The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.Burkholderia pseudomallei, a gram-negative soil bacillus, is the causative agent of melioidosis, a severe and potentially fatal emerging tropical infectious disease. Its intrinsic resistance to many antibiotics is attributed mostly to multiple multidrug efflux pumps, such as AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC, which efflux aminoglycosides, macrolides, chloramphenicol, and trimethoprim (4,12,14). We hypothesized that these pumps also efflux physiological compounds that could compromise the fitness of the bacterium when accumulated to high intracellular concentrations and propose that this exerts a positive selection pressure for persistence even in the absence of antimicrobials (11).B. pseudomallei is reported to produce up to six different types of acyl-homoserine lactones (acyl-HSLs), the composition of which may differ slightly from strain to strain. For instance, strain PP844 secreted N-octanoyl-homoserine lac-, and N-(3-hydroxy)-dodecanoyl-homoserine lactone (3-hydroxy-C12HSL), while strain DD503, an amrAB-oprA efflux pump operon deletion mutant derived from 1026b, secreted only C8HSL, 3-hydroxy-C8HSL, and C10HSL (13,(17)(18)(19). It is unclear how acyl-HSLs move across the B. pseudomallei cell membranes and into the extracellular compartment, although in Pseudomonas aeruginosa, the shorter-chain acylHSLs appear to do so by diffusion while the longer-chain acyl-HSLs are secreted by multidrug efflux pumps (1, 15). In B. pseudomallei KHW, the expression of bpeAB-lacZ was induced by acyl-HSLs, and the bpeAB mutant failed to secrete any extracellular acyl-HSLs when it was cross-streaked against the JB525 reporter strain (3).Extracellular secretion of acyl-HSLs is dependent on BpeAB-OprB. In order to ascertain whether a blockage in the efflux mechanism, an inhibition of acyl-HSL synthesis, or both had contributed to the absence of extracellular acyl-HSLs in the bpeAB mutant, we compared the acyl-HSLs produced in cell supernatants of B. pseudomallei before and after the bacterial cells were permeabilized by a freeze-thaw procedure. Wild-type B. pseudomallei KHW, KHW carrying a plasmid overexpressing the BpeR repressor, the bpeAB null mutant, and a complemented bpeAB mutant were used in the comparison (Table 1), and a modified cross-streak bioassay was used for detection of acyl-HSLs (2). The culture supernatants of unpermeabilized wild-type KHW and the complemented bpeAB mutant contained acyl-HSLs but not those of the bpeAB mutant and the bpeR-overexpressing strain, both of which had impaired BpeAB-OprB function (Fig. 1, lanes U). Acyl-HSLs were detected in the cell sup...

Long YC, Tan TM, Takao I, Tang BL. The biochemistry and cell biology of aging: metabolic regulation through mitochondrial signaling. Am J Physiol Endocrinol Metab 306: E581-E591, 2014. First published January 22, 2014 doi:10.1152/ajpendo.00665.2013.-Cellular and organ metabolism affects organismal lifespan. Aging is characterized by increased risks for metabolic disorders, with age-associated degenerative diseases exhibiting varying degrees of mitochondrial dysfunction. The traditional view of the role of mitochondria generated reactive oxygen species (ROS) in cellular aging, assumed to be causative and simply detrimental for a long time now, is in need of reassessment. While there is little doubt that high levels of ROS are detrimental, mounting evidence points toward a lifespan extension effect exerted by mild to moderate ROS elevation. Dietary caloric restriction, inhibition of insulin-like growth factor-I signaling, and inhibition of the nutrient-sensing mechanistic target of rapamycin are robust longevity-promoting interventions. All of these appear to elicit mitochondrial retrograde signaling processes (defined as signaling from the mitochondria to the rest of the cell, for example, the mitochondrial unfolded protein response, or UPR mt ). The effects of mitochondrial retrograde signaling may even spread to other cells/tissues in a noncell autonomous manner by yet unidentified signaling mediators. Multiple recent publications support the notion that an evolutionarily conserved, mitochondria-initiated signaling is central to the genetic and epigenetic regulation of cellular aging and organismal lifespan.aging; mitochondria; reactive oxygen species; mitochondria retrograde signaling; mitokine ". . . THE SUM OF THE DELETERIOUS free radical reactions going on continuously throughout the cells and tissues constitutes the aging process or is a major contributor to it" was Denham Harman's take on how aging occurs (65), which aptly summarizes the classical "Free Radical Theory of Aging" he proposed (64). The mitochondrial respiratory chain (or the electron transport chain, ETC) is the main cellular source of reactive oxygen species (ROS) such as superoxide, which could be converted to H 2 O 2 by superoxide dismutases (SODs). The latter generates highly damaging hydroxyl radicals via the Fenton reaction (60) in the presence of transition metals like Fe. Among the cumulative deleterious effects of ROS are mutations to somatic mitochondrial DNA (mtDNA), which impair mitochondrial function over time, and form the basis of the modified theory of "Mitochondria Theory of Aging" (105). The central tenet of the theories has some experimental support. For example, overexpression of SOD and catalase extends Drosophila lifespan (130). Deletion of the highly expressed peroxisomal catalase gene ctl-2 in Caenorhabditis elegans accelerated an aging phenotype (140). Key experimental support for cumulative mtDNA damage as a cause of aging is provided by homozygous knockin mice that express a proofreading-deficient version of PolgA...

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It is often associated with neurological complications and has caused high mortality levels in recent outbreaks in the Asia Pacific region. Currently, there is no effective antiviral therapy against EV71 infections. In this study, we have evaluated and compared the efficacies of three different forms of small interfering RNAs (siRNAs) in inhibiting EV71 replication in a murine model. We have shown that both synthetic 19-mer siRNAs and plasmid-borne short hairpin RNAs (shRNAs) targeted at the conserved 3D(pol) region were able to inhibit EV71 infections in suckling mice when delivered with or without lipid carrier via the systemic route. The treated mice did not exhibit hind limb paralysis and weight loss, as was observed in untreated mice. EV71 replication was significantly reduced as revealed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. In addition, no evidence of interferon (IFN) induction was detected in the intestinal tissues harvested from the mice as a result of siRNA administration. However, the chemically synthesized 29-mer shRNA did not protect the suckling mice from EV71 infections despite being more potent in the in vitro system. Our results indicate that RNA interference (RNAi) may be a promising therapeutic approach for fighting EV71 infections.

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation betweenPlasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays forP. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive forP. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript.