Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(?), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.     

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.     

Efficient CNS gene delivery by intravenous injection

We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS.     

The Construction and Use of Log-Odds Substitution Scores for Multiple Sequence Alignment

Most pairwise and multiple sequence alignment programs seek alignments with optimal scores. Central to defining such scores is selecting a set of substitution scores for aligned amino acids or nucleotides. For local pairwise alignment, substitution scores are implicitly of log-odds form. We now extend the log-odds formalism to multiple alignments, using Bayesian methods to construct “BILD” (“Bayesian Integral Log-odds”) substitution scores from prior distributions describing columns of related letters. This approach has been used previously only to define scores for aligning individual sequences to sequence profiles, but it has much broader applicability. We describe how to calculate BILD scores efficiently, and illustrate their uses in Gibbs sampling optimization procedures, gapped alignment, and the construction of hidden Markov model profiles. BILD scores enable automated selection of optimal motif and domain model widths, and can inform the decision of whether to include a sequence in a multiple alignment, and the selection of insertion and deletion locations. Other applications include the classification of related sequences into subfamilies, and the definition of profile-profile alignment scores. Although a fully realized multiple alignment program must rely upon more than substitution scores, many existing multiple alignment programs can be modified to employ BILD scores. We illustrate how simple BILD score based strategies can enhance the recognition of DNA binding domains, including the Api-AP2 domain in Toxoplasma gondii and Plasmodium falciparum.     

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.     

The “ART” of Linkage: Pre-Treatment Loss to Care after HIV Diagnosis at Two PEPFAR Sites in Durban,South Africa

Background

Although loss to follow-up after antiretroviral therapy (ART) initiation is increasingly recognized, little is known about pre-treatment losses to care (PTLC) after an initial positive HIV test. Our objective was to determine PTLC in newly identified HIV-infected individuals in South Africa.

Methodology/Principal Findings

We assembled the South African Test, Identify and Link (STIAL) Cohort of persons presenting for HIV testing at two sites offering HIV and CD4 count testing and HIV care in Durban, South Africa. We defined PTLC as failure to have a CD4 count within 8 weeks of HIV diagnosis. We performed multivariate analysis to identify factors associated with PTLC. From November 2006 to May 2007, of 712 persons who underwent HIV testing and received their test result, 454 (64%) were HIV-positive. Of those, 206 (45%) had PTLC. Infected patients were significantly more likely to have PTLC if they lived ≥10 kilometers from the testing center (RR = 1.37; 95% CI: 1.11–1.71), had a history of tuberculosis treatment (RR = 1.26; 95% CI: 1.00–1.58), or were referred for testing by a health care provider rather than self-referred (RR = 1.61; 95% CI: 1.22–2.13). Patients with one, two or three of these risks for PTLC were 1.88, 2.50 and 3.84 times more likely to have PTLC compared to those with no risk factors.

Conclusions/Significance

Nearly half of HIV-infected persons at two high prevalence sites in Durban, South Africa, failed to have CD4 counts following HIV diagnosis. These high rates of pre-treatment loss to care highlight the urgent need to improve rates of linkage to HIV care after an initial positive HIV test.     

Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background

We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K_d) and gp120-to-CD4 inhibition constants (K_i). The PA analogs were selected to satisfy Lipinski''s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Principal Findings

PA analog 2-bromopalmitate (2-BP) was most efficacious with K_d ∼74 nM and K_i ∼122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher K_d ∼140 nM and K_i ∼354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with K_d ∼364 nM and inhibiting gp120-to-CD4 binding with K_i ∼1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry.

Significance

Considering observed differences between K_i and K_d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.     

Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

Background

Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways.

Methods/Principal Findings

Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm²), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm²) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis.

Conclusions/Significance

We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.