HERITABILITY OF AEROBIC POWER OF INDIVIDUALS IN NORTHEAST BRAZIL

The objective of this study was to evaluate the genetic and environmental contribution to variation in aerobic power in monozygotic (MZ) and dizygotic (DZ) twins. The sample consisted of 20 MZ individuals (12 females and 8 males) and 16 DZ individuals (12 females and 4 males), aged from 8 to 26 years, residents in Natal, Rio Grande do Norte. The twins were assessed by a multistage fitness test. The rate of heritability found for aerobic power was 77%. Based on the results, the estimated heritability was largely responsible for the differences in aerobic power. This implies that such measures are under strong genetic influence.     

Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil

Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K_D = 30 nm), close to its IC₅₀ concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K_D = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612–9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K_D = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.The HIV-1² surface glycoprotein Env promotes viral entry through the fusion of viral and cellular membranes (3). Env consists of three gp120 surface subunits and three gp41 transmembrane subunits arranged as a trimer-of-heterodimers on the virion surface. In the current model of HIV-1 entry, cellular receptor binding to gp120 initiates a series of coordinated structural transformations that stimulate gp41 to extend and insert its N-terminal fusion peptide into target cell membranes (see Fig. 1A) (4, 5). This high energy extended intermediate structure ultimately collapses into a trimer-of-hairpins conformation that juxtaposes the gp41 fusion peptide and transmembrane domain. Because the fusion peptide and transmembrane domain are inserted in target cell and viral membranes, formation of the trimer-of-hairpins is proposed to bring these membranes into the close proximity required for efficient fusion.Open in a separate windowFIGURE 1.HIV-1 gp41 and its role in viral membrane fusion. A, a model of HIV-1 entry (46). In native Env prior to receptor activation, gp41 is held in a metastable conformation by a canopy of gp120 proteins (green). Receptor binding to gp120 stimulates gp41 to extend and insert its fusion peptide segment (red) into the target cell membrane. The N-HR (gray) and C-HR (blue) regions of the gp41 ectodomain are transiently exposed in this prehairpin state. Subsequently, gp41 collapses into the trimer-of-hairpins conformation that brings the gp41 fusion peptides, transmembrane regions (purple), and their associated membranes into the close proximity for membrane fusion. The actual disposition of gp120 in both the prehairpin and trimer-of-hairpins states is uncertain; for clarity, the protein is omitted in the schematic of the trimer-of-hairpins conformation. B, a diagram of HIV-1 gp41 identifying its fusion peptide (FP), N-HR, C-HR, MPER (MP), transmembrane (TM), and cytoplasmic (cyto) domains. Amino acid sequences above and below the diagram are derived from the N-HR and C-HR/MPER regions of Env_HXB2; all but the MPER sequence WNWF (magenta) were used in the design of 5H-ex. The N-HR and C-HR segments found in the original 5-Helix are boxed in gray and blue, respectively, whereas the sequences of C37 and T20 are denoted by lines. The side chains of the C-HR amino acids marked with + pack into the hydrophobic pocket at the C terminus of the N-HR coiled coil.The core of the trimer-of-hairpins is a bundle of six α-helices formed by two hydrophobic heptad repeat sequences in the N- and C-terminal regions of the gp41 ectodomain (N-HR and C-HR, respectively) (6, 7). In the trimer-of-hairpins, the N-HR segments from three gp41 ectodomains form a central trimeric coiled coil, around which the three C-HR segments pack as antiparallel helices into hydrophobic grooves (8–11). In the prehairpin extended conformation, the N-HR and C-HR segments are unassociated and transiently accessible to inhibitors of HIV-1 entry (5, 12). Several such inhibitors are formed from the peptide sequence of the C-HR and adjacent gp41 regions (4, 6, 13, 14). Denoted C-peptides, they work in a dominant negative fashion by binding to the exposed N-HR coiled coil, thereby blocking trimer-of-hairpins formation and inhibiting viral membrane fusion (4, 15–21). One C-peptide, T20 (also called enfuvirtide), has shown antiviral activity in vivo and has been approved for use in the treatment of HIV-1 infection (22, 23).T20 is a 36-amino acid peptide extending from Tyr⁶³⁸ in the middle of the C-HR to Phe⁶⁷³ in the Trp-rich membrane proximal external region (MPER) that precedes the gp41 transmembrane domain (residue numbering is according to the Env_HXB2 sequence; see Fig. 1B) (13). In T20, these C-terminal MPER-derived residues are critical for inhibitory activity, although their structure and function in the gp41-bound state are currently unknown (1, 24, 25). A second class of similarly potent C-peptides includes C34 (residues 628–661) and the slightly larger C37 (residues 625–661) (4, 6, 26, 27). These peptides are derived entirely from the C-HR sequence and thus are shifted in the N-terminal direction compared with T20 (Fig. 1B). The interactions of C34 and C37 with gp41 are greatly stabilized by residues Trp⁶²⁸, Trp⁶³¹, and Ile⁶³⁵ near the C-HR N terminus (4). Their bulky hydrophobic side chains pack into a deep hydrophobic pocket on the surface of the N-HR coiled coil. T20 lacks these pocket binding residues and their stabilizing effect. However, T20 does contain bulky hydrophobic residues (Trp⁶⁷⁰, Trp⁶⁷², and Phe⁶⁷³) at its C terminus that might pack into a similar pocket at the other end of the N-HR coiled coil.High resolution structures of the gp41 trimer-of-hairpins have aided our understanding of the mechanism of C-peptide inhibition. These structures have enabled the design of polypeptides that mimic the gp41 N-HR coiled coil and bind C34/C37, thereby providing a tool to probe the structure-activity relationships of the inhibitors (26, 28–30). No similar tool is available for investigating T20 inhibition in detail. The structures of the gp41 trimer-of-hairpins do not include the T20 C terminus (9 residues) nor the gp41 N-terminal segments that putatively interact with it. Furthermore, gp41 N-HR-derived peptides predicted to interact with T20 are poorly soluble and difficult to use in solution phase interaction assays (6). Here we describe the design of a soluble protein (denoted 5H-ex) that mimics the putative T20-binding site on the N-HR coiled coil. 5H-ex interacts with T20 with an equilibrium dissociation constant (K_D) of 30 nm, close to the T20 50% inhibitory concentration (IC₅₀) of 3 nm. Using this protein, we explored the extent to which the N and C termini of T20 contribute to its binding activity. First, we showed that the MPER-derived residues at the peptide C terminus do not stabilize the 5H-ex/T20 interaction. Second, we identified an N-terminal substitution that significantly enhanced T20 binding affinity and improved peptide inhibitory activity against T20-resistant HIV-1. The results suggest that T20 binding to the N-HR coiled coil is stabilized primarily by residues derived from the C-HR and not the MPER. 5H-ex is likely to be a useful tool in probing the structure-activity relationship of T20.     

