Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.     

Determinants and Experiences of Repeat Pregnancy among HIV-Positive Kenyan Women—A Mixed-Methods Analysis

Objective

To identify factors associated with repeat pregnancy subsequent to an index pregnancy among women living with HIV (WLWH) in western Kenya who were enrolled in a 24-month phase-II clinical trial of triple-ART prophylaxis for prevention of mother-to-child transmission, and to contextualize social and cultural influences on WLWH’s reproductive decision making.

Methods

A mixed-methods approach was used to examine repeat pregnancy within a 24 month period after birth. Counselor-administered questionnaires were collected from 500 WLWH. Forty women (22 with a repeat pregnancy; 18 with no repeat pregnancy) were purposively selected for a qualitative interview (QI). Simple and multiple logistic regression analyses were performed for quantitative data. Thematic coding and saliency analysis were undertaken for qualitative data.

Results

Eighty-eight (17.6%) women had a repeat pregnancy. Median maternal age was 23 years (range 15-43 years) and median gestational age at enrollment was 34 weeks. In multiple logistic regression analyses, living in the same compound with a husband (adjusted odds ratio (AOR): 2.33; 95% confidence interval (CI): 1.14, 4.75) was associated with increased odds of repeat pregnancy (p ≤ 0.05). Being in the 30-43 age group (AOR: 0.25; 95% CI: 0.07, 0.87), having talked to a partner about family planning (FP) use (AOR: 0.53; 95% CI: 0.29, 0.98), and prior usage of FP (AOR: 0.45; 95% CI: 0.25, 0.82) were associated with a decrease in odds of repeat pregnancy. QI findings centered on concerns about modern contraception methods (side effects and views that they ‘ruined the womb’) and a desire to have the right number of children. Religious leaders, family, and the broader community were viewed as reinforcing cultural expectations for married women to have children. Repeat pregnancy was commonly attributed to contraception failure or to lack of knowledge about post-delivery fertility.

Conclusions

In addition to cultural context, reproductive health programs for WLWH may need to address issues related to living circumstances and the possibility that reproductive-decision making may extend beyond the woman and her partner.     

The Impact of Implementing a Test,Treat and Retain HIV Prevention Strategy in Atlanta among Black Men Who Have Sex with Men with a History of Incarceration: A Mathematical Model

Background

Annually, 10 million adults transition through prisons or jails in the United States (US) and the prevalence of HIV among entrants is three times higher than that for the country as a whole. We assessed the potential impact of increasing HIV Testing/Treatment/Retention (HIV-TTR) in the community and within the criminal justice system (CJS) facilities, coupled with sexual risk behavior change, focusing on black men-who-have-sex-with-men, 15–54 years, in Atlanta, USA.

Methods

We modeled the effect of a HIV-TTR strategy on the estimated cumulative number of new (acquired) infections and mortality, and on the HIV prevalence at the end of ten years. We additionally assessed the effect of increasing condom use in all settings.

Results

In the Status Quo scenario, at the end of 10 years, the cumulative number of new infections in the community, jail and prison was, respectively, 9246, 77 and 154 cases; HIV prevalence was 10815, 69 and 152 cases, respectively; and the cumulative number of deaths was 2585, 18 and 34 cases, respectively. By increasing HIV-TTR coverage, the cumulative number of new infections could decrease by 15% in the community, 19% in jail, and 8% in prison; HIV prevalence could decrease by 8%, 9% and 7%, respectively; mortality could decrease by 20%, 39% and 18%, respectively. Based on the model results, we have shown that limited use and access to condoms have contributed to the HIV incidence and prevalence in all settings.

Conclusions

Aggressive implementation of a CJS-focused HIV-TTR strategy has the potential to interrupt HIV transmission and reduce mortality, with benefit to the community at large. To maximize the impact of these interventions, retention in treatment, including during the period after jail and prison release, and increased condom use was vital for decreasing the burden of the HIV epidemic in all settings.     

Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission

Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.     

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

Harnessing Case Isolation and Ring Vaccination to Control Ebola

As a devastating Ebola outbreak in West Africa continues, non-pharmaceutical control measures including contact tracing, quarantine, and case isolation are being implemented. In addition, public health agencies are scaling up efforts to test and deploy candidate vaccines. Given the experimental nature and limited initial supplies of vaccines, a mass vaccination campaign might not be feasible. However, ring vaccination of likely case contacts could provide an effective alternative in distributing the vaccine. To evaluate ring vaccination as a strategy for eliminating Ebola, we developed a pair approximation model of Ebola transmission, parameterized by confirmed incidence data from June 2014 to January 2015 in Liberia and Sierra Leone. Our results suggest that if a combined intervention of case isolation and ring vaccination had been initiated in the early fall of 2014, up to an additional 126 cases in Liberia and 560 cases in Sierra Leone could have been averted beyond case isolation alone. The marginal benefit of ring vaccination is predicted to be greatest in settings where there are more contacts per individual, greater clustering among individuals, when contact tracing has low efficacy or vaccination confers post-exposure protection. In such settings, ring vaccination can avert up to an additional 8% of Ebola cases. Accordingly, ring vaccination is predicted to offer a moderately beneficial supplement to ongoing non-pharmaceutical Ebola control efforts.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Stress-Tolerant Feedstocks for Sustainable Bioenergy Production on Marginal Land

Given the mandated increases in fuel production from alternative sources, limited high-quality production land, and predicted climate changes, identification of stress-tolerant biomass crops will be increasingly important. However, existing literature largely focuses on the responses of a small number of crops to a single source of abiotic stress. Here, we provide a much-needed review of several types of stress likely to be encountered by biomass crops on marginal lands and under future climate scenarios: drought, flooding, salinity, cold, and heat. The stress responses of 17 leading biomass crops of all growth habits (e.g., perennial grasses, short-rotation woody crops, and large trees) are summarized, and we identify several that could be considered “all purpose” for multiple stress types. Importantly, we note that some of these crops are or could become invasive in some landscapes. Therefore, growers must take care to avoid dissemination of plants or propagules outside of cultivation.