Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila

Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3' end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105-154 of dFLASH bind to amino acids 1-78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

How Perceptions of HIV-Related Stigma Affect Decision-Making Regarding Childbirth in Rural Kenya

Introduction

HIV prevalence among pregnant women in Kenya is high. Furthermore, there is a high risk of maternal mortality, as many women do not give birth with a skilled healthcare provider. Previous research suggests that fears of HIV testing and unwanted disclosure of HIV status may be important barriers to utilizing maternity services. We explored relationships between women’s perceptions of HIV-related stigma and their attitudes and intentions regarding facility-based childbirth.

Methods

1,777 pregnant women were interviewed at their first antenatal care visit. We included socio-demographic characteristics, stigma scales, HIV knowledge measures, and an 11-item scale measuring health facility birth attitudes (HFBA). HFBA includes items on cost, transport, comfort, interpersonal relations, and services during delivery at a health facility versus at home. A higher mean HFBA score indicates a more positive attitude towards facility-based childbirth. The mean HFBA score was dichotomized at the median and analyses were conducted with this dichotomized HFBA score using mixed effects logit models.

Results

Women who anticipated HIV-related stigma from their male partner had lower adjusted odds of having positive attitudes about giving birth at the health facility (adjusted OR = .63, 95% CI 0.50–0.78) and less positive attitudes about health facility birth were strongly related to women’s intention to give birth outside a health facility (adjusted OR = 5.56, 95% CI 2.69–11.51).

Conclusions

In this sample of pregnant women in rural Kenya, those who anticipated HIV-related stigma were less likely to have positive attitudes towards facility-based childbirth. Furthermore, negative attitudes about facility-based childbirth were associated with the intention to deliver outside a health facility. Thus, HIV-related stigma reduction efforts might result in more positive attitudes towards facility-based childbirth, and thereby lead to an increased level of skilled birth attendance, and reductions in maternal and infant mortality.     

ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation

ABCA1 exports cholesterol and phospholipids from cells by a multistep pathway that involves forming cell surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Here we used a mutational analysis approach to evaluate the relationship between these events. We prepared seven naturally occurring mutants and one artificial missense mutant of ABCA1 with varying degrees of impaired function, expressed them to similar levels as wild-type ABCA1 on the cell surface of BHK cells, and measured ABCA1-dependent lipid export, apolipoprotein A-I (apoA-I) binding, and signaling activities. Linear regression analyses showed that cholesterol and phospholipid efflux and cellular apoA-I binding correlated significantly with the ability of ABCA1 to form cell surface lipid domains. Lipid export and cellular apoA-I binding activities and formation of lipid domains also correlated with the amount of apoA-I that could be cross-linked to ABCA1. Moreover, each of these lipid export and apoA-I binding activities correlated with apoA-I-induced Janus kinase 2 (JAK2) activation. Thus, these missense mutations in ABCA1 impair lipid export, apoA-I binding, and apoA-I-stimulated JAK2 activities to similar extents, indicating that these processes are highly interactive components of a pathway that functions to export lipids from cells.     

Genetic structure among greater white‐fronted goose populations of the Pacific Flyway

An understanding of the genetic structure of populations in the wild is essential for long‐term conservation and stewardship in the face of environmental change. Knowledge of the present‐day distribution of genetic lineages (phylogeography) of a species is especially important for organisms that are exploited or utilize habitats that may be jeopardized by human intervention, including climate change. Here, we describe mitochondrial (mtDNA) and nuclear genetic (microsatellite) diversity among three populations of a migratory bird, the greater white‐fronted goose (Anser albifrons), which breeds discontinuously in western and southwestern Alaska and winters in the Pacific Flyway of North America. Significant genetic structure was evident at both marker types. All three populations were differentiated for mtDNA, whereas microsatellite analysis only differentiated geese from the Cook Inlet Basin. In sexual reproducing species, nonrandom mate selection, when occurring in concert with fine‐scale resource partitioning, can lead to phenotypic and genetic divergence as we observed in our study. If mate selection does not occur at the time of reproduction, which is not uncommon in long‐lived organisms, then mechanisms influencing the true availability of potential mates may be obscured, and the degree of genetic and phenotypic diversity may appear incongruous with presumed patterns of gene flow. Previous investigations revealed population‐specific behavioral, temporal, and spatial mechanisms that likely influence the amount of gene flow measured among greater white‐fronted goose populations. The degree of observed genetic structuring aligns well with our current understanding of population differences pertaining to seasonal movements, social structure, pairing behavior, and resource partitioning.     

