Highly prevalent Russian HIV-1 V3-loop sequence variants are susceptible to maraviroc.

INTRODUCTION: Maraviroc inhibits CCR5-tropic HIV-1 across different subtypes in vitro and has demonstrated efficacy in clinical trials. V3-loop amino acid variants observed in individual maraviroc-resistant viruses have not been found to be predictive of reduced susceptibility. Sequence-database searches have demonstrated that approximately 7.3% of viruses naturally encode these variants, raising concerns regarding potential pre-existing resistance. A study from Russia reported that combinations of these same amino acids are present in the V3 loops of the Russian variant subtype A (IDU-A, now A6) with a much greater prevalence (range: 74.4%-92.3%) depending on the combination. However, these studies and database searches did not include phenotypic evaluation. METHODS: Sixteen Russian HIV-1 isolates (including sub-subtype A6 viruses) were assessed for V3 loop sequence and phenotypic susceptibility to maraviroc. RESULTS: All 12 of the A6 viruses and 2/4 subtype B isolates encoded V3-loop variants that have previously been identified in individual virus isolates with reduced susceptibility to maraviroc. However, despite the prevalence of these V3-loop amino acid variants among the tested viruses, phenotypic sensitivity to maraviroc was observed in all instances. Similarly, reduced susceptibility to maraviroc was not found in virus from participants who experienced virologic failure in a clinical study of maraviroc in Russia (A4001101, [NCT01275625]). DISCUSSION: Altogether, these data confirm that the presence of individual or combinations of V3-loop amino acid residues in sub-subtype A6 viruses alone does not predict natural resistance to maraviroc and that V3-loop genotype analysis of R5 virus prior to treatment is not helpful in predicting clinical outcome.

Bactericidal activity and mechanism of action of AZD5847, a novel oxazolidinone for treatment of tuberculosis.

Treatment of tuberculosis (TB) is impaired by the long duration and complexity of therapy and the rising incidence of drug resistance. There is an urgent need for new agents with improved efficacy, safety, and compatibility with combination chemotherapies. Oxazolidinones offer a potential new class of TB drugs, and linezolid-the only currently approved oxazolidinone-has proven highly effective against extensively drug-resistant (XDR) TB in experimental trials. However, widespread use of linezolid is prohibited by its significant toxicities. AZD5847, a novel oxazolidinone, demonstrates improved in vitro bactericidal activity against both extracellular and intracellular M. tuberculosis compared to that of linezolid. Killing kinetics in broth media and in macrophages indicate that the rate and extent of kill obtained with AZD5847 are superior to those obtained with linezolid. Moreover, the efficacy of AZD5847 was additive when tested along with a variety of conventional TB agents, indicating that AZD5847 may function well in combination therapies. AZD5847 appears to function similarly to linezolid through impairment of the mycobacterial 50S ribosomal subunit. Future studies should be undertaken to further characterize the pharmacodynamics and pharmacokinetics of AZD5847 in both in vitro and animal models as well is in human clinical trials.

Bacterial adhesion to bisphosphonate coated hydroxyapatite.

Staphylococcus aureus (S. aureus) is commonly associated with microbial infection of orthopaedic implants. Such infections often lead to osteomyelitis, which may result in failure of the implant due to localised bone destruction. Bacterial adhesion and subsequent colonisation of the device may occur as a consequence of contamination during surgery, or by seeding from a distant site through the blood circulation. Coating of the hydroxyapatite (HA) ceramic component of artificial hip joints with the bisphosphonates clodronate (C) and pamidronate (P) has been proposed as a means to minimise osteolysis and thereby prevent loosening of the implant. However, the effect of the bisphosphonate coating on bacterial adhesion to the HA materials must be determined before this approach can be implemented. In this study coated HA materials were incubated with the S. aureus and the number of adherent bacteria determined using the Modified Vortex Device (MVD) method. The number of bacteria adherent to the P coated HA material was significantly greater than that adherent to uncoated HA (60-fold increase) or to the C coated HA (90-fold increase). Therefore, even though earlier studies suggested that P bound to HA may improve osseointegration, the results presented would suggest that the use of this coating may be limited by the potential increased susceptibility of the coated device to infection.

Human immunodeficiency virus type 1 integrase: arrangement of protein domains in active cDNA complexes.