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.     

Minimizing the risk of reporting false positives in large-scale RNAi screens

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.     

Nucleoporin Nup50 stabilizes closed conformation of armadillo repeat 10 in importin α5

The human genome encodes six isoforms of importin α that show greater than 60% sequence similarity and remarkable substrate specificity. The isoform importin α5 can bind phosphorylated cargos such as STAT1 and Epstein-Barr Virus Nuclear Antigen 1, as well as the influenza virus polymerase subunit PB2. In this work, we have studied the interaction of the nucleoporin Nup50 with importin α5. We show that the first 47 residues of Nup50 bind to the C terminus of importin α5 like a "clip," stabilizing the closed conformation of ARM 10. In vitro, Nup50 binds with high affinity either to empty importin α5 or to a preassembled complex of importin α5 bound to the C-terminal domain of the import cargo PB2, resulting in a trimeric complex. By contrast, PB2 can only bind with high affinity to importin α5 in the absence of Nup50. This suggests that Nup50 primary function may not be to actively displace the import cargo from importin α5 but rather to prevent cargo rebinding in preparation for recycling. This is the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor.     

Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from Env_SF162 into the Env_HXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the Env_HXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.     

Outcome of Tuberculosis Treatment in Patients with Diabetes Mellitus Treated in the Revised National Tuberculosis Control Programme in Malappuram District,Kerala, India

Settings

Kerala State, India has reported the greatest dual burden of Tuberculosis (TB) and Diabetes Mellitus (DM). Malappuram district in Kerala has monitored and recorded DM status and its control from 2010 under Revised National Tuberculosis Control Program (RNTCP).

Objectives

To assess, under programme conditions, comprehensiveness of recording DM status among TB cases and the TB treatment outcomes among DM patients (disaggregated by glycemic control) and compare with-non DM patients.

Design

This retrospective record review included 3,116TB patients from April 2010 to September 2011.DM was defined as per international guidelines and TB treatment outcomes were categorized as favourable(cured and treatment completed) and unfavourable(death, default, failure and transfer out). Relative Risk (RR) and 95% confidence intervals(CI) were calculated to assess the risk of unfavourable outcomes.

Results

DM status was recorded in 90% of TB cases and 667 (24%) had DM. 17% of DM patients and 23% of patients with unknown DM status had unfavourable outcomes but this difference was not statistically significant. Unadjusted RR for poor glycemic control or unknown control status for unfavourable outcome were (2.00; 95% CI 0.97–4.13) and (2.14; 95% CI 1.11–4.13).

Conclusion

This study could not confirm an adverse association between DM or its control during treatment and the course of response to TB treatment.DM screening in TB cases and recording of DM care needs to be improved to enable more conclusive evidence.