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Computing the velocity field is an expensive process for mantle convection codes. This has implications for particle methods used to model the advection of quantities such as temperature or composition. A common choice for the numerical treatment of particle trajectories is classical fourth‐order explicit Runge–Kutta (ERK4) integration, which involves a velocity computation at each of its four stages. To reduce the cost per time step, it is possible to evaluate the velocity for a subset of the four time integration stages. We explore two such alternative schemes, in which velocities are only computed for: (a) stage 1 on odd‐numbered time steps and stages 2–4 for even‐numbered time steps, and (b) stage 1 for all time steps. A theoretical analysis of stability and accuracy is presented for all schemes. It was found that the alternative schemes are first‐order accurate with stability regions different from that of ERK4. The efficiency and accuracy of the alternate schemes were compared against ERK4 in four test problems covering isothermal, thermal, and thermochemical flows. Exact solutions were used as reference solutions when available. In agreement with theory, the alternate schemes were observed to be first‐order accurate for all test problems. Accordingly, they may be used to efficiently compute solutions to within modest error tolerances. For small error tolerances, however, ERK4 was the most efficient. Plain Language Summary: Computation of the flow velocity is an expensive process for mantle convection codes. For high‐velocity flows, tracer particle methods can be used to model the transport of mantle material while minimizing model errors such as artificial diffusion. A major element of tracer particle methods is the computation of particle trajectories, which is commonly done using Runge–Kutta (RK) methods. A popular choice is the classical fourth‐order accurate RK method, which is comprised of four stages per model time step. Strictly speaking, flow velocities are to be computed for each RK stage. However, flow velocities can be computed less often in order to reduce computation time. In this study, we examine the impact of how often the flow velocities are computed during time integration on accuracy and efficiency. We find that velocities can be computed less frequently to efficiently compute solutions to modest error tolerances. However, computing the velocities for every time integration stage is the most efficient way to find solutions with tight error tolerances. Specifically, highly accurate results can be achieved with less computation time using the classical fourth‐order explicit RK method with velocity updates at all four stages. Key Points: Computation of the velocity field is expensive for mantle convection codesComputing the velocity for a subset of Runge–Kutta stages can give acceptable results for modest error tolerances at reduced costThe reduced cost from not updating the velocity at each time integration stage is not worth the accuracy lost for tight error tolerances [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Describes the BedLab genotyping assay system. Advantages of the system compared with conventional microarrays; Capabilities of the compact confocal-type imaging system built in the product; Comparison between GoldenGate assay and conventional single nucleotide polymorphism genotyping systems.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

The anterior neck is an unusual location for lipomas. Cervical lipomas can be mistaken for non-functioning thyroid nodules. We report eight cases from our files diagnosed by fine needle aspiration (FNA). Our purpose is to call attention to this simple technique in establishing an accurate diagnosis and how it can contribute to better patient management by avoiding unnecessary thyroid suppressive therapy and/or surgery. We advocate using FNA as the initial diagnostic test on palpable masses in the neck. The differential diagnosis must include thyroid lipomatosis, thyrolipoma or adenolipoma, amyloid goiter with fatty infiltration, and fat-containing thyroidal neoplasms (papillary carcinoma and follicular neoplasms).

Eighty-nine ophthalmologists in the Dallas-Fort Worth area were surveyed to find the methods used to sterilize applanation tonometer tips. Sixteen different methods were in use, with the most popular being alcohol wipes (26%) and diluted sodium hypochlorite soak (23%). Six of the most frequently used sterilization techniques were evaluated for removal of type 8 adenovirus applied to sterile tonometer tips. Adenovirus was removed or inactivated from applanation tonometer tip surfaces by using one of the following techniques: soaking inoculated tips for 15 minutes in diluted sodium hypochlorite (1:10 household bleach), 3% hydrogen peroxide, or 70% isopropyl alcohol; or wiping with alcohol "prep pads", 1:1000 merthiolate, or dry tissues.