Early steps of retroviral replication involve reverse transcription of the viral RNA genome and integration of the resulting cDNA copy into a chromosome of the host cell. The viral-encoded integrase protein carries out the initial DNA breaking and joining reactions that mediate integration. The organization of the active integrase-DNA complex is unknown, though integrase is known to act as a multimer, and high resolution structures of the isolated integrase domains have been determined. Here we use site-specific cross-linking based on disulfide bond formation to map integrase-DNA contacts in active complexes. We establish that the DNA-binding C-terminal domain of one integrase monomer acts with the central catalytic domain from another monomer at each viral cDNA end. These data allow detailed modeling of an integrase tetramer in which pairs of trans interactions link integrase dimers bound to substrate DNA. We also detected a conformational change in integrase- DNA complexes accompanying cleavage of the viral cDNA terminus.

Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection.

Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal.

A quantitative assay for HIV DNA integration in vivo.

Early steps of infection by HIV-1 involve entry of the viral core into cells, reverse transcription to form the linear viral DNA, and integration of that DNA into a chromosome of the host. The unintegrated DNA can also follow non-productive pathways, in which it is circularized by recombination between DNA long-terminal repeats (LTRs), circularized by ligation of the DNA ends or degraded. Here we report quantitative methods that monitor formation of reverse transcription products, two-LTR circles and integrated proviruses. The integration assay employs a novel quantitative form of Alu-PCR that should be generally applicable to studies of integrating viruses and gene transfer vectors.

Task-dependent control of human masseter muscles from ipsilateral and contralateral motor cortex.

Corticotrigeminal projections to human masseter motoneuron pools were investigated with focal transcranial magnetic stimulation (TMS). Responses in left and right masseter muscles were quantified from the surface electromyogram (EMG) during different biting tasks. During bilateral biting, TMS elicited motor evoked potentials (MEPs) in both masseter muscles. On average, the MEP area in the masseter contralateral to the stimulus was 39% larger than in the ipsilateral muscle, despite comparable pre-stimulus EMG in both muscles. MEPs elicited while subjects attempted unilateral activation of one masseter muscle were compared with those obtained in the same muscle during a bilateral bite at an equivalent EMG level. MEPs in the masseter contralateral to the stimulated hemisphere were significantly smaller during unilateral compared with bilateral biting. There was no significant difference in the size of ipsilateral MEPs during ipsilateral and bilateral biting. We conclude that the corticotrigeminal projections to masseter are bilateral, with a stronger contralateral projection. The command for unilateral biting is associated with a reduced excitability of corticotrigeminal neurons in the contralateral, but not the ipsilateral motor cortex. We suggest that this may be accomplished by reduced activity of a population of corticotrigeminal neurons which branch to innervate both masseter motoneuron pools.

Clinical and echocardiographic characteristics of significant pericardial effusions following cardiothoracic surgery and outcomes of echo-guided pericardiocentesis for management: Mayo Clinic experience, 1979-1998.

STUDY OBJECTIVES: This study assessed the clinical features, timing of presentation, and echocardiographic characteristics associated with clinically significant pericardial effusions after cardiothoracic surgery. The outcomes of echocardiographically (echo-) guided pericardiocentesis for the management of these effusions were evaluated. DESIGN: From the prospective Mayo Clinic Registry of Echo-guided Pericardiocentesis (February 1979 to June 1998), 245 procedures performed for clinically significant postoperative effusions were identified. Clinical features, effusion causes, echocardiographic findings, and management outcomes were studied and analyzed. Cross-referencing the registry with the Mayo Clinic surgical database provided an estimate of the incidence of significant postoperative effusions and the number of cases in which primary surgical management was chosen instead of pericardiocentesis. RESULTS: Use of anticoagulant therapy was considered a significant contributing factor in 86% and 65% of early effusions (< or =7 days after surgery) and late effusions (>7 days after surgery), respectively. Postpericardiotomy syndrome was an important factor in the development of late effusions (34%). Common presenting symptoms included malaise (90%), dyspnea (65%), and chest pain (33%). Tachycardia, fever, elevated jugular venous pressure, hypotension, and pulsus paradoxus were found in 53%, 40%, 39%, 27%, and 17% of cases, respectively. Transthoracic echocardiography permitted rapid diagnosis and hemodynamic assessment of all effusions except for three cases that required transesophageal echocardiography for confirmation. Echo-guided pericardiocentesis was successful in 97% of all cases and in 96% of all loculated effusions. Major complications (2%), including chamber lacerations (n = 2) and pneumothoraces (n = 3), were successfully treated by surgical repair and chest tube reexpansion, respectively. Median follow-up duration for the study population was 3.8 years (range, 190 days to 16.4 years). The use of extended catheter drainage was associated with reduction in recurrence for early and late postoperative effusions by 46% and 50%, respectively. CONCLUSIONS: The symptoms and physical findings of clinically significant postoperative pericardial effusions are frequently nonspecific and may be inadequate for a decision regarding intervention. Echocardiography can quickly confirm the presence of an effusion, and pericardiocentesis under echocardiographic guidance is safe and effective. The use of a pericardial catheter for extended drainage is associated with lower recurrence rates, and the majority of patients so treated do not require further intervention.

