Parasitism byEphedrus cerasicola [Hym.: Aphidiidae] developing in different stages ofMyzus persicae [Hom.: Aphididae]

The parasitoidEphedrus cerasicola Stary oviposited in the 4 nymphal instars and in newly moulted apterous adults ofMyzus persicae (Sulzer). Development and reproduction of unparasitized and parasitized aphids at 21°C were compared. Unparasitized aphids developed to adults in 6.5 days and started to reproduce after 7 days. Longevity varied between 7 and 42 days. Net reproductive rate (R₀) was 40.7. In contrast to older nymphs, aphids parasitized in the 1 st instar almost never reached the adult stage before mummification. Aphids parasitized in 2nd, 3rd and 4th instar and as newly moulted adults produced respectively 0.07 %, 2 %, 23 % and 32 % of offspring produced by unparasitized aphids. Corresponding reproductive periods were 1, 1.4, 3 and 4 days. Host age at parasitization had a slight effect on the parasitoid's developmental rate and had no effect on egg or pupal survival, or on the sex ratio of the emerging parasitoids.     

Genetic determination of telomere size in humans: a twin study of three age groups.

Reduction of telomere length has been postulated to be a causal factor in cellular aging. Human telomeres terminate in tandemly arranged repeat arrays consisting of the (TTAGGG) motif. The length of these arrays in cells from human mitotic tissues is inversely related to the age of the donor, indicating telomere reduction with age. In addition to telomere length differences between different age cohorts, considerable variation is present among individuals of the same age. To investigate whether this variation can be ascribed to genetic influences, we have measured the size of terminal restriction fragments (TRFs) in HaeIII-digested genomic DNA from 123 human MZ and DZ twin pairs 2-95 years of age. The average rate of telomere shortening was 31 bp/year, which is similar to that observed by others. Statistical analysis in 115 pairs 2-63 years of age indicates a 78% heritability for mean TRF length in this age cohort. The individual differences in mean TRF length in blood, therefore, seem to a large extent to be genetically determined.     

Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process—indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.     

Targeting the Non-structural Protein 1 from Dengue Virus to a Dendritic Cell Population Confers Protective Immunity to Lethal Virus Challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4⁺ and CD8⁺ T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205⁺ DC population with poly (I:C) opens perspectives for dengue vaccine development.     

Expanding ART for treatment and prevention of HIV in South Africa: estimated cost and cost-effectiveness 2011-2050

Background

Antiretroviral Treatment (ART) significantly reduces HIV transmission. We conducted a cost-effectiveness analysis of the impact of expanded ART in South Africa.

Methods

We model a best case scenario of 90% annual HIV testing coverage in adults 15–49 years old and four ART eligibility scenarios: CD4 count <200 cells/mm³ (current practice), CD4 count <350, CD4 count <500, all CD4 levels. 2011–2050 outcomes include deaths, disability adjusted life years (DALYs), HIV infections, cost, and cost per DALY averted. Service and ART costs reflect South African data and international generic prices. ART reduces transmission by 92%. We conducted sensitivity analyses.

Results

Expanding ART to CD4 count <350 cells/mm³ prevents an estimated 265,000 (17%) and 1.3 million (15%) new HIV infections over 5 and 40 years, respectively. Cumulative deaths decline 15%, from 12.5 to 10.6 million; DALYs by 14% from 109 to 93 million over 40 years. Costs drop $504 million over 5 years and $3.9 billion over 40 years with breakeven by 2013. Compared with the current scenario, expanding to <500 prevents an additional 585,000 and 3 million new HIV infections over 5 and 40 years, respectively. Expanding to all CD4 levels decreases HIV infections by 3.3 million (45%) and costs by $10 billion over 40 years, with breakeven by 2023. By 2050, using higher ART and monitoring costs, all CD4 levels saves $0.6 billion versus current; other ART scenarios cost $9–194 per DALY averted. If ART reduces transmission by 99%, savings from all CD4 levels reach $17.5 billion. Sensitivity analyses suggest that poor retention and predominant acute phase transmission reduce DALYs averted by 26% and savings by 7%.

Conclusion

Increasing the provision of ART to <350 cells/mm3 may significantly reduce costs while reducing the HIV burden. Feasibility including HIV testing and ART uptake, retention, and adherence should be evaluated.     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.     

PCSK9 SNP rs11591147 is associated with low cholesterol levels but not with cognitive performance or noncardiovascular clinical events in an elderly population

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a protein involved in LDL-cholesterol metabolism. The single-nucleotide polymorphism (SNP) rs11591147 has been associated with lower LDL-cholesterol and a lower risk of coronary heart disease. Because PCSK9 has high affinity to the LDL receptor, inhibiting PCSK9 is a testable therapeutic target for lipid-lowering therapy. Currently, several approaches to inhibit PCSK9 are under development, but it is unknown what the effects of those inhibitors will be on cognition or noncardiovascular clinical events. In this study, we assessed the association between rs11591147 and cognitive performance, activities of daily living (ADL), and noncardiovascular clinical events within 5,777 participants of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). Rs11591147 was associated with 10% to 16% lower LDL cholesterol levels (P = 3.62 × 10⁻¹²), but was not associated with cognitive performance, ADL, or noncardiovascular clinical events in the PROSPER study. Our findings suggest that lower cholesterol levels due to genetic variation in the PCSK9 gene are not associated with cognitive performance, functional status, or noncardiovascular clinical events.