3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes

Crude incidence in two-phase designs in the presence of competing risks.

BackgroundIn many studies, some information might not be available for the whole cohort, some covariates, or even the outcome, might be ascertained in selected subsamples. These studies are part of a broad category termed two-phase studies. Common examples include the nested case-control and the case-cohort designs. For two-phase studies, appropriate weighted survival estimates have been derived; however, no estimator of cumulative incidence accounting for competing events has been proposed. This is relevant in the presence of multiple types of events, where estimation of event type specific quantities are needed for evaluating outcome.MethodsWe develop a non parametric estimator of the cumulative incidence function of events accounting for possible competing events. It handles a general sampling design by weights derived from the sampling probabilities. The variance is derived from the influence function of the subdistribution hazard.ResultsThe proposed method shows good performance in simulations. It is applied to estimate the crude incidence of relapse in childhood acute lymphoblastic leukemia in groups defined by a genotype not available for everyone in a cohort of nearly 2000 patients, where death due to toxicity acted as a competing event. In a second example the aim was to estimate engagement in care of a cohort of HIV patients in resource limited setting, where for some patients the outcome itself was missing due to lost to follow-up. A sampling based approach was used to identify outcome in a subsample of lost patients and to obtain a valid estimate of connection to care.ConclusionsA valid estimator for cumulative incidence of events accounting for competing risks under a general sampling design from an infinite target population is derived

Depressive Symptoms at HIV Testing and Two-Year All-Cause Mortality Among Men Who Inject Drugs in Vietnam

People who inject drugs (PWID) with HIV experience an elevated risk of death. A potentially important determinant of survival is the high burden of depression. This study examined the relationship of depressive symptoms at HIV testing with 2-year all-cause mortality among newly diagnosed HIV-positive PWID in Vietnam. At HIV testing, 141 PWID (42%) experienced severe depressive symptoms, and over the 2 years following diagnosis, 82 PWID (24%) died. Controlling for potential confounders, the 2-year risk of death among those with depressive symptoms was 9.7% (95% CI − 1.2, 20.6%) higher than the risk among those without depressive symptoms. This increased risk of mortality for PWID with depressive symptoms was relatively consistent throughout the 2-year period: at 6, 12, and 18 months, the risk difference was 12.6% (5.5–19.7%), 13.9% (4.6–23.2%), and 11.0% (0.9–21.1%), respectively. HIV diagnosis may provide an important opportunity for depression screening and treatment, subsequently improving survival in this key population. Trial registry: ClinicalTrials.gov NCT01689545

Sampling-Based Approaches to Improve Estimation of Mortality among Patient Dropouts: Experience from a Large PEPFAR-Funded Program in Western Kenya

Monitoring and evaluation (M&E) of HIV care and treatment programs is impacted by losses to follow-up (LTFU) in the patient population. The severity of this effect is undeniable but its extent unknown. Tracing all lost patients addresses this but census methods are not feasible in programs involving rapid scale-up of HIV treatment in the developing world. Sampling-based approaches and statistical adjustment are the only scaleable methods permitting accurate estimation of M&E indices.In a large antiretroviral therapy (ART) program in western Kenya, we assessed the impact of LTFU on estimating patient mortality among 8,977 adult clients of whom, 3,624 were LTFU. Overall, dropouts were more likely male (36.8% versus 33.7%; p = 0.003), and younger than non-dropouts (35.3 versus 35.7 years old; p = 0.020), with lower median CD4 count at enrollment (160 versus 189 cells/ml; p<0.001) and WHO stage 3-4 disease (47.5% versus 41.1%; p<0.001). Urban clinic clients were 75.0% of non-dropouts but 70.3% of dropouts (p<0.001). Of the 3,624 dropouts, 1,143 were sought and 621 had their vital status ascertained. Statistical techniques were used to adjust mortality estimates based on information obtained from located LTFU patients. Observed mortality estimates one year after enrollment were 1.7% (95% CI 1.3%-2.0%), revised to 2.8% (2.3%-3.1%) when deaths discovered through outreach were added and adjusted to 9.2% (7.8%-10.6%) and 9.9% (8.4%-11.5%) through statistical modeling depending on the method used. The estimates 12 months after ART initiation were 1.7% (1.3%-2.2%), 3.4% (2.9%-4.0%), 10.5% (8.7%-12.3%) and 10.7% (8.9%-12.6%) respectively. CONCLUSIONS/SIGNIFICANCE ABSTRACT: Assessment of the impact of LTFU is critical in program M&E as estimated mortality based on passive monitoring may underestimate true mortality by up to 80%. This bias can be ameliorated by tracing a sample of dropouts and statistically adjust the mortality estimates to properly evaluate and guide large HIV care and treatment programs

