the leaflets of short-chain lipid bilayers, resulting in an asymmetric distribution of cholesterol molecules. This asymmetry was observed experimentally in a case of unilamellar vesicles (ULVs), and reproduced through a set of novel asymmetric simulations. In contrast to ULVs, experimental data for oriented multilamellar stacks does not show the asymmetry, suggesting that it results from the curvature of the ULV bilayers.

An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway.

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.