The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.

Motor cortical control of human masticatory muscles.

The corticotrigeminal projections to masseter and anterior digastric motoneuron pools that are activated by TMS are bilateral, but not symmetrical. This conclusion is supported by whole-muscle data showing larger MEPs in the contralateral muscle with unilateral focal TMS, as well as evidence that TMS stimulation of one hemisphere may produce excitation in a masseter or digastric single motor unit while stimulation of the opposite hemisphere produced inhibition of the same motor unit. The asymmetry is particularly marked for masseter, in which the low-threshold motor units were most commonly excited with contralateral TMS and inhibited with ipsilateral TMS. Spike-triggered averaging of digastric motor unit activity revealed cross-talk in surface EMG recordings from digastric muscles, and no evidence that muscle fibres in both digastric muscles were innervated by a common motor axon. Narrow excitatory peaks in the PSTH of motor unit discharge elicited by TMS in masseter (either hemisphere) and digastric motor units (ipsilateral hemisphere) suggest a direct corticomotoneuronal projection. The contralateral projection to digastric motoneurons may include additional oligosynaptic connections, as judged by the broader peaks in the PSTH with contralateral TMS. The organisation of bilateral corticotrigeminal inputs revealed with TMS suggests that: (a) the contralateral hemisphere provides relatively more of the excitatory input delivered via the fast corticotrigeminal pathway for both masseter and digastric motoneuron pools, and (b) corticotrigeminal projections from either hemisphere are capable of contributing to the voluntary command mediating activation of masseter, and (to a lesser extent) anterior digastric muscles on one side, that is independent of the homologous muscles on the other side.

Cysteine and disulfide scanning reveals two amphiphilic helices in the linker region of the aspartate chemoreceptor.

The transmembrane aspartate receptor of E. coli and S. typhimurium mediates cellular chemotaxis toward aspartate by regulating the activity of the cytoplasmic histidine kinase, CheA. Ligand binding results in transduction of a conformational signal through the membrane to the cytoplasmic domain where both kinase regulation and adaptation occur. Of particular interest is the linker region, E213 to Q258, which connects and transduces the conformational signal between the cytoplasmic end of the transmembrane signaling helix (alpha 4/TM2) and the major methylation helix of the cytoplasmic domain (alpha 6). This linker is crucial for stable folding and function of the homodimeric receptor. The present study uses cysteine and disulfide scanning mutagenesis to investigate the secondary structure and packing surfaces within the linker region. Chemical reactivity assays reveal that the linker consists of three distinct subdomains: two alpha-helices termed alpha 4 and alpha 5 and, between them, an ordered region of undetermined secondary structure. When cysteine is scanned through the helices, characteristic repeating patterns of solvent exposure and burial are observed. Activity assays, both in vivo and in vitro, indicate that each helix possesses a buried packing face that is crucial for proper receptor function. The interhelical subdomain is at least partially buried and is also crucial for proper receptor function. Disulfide scanning places helix alpha 4 distal to the central axis of the homodimer, while helix alpha 5 is found to lie at the subunit interface. Finally, sequence alignments suggest that all three linker subdomains are highly conserved among the large subfamily of histidine kinase-coupled sensory receptors that possess methylation sites for use in covalent adaptation.

The two-component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes.

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

Interstitial fluid flow in tendons or ligaments: a porous medium finite element simulation.

The purpose of this study is to describe interstitial fluid flow in axisymmetric soft connective tissue (ligaments or tendons) when they are loaded in tension. Soft hydrated tissue was modelled as a porous medium (using Darcy's Law), and the finite element method was used to solve the resulting equations governing fluid flow. A commercially available computer program (FiDAP) was used to create an axisymmetric model of a biomechanically tested rat ligament. The unknown variables at element nodes were pressure and velocity of the interstitial fluid (Newtonian and incompressible). The effect of variations in fluid viscosity and permeability of the solid matrix was parametrically explored. A transient loading state mimicking a rat ligament mechanical experiment was used in all simulations. The magnitude and distribution of pressure, stream lines, shear (stress) rate, vorticity and velocity showed regular patterns consistent with extension flow. Parametric changes of permeability and viscosity strongly affected fluid flow behaviour. When the radial permeability was 1000 times less than the axial permeability, shear rate and vorticity increased (approximately 5-fold). These effects (especially shear stress and pressure) suggested a strong interaction with the solid matrix. Computed levels of fluid flow suggested a possible load transduction mechanism for cells in the tissue.

