A comparison of two bias-corrected covariance estimators for generalized estimating equations

Mancl and DeRouen (2001, Biometrics57, 126-134) and Kauermann and Carroll (2001, JASA96, 1387-1398) proposed alternative bias-corrected covariance estimators for generalized estimating equations parameter estimates of regression models for marginal means. The finite sample properties of these estimators are compared to those of the uncorrected sandwich estimator that underestimates variances in small samples. Although the formula of Mancl and DeRouen generally overestimates variances, it often leads to coverage of 95% confidence intervals near the nominal level even in some situations with as few as 10 clusters. An explanation for these seemingly contradictory results is that the tendency to undercoverage resulting from the substantial variability of sandwich estimators counteracts the impact of overcorrecting the bias. However, these positive results do not generally hold; for small cluster sizes (e.g., <10) their estimator often results in overcoverage, and the bias-corrected covariance estimator of Kauermann and Carroll may be preferred. The methods are illustrated using data from a nested cross-sectional cluster intervention trial on reducing underage drinking.     

Automatic prediction of protein interactions with large scale motion

Proteins often change their conformation upon binding to other molecules. Taking these conformational changes into account in docking is an extremely difficult task: the larger the scale of the motion the harder it is to predict the structure of the association complex. Here, we present a fully automated method for flexible docking with large scale motion in one of the docked molecules. The method automatically identifies hinge regions and rigid parts and then docks the input molecules while explicitly considering the hinges and possible protein motions.     

Adjudicated Morbidity and Mortality Outcomes by Age among Individuals with HIV Infection on Suppressive Antiretroviral Therapy

Background

Non-AIDS conditions such as cardiovascular disease and non-AIDS defining cancers dominate causes of morbidity and mortality among persons with HIV on suppressive combination antiretroviral therapy. Accurate estimates of disease incidence and of risk factors for these conditions are important in planning preventative efforts.

Methods

With use of medical records, serious non-AIDS events, AIDS events, and causes of death were adjudicated using pre-specified criteria by an Endpoint Review Committee in two large international trials. Rates of serious non-AIDS which include cardiovascular disease, end-stage renal disease, decompensated liver disease, and non-AIDS cancer, and other serious (grade 4) adverse events were determined, overall and by age, over a median follow-up of 4.3 years for 3,570 participants with CD4⁺ cell count ≥300 cells/mm³ who were taking antiretroviral therapy and had an HIV RNA level ≤500 copies/mL. Cox models were used to examine the effect of age and other baseline factors on risk of a composite outcome of all-cause mortality, AIDS, or serious non-AIDS.

Results

Five-year Kaplan-Meier estimates of the composite outcome, overall and by age were 8.3% (overall), 3.6% (<40), 8.7% (40–49) and 16.1% (≥50), respectively (p<0.001). In addition to age, smoking and higher levels of interleukin-6 and D-dimer were significant predictors of the composite outcome. The composite outcome was dominated by serious non-AIDS events (overall 65% of 277 participants with a composite event). Most serious non-AIDS events were due to cardiovascular disease and non-AIDS cancers.

Conclusions

To date, few large studies have carefully collected data on serious non-AIDS outcomes. Thus, reliable estimates of event rates are scarce. Data cited here, from a geographically diverse cohort, will be useful for planning studies of interventions aimed at reducing rates of serious non-AIDS events among people with HIV.     

Intermediate-Type Vancomycin Resistance (VISA) in Genetically-Distinct Staphylococcus aureus Isolates Is Linked to Specific,Reversible Metabolic Alterations

Intermediate (VISA-type) vancomycin resistance in Staphylococcus aureus has been associated with a range of physiologic and genetic alterations. Previous work described the emergence of VISA-type resistance in two clonally-distinct series of isolates. In both series (the first belonging to MRSA clone ST8-USA300, and the second to ST5-USA100), resistance was conferred by a single mutation in yvqF (a negative regulator of the vraSR two-component system associated with vancomycin resistance). In the USA300 series, resistance was reversed by a secondary mutation in vraSR. In this study, we combined systems-level metabolomic profiling with statistical modeling techniques to discover specific, reversible metabolic alterations associated with the VISA phenotype.     

A Novel Mechanism for Antibody-based Anthrax Toxin Neutralization: INHIBITION OF PREPORE-TO-PORE CONVERSION

Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA₆₃, oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 2α₁ loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.     

Genome structure of Tetrahymena pyriformiis

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0×10⁸ base pairs (or 1.2×10¹¹ daltons). About 90% of the macronuclear DNA fragments 200–300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7–9% at a rate corresponding to moderate repetitive sequences; 3–4% of such DNA fragments reassociate at C₀t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200–300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called zero time binding or foldback DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.