Surgical myectomy versus alcohol septal ablation for obstructive hypertrophic cardiomyopathy: A propensity score–matched cohort

Objectives

In patients with hypertrophic cardiomyopathy, obstruction of the left ventricular outflow tract can be relieved by surgical septal myectomy or alcohol septal ablation, but uncertainty remains regarding long-term results and comparative effectiveness of alcohol septal ablation. This study aims to compare short- and long-term outcomes of the 2 procedures.

A novel tetrameric gp350 as a potential Epstein–Barr virus vaccine

Infectious mononucleosis and B-cell transformation in response to infection with Epstein–Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1–470) gp350 protein (gp350^1–470). Tetrameric gp350^1–470 induced ∼20-fold higher serum titers of gp350^1–470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350^1–470. Further, epidermal immunization with plasmid DNA encoding gp350^1–470 tetramer induced 8-fold higher serum titers of gp350^1–470-specific IgG relative to monomer. Tetrameric gp350^1–470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350^1–470 had no effect on the gp350^1–470-specific IgG response in naïve mice, and resulted in suppressed gp350^1–470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350^1–470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.     

Force-dependent polymorphism in type IV pili reveals hidden epitopes

Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.     

Micropatterning of costimulatory ligands enhances CD4+ T cell function

Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell–APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naïve T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naïve CD4⁺ T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-κB translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell–cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/segregation.     

Heterogeneous phenotypes of platelet and plasma von Willebrand factor in obligatory heterozygotes for severe von Willebrand disease

To characterize the heterogeneity of severe (type III) von Willebrand disease (vWD), plasma and platelet von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (Ricof) were measured in 28 obligatory heterozygotes (ie, parents or children of probands from 15 different kindreds with severe vWD). On the average, heterozygotes had low levels of vWF in both platelets and plasma. There was, however, considerable heterogeneity, with four distinct patterns. Eleven heterozygotes had concordant reduction of vWF:Ag and Ricof in both plasma and platelets; five had low levels of vWF:Ag and Ricof in plasma contrasting with normal levels in platelets; eight had a peculiar pattern, the reverse of the above (ie, low levels in platelets and normal levels in plasma); and in one, both vWF measurements were normal in plasma and platelets. These patterns were genetically determined: they were consistent in four couples of consanguineous heterozygotes and in two couples carrying the same gene deletion. Only the remaining three heterozygotes had no clearly identifiable pattern. Other findings of this study were that although most of the heterozygotes had normal bleeding times, the 7 of 28 who had prolonged bleeding times had concordantly low levels of vWF measurements in both plasma and platelets. In conclusion, this large series of obligatory heterozygotes provides evidence for phenotypic and genotypic heterogeneity of severe vWD.     

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.

The polyprotein Gag is the main structural component of HIV-1, consisting of the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 domains as well as the spacer peptides SP1 and SP2 (1). Gag is produced in infected host cells and trafficked to the plasma membrane, where it assembles into a hexagonal lattice via its CA domain and recruits other viral proteins and the viral RNA genome (1, 2). Assembly of the curved Gag lattice is commensurate with membrane bending at the site of assembly, after which recruitment of Endosomal Sorting Complex Required for Transport III (ESCRT-III) components by the p6 domain of Gag induces membrane scission and release of the immature virus particle (2). The hexagonal Gag lattice accommodates curvature in the growing bud by incorporating vacancy defects (3). The activity of ESCRT-III is timed such that the final immature lattice is incomplete, giving rise to an additional large gap in the lattice, resulting in a truncated spherical shape (4–6).During or after budding, the viral protease is activated and cleaves this immature Gag lattice into its component domains, which leads to structural rearrangement within the virus particle (2). The released CA domains assemble to form a closed, conical capsid around the condensed ribonucleoprotein (RNP) complex of the mature virus (1, 7). Maturation is required for the virion to become infectious (1).Within the immature virus particle, the N-terminal domain of CA (CA_NTD) forms trimeric interactions linking three Gag hexamers while the C-terminal domain of CA (CA_CTD) forms dimeric interactions mediated by helix 9 of CA, linking two Gag hexamers together (8). The CA_CTD additionally forms intrahexamer interactions around the sixfold axis of the hexamer (8, 9). Amphipathic helices formed by the C-terminal residues of CA_CTD and the N-terminal residues of SP1 junction assemble into a six-helix bundle (6HB), thereby imposing hexagonal order on the CA domains, via classical knobs-in-holes packing mediated by exposed hydrophobic side chains, as also seen in coiled coils (9, 10). In combination, these relatively weak interactions give rise to a very dynamic, reversible assembly process that prevents the assembling lattice from becoming trapped in kinetically unfavorable states (11). This robust assembly behavior is consistent with icosahedral viruses (12–15). It is not surprising, therefore, that the energetics of Gag assembly are tightly controlled and highly dependent on scaffolding effects from the viral RNA and the membrane-interacting MA domain of Gag in order to ensure productive viral assembly (11, 16). Analysis of the diffusion pattern of fluorescently labeled Gag supports the notion that Gag is trafficked to the site of assembly as low-order multimers, although it is still unclear whether these are Gag dimers, trimers, or other multimeric forms of Gag (16, 17).The primary assembly unit of the Gag lattice remains largely unknown. We can identify two hypothetical ways in which the lattice could assemble. First, the lattice could grow by addition of Gag hexamers (or sets of six component monomers), such that the CA-SP1 junction is assembled within a hexameric 6HB at all positions in the lattice. In this case, interfaces between hexamers would be unoccupied at the edge of the lattice. From a purely energetic perspective, this appears most reasonable. Second, the lattice could form via addition of Gag dimers or Gag trimers (or equivalently from sets of either two or three component monomers). This would maintain, for example, the dimeric CA-CA interhexamer interactions but leave incomplete hexamers at the lattice edges, including unoccupied hexamer-forming interfaces along the CA-SP1 bundle. It additionally remains unclear whether the unoccupied Gag-Gag interfaces at the lattice edges are simply exposed, or whether they are stabilized by alternative conformations of individual domains or proteins, or by other binding partners. Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus assembly.Viral assembly, budding, and maturation are tightly linked, and disrupting the kinetics of any of these processes can give rise to defects in maturation and formation of noninfectious viral particles (1, 18, 19). The rate-limiting proteolytic cleavage site in the maturation process resides within the CA-SP1 6HB (20). Unfolding of the helical bundle is required to allow proteolytic cleavage to proceed (21–23), but the exact mechanism for protease access to this site is not known. The spatial localization of proteolytic processing within the context of the immature Gag lattice is relevant: Does the protease act on Gag within the lattice, or does it act on the edges of the Gag lattice, causing a cascade of lattice disruption? At the lattice edge, is the substrate for the protease with a 6HB or within an incomplete hexamer? Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus maturation.High-resolution immature Gag structures have previously been determined directly from purified viruses by cryo-electron tomography (cryo-ET) and subtomogram averaging (10). These structures represent an average hexamer within the immature lattice, with a full complement of six Gag hexamer neighbors. Here, we have applied subtomogram classification and averaging approaches to an existing immature virus dataset (10) in order to determine the structures of Gag assemblies at lattice edges. We also applied atomistic molecular dynamics simulations to assess the roles of the different CA-CA interactions in immature lattice stabilization and predict the properties of the structures we observe at lattice edges. Together, our results suggest that the basic unit of immature HIV-1 assembly is a Gag dimer and partial CA-SP1 helical bundles are present at the edges of the assembled lattice and may be substrates for initiation of maturation.