Research questions and priorities for tuberculosis: a survey of published systematic reviews and meta-analyses

Background

Systematic reviews are increasingly informing policies in tuberculosis (TB) care and control. They may also be a source of questions for future research. As part of the process of developing the International Roadmap for TB Research, we did a systematic review of published systematic reviews on TB, to identify research priorities that are most frequently suggested in reviews.

Methodology/Principal Findings

We searched EMBASE, MEDLINE, Web of Science, and the Cochrane Library for systematic reviews and meta-analyses on any aspect of TB published between 2005 and 2010. One reviewer extracted data and a second reviewer independently extracted data from a random subset of included studies. In total, 137 systematic reviews, with 141 research questions, were included in this review. We used the UK Health Research Classification System (HRCS) to help us classify the research questions and priorities. The three most common research topics were in the area of detection, screening and diagnosis of TB (32.6%), development and evaluation of treatments and therapeutic interventions (23.4%), and TB aetiology and risk factors (19.9%). The research priorities determined were mainly focused on the discovery and evaluation of bacteriological TB tests and drug-resistant TB tests and immunological tests. Other important topics of future research were genetic susceptibility linked to TB and disease determinants attributed to HIV/TB. Evaluation of drug treatments for TB, drug-resistant TB and HIV/TB were also frequently proposed research topics.

Conclusions

Systematic reviews are a good source of key research priorities. Findings from our survey have informed the development of the International Roadmap for TB Research by the TB Research Movement.     

Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,¹ water disinfectants,² therapeutics,^{1, 3} and biomedical devices⁴ due to their powerful antimicrobial properties.^3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication⁷ and size-based isolation^4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.^7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).^10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 μg ml^-1 down to 10 ml of 198.7 μg ml^-1) than conventional methods of isolation such as ultracentrifugation.¹⁹TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.^20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 μg ml^-1) were synthesized using the well-established Creighton method^22,23 by the reduction of AgNO₃ with NaBH₄. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm²) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm² and 20 cm²) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 μg ml^-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.     

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E^rns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E^rns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of E^rns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, E^rnsE1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of E^rns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase.     

A simplified 2-D electrophoresis protocol with the aid of an organic disulfide

2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.