New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings.

Point-of-care diagnosis based on nucleic acid testing aims to incorporate all the analytical steps, from sample preparation to nucleic acid amplification and detection, in a single device. This device needs to provide a low-cost, robust, sensitive, specific, and easily readable analysis. Microfluidics has great potential for handling small volumes of fluids on a single platform. Microfluidic technology has recently been applied to paper, which is already used in low-cost lateral flow tests. Nucleic acid extraction from a biological specimen usually requires cell filtration and lysis on specific membranes, while affinity matrices, such as chitosan or polydiacetylene, are well suited to concentrating nucleic acids for subsequent amplification. Access to electricity is often difficult in resource-limited areas, so the amplification step needs to be equipment-free. Consequently, the reaction has to be isothermal to alleviate the need for a thermocycler. LAMP, NASBA, HDA, and RPA are examples of the technologies available. Nucleic acid detection techniques are currently based on fluorescence, colorimetry, or chemiluminescence. For point-of-care diagnostics, the results should be readable with the naked eye. Nowadays, interpretation and communication of results to health professionals could rely on a smartphone, used as a telemedicine device. The major challenge of creating an "all-in-one" diagnostic test involves the design of an optimal solution and a sequence for each analytical step, as well as combining the execution of all these steps on a single device. This review provides an overview of available materials and technologies which seem to be adapted to point-of-care nucleic acid-based diagnosis, in low-resource areas.

Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

vimA gene downstream of recA is involved in virulence modulation in Porphyromonas gingivalis W83.

A 0.9-kb open reading frame encoding a unique 32-kDa protein was identified downstream of the recA gene of Porphyromonas gingivalis. Reverse transcription-PCR and Northern blot analysis showed that both the recA gene and this open reading frame are part of the same transcriptional unit. This cloned fragment was insertionally inactivated using the ermF-ermAM antibiotic resistance cassette to create a defective mutant by allelic exchange. When plated on Brucella blood agar, the mutant strain, designated P. gingivalis FLL92, was non-black pigmented and showed significant reduction in beta-hemolysis compared with the parent strain, P. gingivalis W83. Arginine- and lysine-specific cysteine protease activities, which were mostly soluble, were approximately 90% lower than that of the parent strain. Expression of the rgpA, rgpB, and kgp protease genes was the same in P. gingivalis FLL92 as in the wild-type strain. In contrast to the parent strain, P. gingivalis FLL92 showed increased autoaggregration in addition to a significant reduction in hemagglutinating and hemolysin activities. In in vivo experiments using a mouse model, P. gingivalis FLL92 was dramatically less virulent than the parent strain. A molecular survey of this mutant and the parent strain using all known P. gingivalis insertion sequence elements as probes suggested that no intragenomic changes due to the movement of these elements have occurred in P. gingivalis FLL92. Taken together, these results suggest that the recA downstream gene, designated vimA (virulence-modulating gene), plays an important role in virulence modulation in P. gingivalis W83, possibly representing a novel posttranscriptional or translational regulation of virulence factors in P. gingivalis.

Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis.

Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene.

Unaltered expression of the major protease genes in a non-virulent recA-defective mutant of Porphyromonas gingivalis W83.

Porphyromonas gingivalis FLL32, a recA mutant, was isolated during construction of a recA defective mutant of P. gingivalis W83 by allelic exchange mutagenesis. In contrast to W83 and FLL33, the typical recA- mutant previously reported, FLL32 was non-pigmented, lacked beta-hemolytic activity on blood agar and produced significantly less proteolytic activity. The proteolytic activity in FLL32 was mostly soluble. Expression of the rgpA, rgpB and kgp protease genes was unaltered in FLL32 when compared to FLL33 and the wild-type strain. FLL32 exhibited reduced virulence in a murine model and partially protected the animals immunized with that strain against a subsequent lethal challenge by the wild-type strain. These results indicate that the reduced level of proteolytic activity in FLL32 may be due to a defect in the processing of the proteases. Further, immunization with a non-virulent recA defective mutant of P. gingivalis can partially protect against a lethal wild-type challenge. The results from this study suggest that the recA locus may be involved in expression and regulation of proteolytic activity in P. gingivalis.

Topological analysis of the aerobic membrane-bound formate dehydrogenase of Escherichia coli.

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, alpha (FdoG), beta (FdoH), and gamma (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (beta-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the alphabeta catalytic dimer is located in the cytoplasm, with a C-terminal anchor for beta protruding into the periplasm. As expected, the gamma subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the 35S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.

Expression and characterization of the Escherichia coli fdo locus and a possible physiological role for aerobic formate dehydrogenase.

In the presence of nitrate, the major anaerobic respiratory pathway includes formate dehydrogenase (FDH-N) and nitrate reductase (NAR-A), which catalyze formate oxidation coupled to nitrate reduction. Two aerobically expressed isoenzymes, FDH-Z and NAR-Z, have been recently characterized. Enzymatic analysis of plasmid subclones carrying min 88 of the Escherichia coli chromosome was consistent with the location of the fdo locus encoding FDH-Z between the fdhD and fdhE genes which are necessary for the formation of both formate dehydrogenases. The fdo locus produced three proteins (107, 34, and 22 kDa) with sizes similar to those of the subunits of the purified FDH-N. In support to their structural role, these polypeptides were recognized by antibodies specific to FDH-N. Expression of a chromosomal fdo-uidA operon fusion was induced threefold by aerobic growth and about twofold by anaerobic growth in the presence of nitrate. However, it was independent of the two global regulatory proteins FNR and ArcA, which control genes for anaerobic and aerobic functions, respectively, and of the nitrate response regulator protein NARL. In contrast, a mutation affecting either the nucleoid-associated H-NS protein or the CRP protein abolished the aerobic expression. A possible role for FDH-Z during the transition from aerobic to anaerobic conditions was examined. Synthesis of FDH-Z was maximal at the end of the aerobic growth and remained stable after a shift to anaerobiosis, whereas FDH-N production developed only under anaerobiosis. Furthermore, in an fnr strain deprived of both FDH-N and NAR-A activities, aerobically expressed FDH-Z and NAR-Z enzymes were shown to reduce nitrate at the expense of formate under anaerobic conditions, suggesting that this pathway would allow the cell to respond quickly to anaerobiosis.

Osmotic repression of anaerobic metabolic systems in Escherichia coli.

The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed. The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes. While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably. An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former. fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF. This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.