Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a highthroughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.
The single cell gel electrophoresis (SCGE) or comet assay is based on the assumption that comet images result from genotoxic damage that ultimately generate DNA single- or double-strand breaks. A criticism of the assay is that some or all of the comet images may be the result of apoptosis-mediated nuclear fragmentation. The objective of this study was to determine if mutagen-induced DNA damage leading to strand breakage observed in the SCGE assay was repairable or was due to nonrepairable nuclear fragmentation. Chinese hamster ovary cells were treated with ethylmethanesulfonate, 2-acetoxyacetylaminofluorene, or H(2)O(2). These mutagens induce genetic damage by different molecular mechanisms. One group of SCGE slides was prepared immediately after treatment, while parallel treated cultures were repeatedly washed and allowed to undergo liquid holding recovery for DNA repair. It was hypothesized that cells with genotoxic damage can repair their genomic DNA, while apoptotic cells cannot reverse nuclear fragmentation. We found a significant decrease in the tail moments of nuclei from mutagen-treated cells after 4 hr of liquid holding. However, this measurement may represent only those cells capable of repair. Apoptotic cells may continue DNA fragmentation during the recovery time and this DNA may become so diffuse that the nuclei disappear after electrophoresis. To overcome this possible artifact, images of nuclei were captured before and after alkaline electrophoresis. Constellations of nuclei were located on SCGE slides by their coordinates on the microscope stage. We found that no nuclei were lost due to apoptotic nuclear fragmentation and DNA migration. Even the so-called "hedgehog" comet images with extreme DNA damage were not lost during liquid holding. These data support the conclusion that mutagen-induced DNA damage is the principal cause of the damage measured in the comet assay.
The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was The dengue virus (DENV), a mosquito-borne flavivirus, consists of four closely related but genetically distinct antigenic serotypes: DENV serotype 1 (DENV-1), DENV-2, DENV-3, and DENV-4. It is tropical and subtropical in distribution and is prevalent in Asia, Africa, and Central and South America (45). Infection with any of the four serotypes of DENV may cause a mild febrile illness, dengue fever (DF). In some cases, however, more-severe manifestations, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), occur; these may prove fatal without proper early intervention (15).Geographic spread of both the mosquito vector and the virus over the past 25 years has led to the increased occurrence of epidemic DF/DHF/DSS, making dengue a major global health problem. The disease is endemic in more than 100 countries, with an estimated 2.5 billion people at risk of infection. It is estimated that 50 million DENV infections occur each year, with 500,000 cases of DHF and at least 22,000 deaths, mainly in children (31,32,45; WHO/WPRO/SEARO meeting on DengueNet implementation in Southeast Asia and the Western Pacific, Kuala Lumpur, Malaysia, 11 to 13 December 2003).DENV infection confers lifelong serotype-specific immunity. Multiple infections with different DENV serotypes occur in regions of hyperendemicity (31, 35). Secondary infections with a different DENV serotype are major risk factors for DHF and DSS (13, 14, 39) due to antibody-dependent enhancement of disease (35). Serotype identification and the differentiation of primary and secondary infections are therefore important both for patient management and for the implementation of public health measures (26,33).The diagnosis of DENV infection and the typing of DENV serotypes can be confirmed using viral isolation techniques, serology, or molecular methods. Virus isolation is the gold standard for detection but requires 7 to 10 days and is often insensitive (26). Serological tests for the detection of viral antibodies, such as immunoglobulin M and immunoglobulin G antibody capture enzyme-linked immunosorbent assays, require the demonstration of a rise in antibody titer from an acute-phase to a convalescent-phase serum sample and therefore have little impact on patient management (24,...
We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR
We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assayfor the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.
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