The development and impact of tuberculosis vaccines

Vaccination is expected to make a major contribution to the goal of eliminating tuberculosis worldwide by 2050. But developing a new effective vaccine will require innovation in scientific research, a proactive approach to clinical trials of new vaccine candidates, and application of vaccines as part of an integrated approach to disease control.     

Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication

Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.     

HIV Treatment as Prevention: Debate and Commentary—Will Early Infection Compromise Treatment-as-Prevention Strategies?

Universal HIV testing and immediate antiretroviral therapy for infected individuals has been proposed as a way of reducing the transmission of HIV and thereby bringing the HIV epidemic under control. It is unclear whether transmission during early HIV infection—before individuals are likely to have been diagnosed with HIV and started on antiretroviral therapy—will compromise the effectiveness of treatment as prevention. This article presents two opposing viewpoints by Powers, Miller, and Cohen, and Williams and Dye, followed by a commentary by Fraser.     

Chemical activation of a food deprivation signal extends lifespan

Model organisms subject to dietary restriction (DR) generally live longer. Accompanying this lifespan extension are improvements in overall health, based on multiple metrics. This indicates that pharmacological treatments that mimic the effects of DR could improve health in humans. To find new chemical structures that extend lifespan, we screened 30 000 synthetic, diverse drug‐like chemicals in Caenorhabditis elegans and identified several structurally related compounds that acted through DR mechanisms. The most potent of these NP1 impinges upon a food perception pathway by promoting glutamate signaling in the pharynx. This results in the overriding of a GPCR pathway involved in the perception of food and which normally acts to decrease glutamate signals. Our results describe the activation of a dietary restriction response through the pharmacological masking of a novel sensory pathway that signals the presence of food. This suggests that primary sensory pathways may represent novel targets for human pharmacology.     

Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc

Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET‐based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady‐state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.     

A qualitative assessment of participation in a rapid scale-up, diagonally-integrated MDG-related disease prevention campaign in Rural Kenya

Background

Many countries face severe scale-up barriers toward achievement of MDGs. We ascertained motivational and experiential dimensions of participation in a novel, rapid, “diagonal” Integrated Prevention Campaign (IPC) in rural Kenya that provided prevention goods and services to 47,000 people within one week, aimed at rapidly moving the region toward MDG achievement. Specifically, the IPC provided interventions and commodities targeting disease burden reduction in HIV/AIDS, malaria, and water-borne illness.

Methods

Qualitative in-depth interviews (IDI) were conducted with 34 people (18 living with HIV/AIDS and 16 not HIV-infected) randomly selected from IPC attendees consenting to participate. Interviews were examined for themes and patterns to elucidate participant experience and motivation with IPC.

Findings

Participants report being primarily motivated to attend IPC to learn of their HIV status (through voluntary counseling and testing), and with receipt of prevention commodities (bednets, water filters, and condoms) providing further incentive. Participants reported that they were satisfied with the IPC experience and offered suggestions to improve future campaigns.

Interpretation

Learning their HIV status motivated participants along with the incentive of a wider set of commodities that were rapidly deployed through IPC in this challenging region. The critical role of wanting to know their HIV status combined with commodity incentives may offer a new model for rapid scaled-up of prevention strategies that are wider in scope in rural Africa.     

Regulation of the pharynx of Caenorhabditis elegans by 5‐HT,octopamine, and FMRFamide‐like neuropeptides

More than fifty FMRFamide‐like neuropeptides have been identified in nematodes. We addressed the role of a subset of these in the control of nematode feeding by electrophysiological recording of the activity of C. elegans pharynx. AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), AF8 (KSAYMRFamide), and GAKFIRFamide (encoded by the C. elegans genes flp‐8, flp‐14, flp‐6, and flp‐5, respectively) increased pharyngeal action potential frequency, in a manner similar to 5‐HT. In contrast, SDPNFLRFamide, SADPNFLRFamide, SAEPFGTMRFamide, KPSVRFamide, APEASPFIRFamide, and AQTVRFamide (encoded by the C. elegans genes flp‐1; flp‐1; flp‐3; flp‐9; flp‐13, and flp‐16, respectively) inhibited the pharynx in a manner similar to octopamine. Only three of the neuropeptides had potent effects at low nanomolar concentrations, consistent with a physiological role in pharyngeal regulation. Therefore, we assessed whether these three peptides mediated their actions either directly on the pharynx or indirectly via the neural circuit controlling its activity by comparing actions between wild‐type and mutants with deficits in synaptic signaling. Our data support the conclusion that AF1 and SAEPFGTMRFamide regulate the activity of the pharynx indirectly, whereas APEASPFIRFamide exerts its action directly. These results are in agreement with the expression pattern for the genes encoding the neuropeptides (Kim and Li, 1999) as both flp‐8 and flp‐3 are expressed in extrapharyngeal neurons, whereas flp‐13 is expressed in I5, a neuron with synaptic output to the pharyngeal muscle. These results provide the first, direct, functional information on the action of neuropeptides in C. elegans. Furthermore, we provide evidence for a putative inhibitory peptidergic synapse, which is likely to have a role in the control of feeding. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 235–244, 2001     