Effects of protein stabilizing agents on thermal backbone motions: a disulfide trapping study.

Chemical stabilizers are widely used to enhance protein stability, both in nature and in the laboratory. Here, the molecular mechanism of chemical stabilizers is studied using a disulfide trapping assay to measure the effects of stabilizers on thermal backbone dynamics in the Escherichia coli galactose/ glucose binding protein. Two types of backbone fluctuations are examined: (a) relative movements of adjacent surface alpha-helices within the same domain and (b) interdomain twisting motions. Both types of fluctuations are significantly reduced by all six stabilizers tested (glycerol, sucrose, trehalose, L-glucose, D-glucose, and D-galactose), and in each case larger amplitude motions are inhibited more than smaller ones. Motional inhibition does not require a high-affinity stabilizer binding site, indicating that the effects of stabilizers are nonspecific. Overall, the results support the theory that effective stabilizing agents act by favoring the most compact structure of a protein, thereby reducing local backbone fluctuations away from the fully folded state. Such inhibition of protein backbone dynamics may be a general mechanism of protein stabilization in extreme thermal or chemical environments.

Burkholderia cepacia and cystic fibrosis: do natural environments present a potential hazard?

An environmental survey of 55 sites yielded only 12 Burkholderia cepacia isolates, none of which displayed the phenotypic properties of a multiresistant epidemic strain associated with pulmonary colonization in patients with cystic fibrosis. Although the environment probably poses a low risk for patients with cystic fibrosis as a source of B. cepacia, the pathogenic potential of individual environmental strains remains unclear. We advise caution in the development of B. cepacia as a biocontrol agent.

Multi-resistance isolates possessing characteristics of both Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic fibrosis.

Multi-resistant strains from three UK centres, previously identified as Burkholderia (formerly Pseudomonas) cepacia, and associated with morbidity, mortality and transmission among patients with cystic fibrosis have been further characterised. Biochemical tests and fatty acid analyses indicate these strains to possess some characteristics atypical of B. cepacia but bearing close resemblance to Burkholderia gladioli, an organism previously regarded solely as a plant pathogen and a hindrance to the identification of B. cepacia. In contrast to the majority of reference strains, all multi-resistant clinical isolates possessed rough lipopolysaccharide which may be a major factor responsible for their increased antibiotic resistance and virulence. In view of the potential clinical and social problems in CF patients posed by these multi-resistant strains, it would seem prudent to consider the isolation of either B. cepacia or B. gladioli as of equal significance.

Serum sensitivity of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis.

Bacterial strains which are sensitive to the bactericidal activity of serum are generally considered to be less virulent than serum-resistant strains and are seldom associated with bacteraemia. Burkholderia (Pseudomonas) cepacia is an important pathogen in cystic fibrosis and is associated with rapid fatal pulmonary decline and bacteraemia in 20% of colonised patients. In this study 19 isolates of B. cepacia expressing either rough or smooth LPS were investigated to determine the degree of serum sensitivity. Strains expressing rough-LPS were serum-sensitive: these included a highly transmissible strain of B. cepacia isolated from approximately 50 cystic fibrosis patients attending various U.K. regional centres and associated with cases of bacteraemia.

Serum IgG and sputum IgA antibody to core lipopolysaccharide antigen from Pseudomonas cepacia in patients with cystic fibrosis.

The immunological response of cystic fibrosis (CF) patients to lipopolysaccharide (LPS) antigens of Pseudomonas cepacia was investigated. Enzyme-linked immunosorbent assays (ELISA) with either P. cepacia whole cells or extracted core LPS from a clinical isolate of P. cepacia as antigen were used to measure serum IgG and sputum IgA anti-P. cepacia antibodies. The ELISA with core LPS distinguished nine CF patients colonised by P. cepacia from nine age- and sex-matched non-colonised CF patients. The rate of increase of anti-P. cepacia IgG antibodies after bacteriologically proven P. cepacia colonisation varied in individual patients: in some patients the first isolation of P. cepacia was preceded or accompanied by a two-to-four-fold rise in anti-P. cepacia LPS IgG titres. Absorption studies and immunoblot analysis of serum from patients colonised with P. cepacia demonstrated that a significant component of the anti-P. cepacia core LPS antibodies was specific for P. cepacia and did not react with the core LPS of P. aeruginosa. Immunoblotting also illustrated that there may be a degree of core heterogeneity between different isolates of P. cepacia. Detection of P. cepacia LPS specific antibodies in serum (IgG) and sputum (IgA) from CF patients is recommended to assist the identification of P. cepacia colonisation in CF patients.