Effects of growth rate, size, and light availability on tree survival across life stages: a demographic analysis accounting for missing values and small sample sizes.

The data set supporting the results of this article is available in the Dryad repository, http://dx.doi.org/10.5061/dryad.6f4qs. Moustakas, A. and Evans, M. R. (2015) Effects of growth rate, size, and light availability on tree survival across life stages: a demographic analysis accounting for missing values.Plant survival is a key factor in forest dynamics and survival probabilities often vary across life stages. Studies specifically aimed at assessing tree survival are unusual and so data initially designed for other purposes often need to be used; such data are more likely to contain errors than data collected for this specific purpose

Simple estimators of the intensity of seasonal occurrence

<p>Abstract</p> <p>Background</p> <p>Edwards's method is a widely used approach for fitting a sine curve to a time-series of monthly frequencies. From this fitted curve, estimates of the seasonal intensity of occurrence (i.e., peak-to-low ratio of the fitted curve) can be generated.</p> <p>Methods</p> <p>We discuss various approaches to the estimation of seasonal intensity assuming Edwards's periodic model, including maximum likelihood estimation (MLE), least squares, weighted least squares, and a new closed-form estimator based on a second-order moment statistic and non-transformed data. Through an extensive Monte Carlo simulation study, we compare the finite sample performance characteristics of the estimators discussed in this paper. Finally, all estimators and confidence interval procedures discussed are compared in a re-analysis of data on the seasonality of monocytic leukemia.</p> <p>Results</p> <p>We find that Edwards's estimator is substantially biased, particularly for small numbers of events and very large or small amounts of seasonality. For the common setting of rare events and moderate seasonality, the new estimator proposed in this paper yields less finite sample bias and better mean squared error than either the MLE or weighted least squares. For large studies and strong seasonality, MLE or weighted least squares appears to be the optimal analytic method among those considered.</p> <p>Conclusion</p> <p>Edwards's estimator of the seasonal relative risk can exhibit substantial finite sample bias. The alternative estimators considered in this paper should be preferred.</p

Longitudinal analysis of alcohol use and intimate partner violence perpetration among men with HIV in northern Vietnam

Background: Alcohol use is a known risk factor for male-perpetrated intimate partner violence (IPV), although few studies have been conducted globally and among men with HIV (MWH). We estimated the longitudinal effects of alcohol use on IPV perpetration among MWH. Methods: This study is a secondary analysis of randomized controlled trial data among male and female antiretroviral treatment patients with hazardous alcohol use in Thai Nguyen, Vietnam. Analyses were restricted to male participants who were married/cohabitating (N = 313). Alcohol use was assessed as proportion days alcohol abstinent, heavy drinking, and alcohol use disorder (AUD) using the Timeline Followback and Mini International Neuropsychiatric Interview questionnaire. Multilevel modeling was used to estimate the effects of higher versus lower average alcohol use on IPV perpetration (between-person effects) and the effects of time-specific deviations in alcohol use on IPV perpetration (within-person effects). Results: Participants with higher average proportion days alcohol abstinent had decreased odds of IPV perpetration (adjusted Odds Ratio [aOR] = 0.43, p = 0.03) and those with higher average heavy drinking and AUD had increased odds of IPV perpetration (Heavy drinking: aOR = 1.05, p = 0.002; AUD: aOR = 4.74, p < 0.0001). Time-specific increases in proportion days alcohol abstinent were associated with decreased odds of IPV perpetration (aOR = 0.39, p = 0.02) and time-specific increases in AUD were associated with increased odds of IPV perpetration (aOR = 2.95, p = 0.001). Within-person effects for heavy drinking were non-significant. Conclusions: Alcohol use is associated with IPV perpetration among Vietnamese men with HIV. In this context, AUD and frequent drinking are stronger correlates of IPV perpetration as compared to heavy drinking