The use of principal component analysis to resolve the spectra and kinetics of cytochrome c oxidase reduction by 5,10-dihydro-5-methyl phenazine.

The method of principal component analysis (PCA) was applied to the absorption-wavelength-time surfaces generated by rapid scanning stopped-flow spectrophotometry (RSSFS). The method was used to resolve the absorption surfaces generated during the reduction of cytochrome c oxidase by 5,10-dihydro-5-methyl phenazine (MPH) into the individual spectral shapes and time courses of the component chromophores. Two forms of resting cytochrome oxidase were used in these analyses: one that has its maximum absorption in the Soret region at 418 nm (418-nm species) and the other has its absorption maximum at 424 nm (424-nm species). A weighting scheme suitable for RSSFS data was developed. The optical absorption spectra obtained by W.H. Vanneste (1966, Biochemistry, 5:838-848) for the oxidase components were found to fit adequately as components of the experimental surfaces. Among these spectra were the oxidized forms of cytochromes a and a3 in the wavelength region 330-520 nm for the 418-nm species. Vanneste's spectral shape for the oxidized cytochrome a3 did not fit as a component in the spectrum of the 424-nm species. After accounting for the spectral shape of all components present, PCA provided a straightforward method for determining the separate time courses of each chromophore. We have found for both forms used that cytochrome a is reduced by MPH in the initial stages of the reaction, while cytochrome a3 is reduced in subsequent, slow phases. An important aspect of PCA is that it provided confirmation of the spectra of the various oxidase components without requiring the use of inhibitors or the use of simplifying mechanistic assumptions. The resolution of time profiles of strongly overlapping chromophores is also demonstrated.     

Reduction of cytochrome oxidase by 5,10-dihydro-5-methylphenazine: kinetic parameters from rapid-scanning stopped-flow experiments

The kinetics of the reduction of resting cytochrome oxidase and of its cyanide complex by 5,10-dihydro-5- methylphenazine (MPH) have been characterized by rapid-scan and fixed-wavelength stopped-flow spectrophotometry in the Soret, visible, and near-IR spectral regions. In this study, we focused on a form of the resting enzyme that is characterized by a Soret absorption maximum at 424 nm. These experiments complement earlier work on the reduction of a 418 nm absorbing form of the resting enzyme [ Halaka , F.G., Babcock , G. T., & Dye, J. L. (1981) J. Biol. Chem. 256, 1084-1087]. The reduction of cytochrome a is accomplished in a second-order reaction with a rate constant of 3 X 10(5) M-1 s-1. The reduction of the 830-nm absorber, Cua, is closely coupled to but lags the reduction of cytochrome a; we have resolved a rate constant of about 20 s-1 for the copper reduction. The reduction of cytochrome a proceeds with a rate constant that is nearly independent of the spectral properties of the resting enzyme and of the ligation state of cytochrome a3. The reduction of cytochrome a3 occurs by slow, intramolecular electron transfer. We have resolved two phases for this process that have rate constants of approximately 0.2 s-1 and approximately 0.02 s-1 for both the 418- and 424-nm forms of the resting enzyme. It appears, therefore, that spectroscopic heterogeneity at the cytochrome a3 site in the resting enzyme exerts very little influence on the kinetics of the anaerobic reduction of the oxidase metal centers. From this we conclude that the rate of electron transfer to the a3 site is probably controlled by the protein conformation and not primarily by local factors within the a3 environment.