Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ₃, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.     

Implementation of an insecticide-treated net subsidy scheme under a public-private partnership for malaria control in Tanzania – challenges in implementation

Background

In the past decade there has been increasing visibility of malaria control efforts at the national and international levels. The factors that have enhanced this scenario are the availability of proven interventions such as artemisinin-based combination therapy, the wide scale use of insecticide-treated nets (ITNs) and a renewed emphasis in indoor residual house-spraying. Concurrently, there has been a window of opportunity of financial commitments from organizations such as the Global Fund for HIV/AIDS, Tuberculosis and Malaria (GFATM), the President's Malaria Initiative and the World Bank Booster programme.

Methods

The case study uses the health policy analysis framework to analyse the implementation of a public-private partnership approach embarked upon by the government of Tanzania in malaria control – 'The Tanzania National Voucher Scheme'- and in this synthesis, emphasis is on the challenges faced by the scheme during the pre-implementation (2001 – 2004) and implementation phases (2004 – 2005). Qualitative research tools used include: document review, interview with key informants, stakeholder's analysis, force-field analysis, time line of events, policy characteristic analysis and focus group discussions. The study is also complemented by a cross-sectional survey, which was conducted at the Rufiji Health Demographic Surveillance Site, where a cohort of women of child-bearing age were followed up regarding access and use of ITNs.

Results

The major challenges observed include: the re-introduction of taxes on mosquito nets and related products, procurement and tendering procedures in the implementation of the GFATM, and organizational arrangements and free delivery of mosquito nets through a Presidential initiative.

Conclusion

The lessons gleaned from this synthesis include: (a) the consistency of the stakeholders with a common vision, was an important strength in overcoming obstacles, (b) senior politicians often steered the policy agenda when the policy in question was a 'crisis event', the stakes and the visibility were high, (c) national stakeholders in policy making have an advantage in strengthening alliances with international organizations, where the latter can become extremely influential in solving bottlenecks as the need arises, and (d) conflict can be turned into an opportunity, for example the Presidential initiative has inadvertently provided Tanzania with important lessons in the organization of 'catch-up' campaigns.     

Endothelial Progenitor Cell Function,Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle‐aged and older adults (42–67 years) were studied: 25 normal weight (normal weight; BMI: 18.5–24.9 kg/m²) and 42 overweight/obese (overweight/obese; BMI: 25.0–34.9 kg/m²). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony–stimulating factor (G‐CSF) was determined in the absence and presence of phytohemagglutinin (10 µg/ml). Intracellular active caspase‐3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell–derived factor‐1α (SDF‐1α; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin‐stimulated release of VEGF (90.6 ± 7.6 vs. 127.2 ± 11.6 pg/ml) and G‐CSF (896.1 ± 77.4 vs. 1,176.3 ± 126.3 pg/ml) was ～25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ～30% greater (P < 0.05) increase in active caspase‐3 in EPCs from overweight/obese (2.8 ± 0.2 ng/ml) compared with normal weight (2.2 ± 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF‐1α. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Effects of penicillin on a moderately halophilic bacterium,Vibrio costicola

Penicillin inhibited growth and killed growing cells of the moderate halophileVibrio costicola. Cells were more susceptible to penicillin at lower than at higher NaCl concentrations. Inhibition of growth by KCN prevented the lethal action of penicillin. Envelopes of treated cells showed structural damage. Chains of cells separated under the action of the drug. Treated cells swelled rapidly, an unexpected reaction in a bacterium whose internal and external osmolarities are thought to be the same.     

Physical characterization of the cloned protease III gene from Escherichia coli K-12.

Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.     

Extracellular fibril production by freshwater algae and cyanobacteria

In order to study the ability of freshwater algae and cyanobacteria to form extracellular fibrils, a screening test using ruthenium red (RR) staining was carried out on 28 species. Five of these were examined for growth and production of fibrillar material in culture media of different phosphate (P;) contents. RR-staining and uronic acid determinations at various stages of algal growth were complemented by electron microscopy of the cells and of fibrillar material released into the medium. The lower P_i concentrations enhanced growth of Micrasterias radiata, Eremosphaera sp., and Microcystis aeruginosa, and had little or no effect on growth of a Xanthidium sp. and Scenedesmus quadricauda. Extracellular uronic acid production, which was higher in low P_i medium in M. radiata, M. aeruginosa, and Xanthidium sp., could reach levels of 50 mg/liter or more. Algae with high proportions of RR-positive cells (M. radiata, Eremosphaera sp., Xanthidium sp., and M. aeruginosa) produced high levels of slime-like material and distinct fibrils that were often seen attached to the cell surface and only slowly released into the medium. No such material was found in cultures (or supernatants) of Sc. quadricauda, which also produced relatively low amounts of polyuronic acids. Specific types of filaments, often forming fascicles with rectangular arrays of globular particles were observed by negative staining electron microscopy of some algal cultures. RR-positive material was also observed in the cytoplasm and on the cell walls and surfaces of M. radiata and M. aeruginosa.Offprint requests to: D. J. Kushner.