Characteristics of persons who inject drugs and who witness opioid overdoses in Vietnam: a cross-sectional analysis to inform future overdose prevention programs

Abstract Background Persons who use opioids have a high risk of overdose and associated mortality. In Vietnam, little is known about the characteristics of this population and the persons who are witness to those overdoses. One approach to combatting fatal overdose has been the use of peer interventions in which a friend or injecting partner administers overdose reversal medication, but availability in Vietnam of these medications is limited to pilot programs with aims to expand in the future (Le Minh and V.F. Go, Personal Communication, 2016). The primary objective of this paper is to explore the characteristics associated with witnessing three or more overdoses in a lifetime. Methods This cross-sectional analysis used baseline data from a four-arm randomized control trial conducted in Thai Nguyen, Vietnam, known as the Prevention for Positives project. One thousand six hundred seventy-three PWID were included in the analysis. We conducted bivariable and multivariable logistic regression to identify characteristics associated with witnessing three or more overdoses in a lifetime. Characteristics explored included education, employment, marital status, risky drug use behaviors, locations for accessing syringes, recent overdose, history of incarceration, drug treatment, and having slept outside in the past 3 months. Results Seventy-two percent (n = 1203) of participants had witnessed at least one overdose in their lifetime, and 46% had witnessed three or more overdoses (n = 765). In the multivariable model, having less than secondary education (AOR 0.70; 95% CI 0.57, 0.86), having slept outside in the past 3 months (AOR 1.77; 95% CI 1.31, 2.40), having a history of incarceration (AOR 1.33; 95% CI 1.07, 1.65), having a history of drug treatment (AOR 1.41; 95% CI 1.12, 1.77), experiencing a recent non-fatal overdose (AOR 3.84; 95% CI 2.36, 6.25), injecting drugs daily (AOR 1.79; 95% CI 1.45, 2.20), receptive needle sharing (AOR 1.30; 95% CI 1.04, 1.63), and number of years injecting (AOR 1.04; 95% CI 1.02, 1.07) were significantly associated with witnessing three or more overdoses. Conclusions Targeted interventions are needed to train persons witnessing an overdose to administer overdose-reversal medication. This includes targeting persons prior to release from prisons, drug treatment centers, and those accessing syringe exchange programs. Additional research should assess the burden of witnessing an overdose as well as locations for medication distribution. Assessments of the training capacity and needs for implementing these programs among drug using peers in Vietnam are of the utmost importance

Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation

AbstractCovalent attachment of surface proteins to the cell wall of Gram-positive bacteria requires a sortase-mediated transpeptidation reaction. In almost all Gram-positive bacteria, the housekeeping sortase, sortase A, recognizes the canonical recognition sequence LPXTG (X=any amino acid). The human pathogen Clostridium difficile carries a single putative sortase gene (cd2718) but neither transpeptidation activity nor specificity of CD2718 has been investigated. We produced recombinant CD2718 and examined its transpeptidation activity in vitro using synthetic peptides and MALDI-ToF(-ToF) MS analysis. We demonstrate that CD2718 has sortase activity with specificity for a (S/P)PXTG motif and can accommodate diaminopimelic acid as a substrate for transpeptidation