The elastic properties of valve interstitial cells undergoing pathological differentiation

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.     

Ingestion without inactivation of bacteriophages by Tetrahymena

Tetrahymena has been shown to ingest and inactivate bacteriophages, such as T4, in co-incubation experiments. In this study, Tetrahymena thermophila failed to inactivate phages PhiX174 and MS2 in co-incubations, although PhiX174 were ingested by T. thermophila, as demonstrated by: (1) recovery at defecation in a pulse-chase experiment, (2) recovery from Tetrahymena by detergent lysis, and (3) transmission electron microscopy. We conclude, therefore, that the phages must be digestion-resistant. Internalized PhiX174 were further shown to be partially protected from lethal damage by ultraviolet (UV) C and UVB irradiation. Finally, ingested PhiX174 were shown to be rapidly transported through buffer in a horizontal swimming, race tube-like assay. The transport and protection of phages may confer evolutionary advantages that explain the acquisition of digestion-resistance by some phages.     

Application of a two‐pool model to soil carbon dynamics under elevated CO2

Elevated atmospheric CO₂ concentrations increase plant productivity and affect soil microbial communities, with possible consequences for the turnover rate of soil carbon (C) pools and feedbacks to the atmosphere. In a previous analysis (Van Groenigen et al., 2014), we used experimental data to inform a one‐pool model and showed that elevated CO₂ increases the decomposition rate of soil organic C, negating the storage potential of soil. However, a two‐pool soil model can potentially explain patterns of soil C dynamics without invoking effects of CO₂ on decomposition rates. To address this issue, we refit our data to a two‐pool soil C model. We found that CO₂ enrichment increases decomposition rates of both fast and slow C pools. In addition, elevated CO₂ decreased the carbon use efficiency of soil microbes (CUE), thereby further reducing soil C storage. These findings are consistent with numerous empirical studies and corroborate the results from our previous analysis. To facilitate understanding of C dynamics, we suggest that empirical and theoretical studies incorporate multiple soil C pools with potentially variable decomposition rates.     

Recycling of CO2 via Crassulacean acid metabolism in the rock outcrop succulent Sedum pulchellum Michx. (Crassulaceae)

Under well-watered conditions in the laboratory, Sedum pulchellum assimilated CO₂ only during the day, yet exhibited small nocturnal increases in tissue acid content followed by deacidification in the light (CAM-cycling). When drought-stressed, little CO₂ was fixed in the day and none at night, yet even greater acid fluctuations were observed (CAM-idling). Calculations indicate that water savings associated with CAM-cycling when water is available are small. Water saving is more likely to be significant during CAM-idling when water supply is limited and stomata are closed day and night. Thus, in this species, CAM-idling may be of greater benefit to the plant, relative to CAM-cycling, in surviving habitats prone to frequent drought stress.Abbreviations A CO₂ exchange rate - CAM Crassulacean acid metabolism - c_i shoot internal CO₂ concentration - g_c shoot conductance to CO₂ - PPFD photosynthetic photon flux density - WUE water-use efficiency Supported by National Science Foundation Grant No. DMB 8506093.     

Influence of central and extremity circumferences on all-cause mortality in men and women

For a given level of adiposity, greater lower body circumferences appear to exert a protective effect on several disease outcomes including cardiovascular disease and diabetes; however, the independent associations between extremity circumferences and mortality have not been widely investigated. The purpose of this study was to determine the independent and shared influences of upper- and lower-body circumferences on the risk of mortality in a population-based sample of adults. The sample included 10,638 adults 20-69 years of age (5,012 men; 5,626 women) from the nationally representative 1981 Canada Fitness Survey (CFS), who were monitored for over 12 years for mortality. BMI was calculated from measured height and weight. Waist, hip, thigh, calf, and upper arm circumferences were measured using a flexible, nonelastic anthropometric tape. Sex-specific proportional hazards regression models were used to evaluate the relationship between standardized values (Z-scores) of extremity circumference measures, waist circumference (WC) and mortality. Age, smoking status, alcohol consumption, and leisure-time physical activity were collected by questionnaire and were included as covariates. During 131,563 person-years of follow-up, there were 340 deaths in men and 231 in women. After mutual adjustment, WC was positively associated with mortality whereas arm, thigh, and calf circumferences were significantly protective in men, and arm and thigh circumferences were protective in women. In conclusion, waist and extremity circumferences appear to have opposite, independent effects on mortality in this sample of Canadians. Independent of BMI and WC, men and women with larger extremity circumferences had a lower risk of mortality.