The Public Health Research Institute
The Public Health Research Institute
Qiao J.,The Public Health Research Institute |
Mindich L.,The Public Health Research Institute
Journal of Virology | Year: 2013
BacteriophageΦ6 contains three double-stranded RNA (dsRNA) genomic segments, L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segmentsMand S in vitro. Segment L is transcribed poorly because its transcript starts with GU instead of GG found on segments S and M. Transcription in vivo is modified by the binding of host protein YajQ to the outside the core particle so that segment L is transcribed well. This mechanism is the determinant of the temporal control of gene expression inΦ6. Mutants ofΦ6 have been isolated that are independent of YajQ for transcription of segment L. The mutations are found in the gene of the viral polymerase or the major capsid protein or both. These mutants are capable of transcribing segment L with the GU start or GA or GC. The same is found to be true when YajQ is added to wild-type particles. Minus-strand synthesis has restrictions that are different from that of plusstrand synthesis, and YajQ or mutations to independence do not modify minus-strand synthesis behavior. Purified polymerase P2 is able to transcribe dsRNA, but transcription behavior of segment L by both wild-type and mutant polymerases is different from that seen in capsid structures. Adding YajQ to purified polymerase does not change its transcription specificity. © 2013, American Society for Microbiology.
Wang Y.,Tianjin Medical University |
Wang Y.,The Public Health Research Institute |
Liu T.-B.,The Public Health Research Institute |
Patel S.,The Public Health Research Institute |
And 4 more authors.
Eukaryotic Cell | Year: 2011
Casein kinases regulate a wide range of cellular functions in eukaryotes, including phosphorylation of proteins that are substrates for degradation via the ubiquitin-proteasome system (UPS). Our previous studydemonstrated that Fbp1, a component of the SCF FBP1 E3 ligase complex, was essential for Cryptococcus virulence. Because the Saccharomyces cerevisiae homolog of Fbp1, Grr1, requires casein kinase I (Yck1 and Yck2) to phosphorylate its substrates, we investigated the function of casein kinase I in Cryptococcus neoformans. In this report, we identified a C. neoformans casein kinase I protein homolog, Cck1. Similar to Fbp1, the expression of Cck1 is negatively regulated by glucose and during mating. cck1 null mutants showed significant virulence attenuation in a murine systemic infection model, but Cck1 was dispensable for the development of classical virulence factors (capsule, melanin, and growth at 37°C). cck1 mutants were hypersensitive to SDS treatment, indicating that Cck1 is required for cell integrity. The functional overlap between Cck1 and Fbp1 suggests that Cck1 may be required for the phosphorylation of Fbp1 substrates. Interestingly, the cck1 mutant also showed increased sensitivity to osmotic stress and oxidative stress, suggesting that Cck1 regulates both cell integrity and the cellular stress response. Our results show that Cck1 regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases (MAPKs), demonstrating that Cck1 regulates cell integrity via the Mpk1 pathway and regulates cell adaptation to stresses via the Hog1 pathway. Overall, our study revealed that Cck1 plays important roles in regulating multiple signaling pathways and is required for fungal pathogenicity. © 2011, American Society for Microbiology. All Rights Reserved.
DuMont A.L.,New York University |
Nygaard T.K.,Montana State University |
Watkins R.L.,Montana State University |
Smith A.,New York University |
And 6 more authors.
Molecular Microbiology | Year: 2011
Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis. © 2010 Blackwell Publishing Ltd.
PubMed | The Public Health Research Institute
Type: | Journal: Methods in molecular medicine | Year: 2011
Molecular beacons are a novel family of hybridization probes, which emit fluorescence upon interaction with their target. They are hairpin-shaped oligonucleotides with a central part complementary to the target, flanked by two 5 6 base pair (bp) inverted repeats, which can form a stable stem. A fluorescent moiety is covalently linked to the 5 end of the molecule, whereas the quenching moiety, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), is covalently linked to the 3 end. The stem keeps the two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When molecular beacons bind to their target, they undergo a conformational change that results in the restoration of fluorescence of the internally quenched fluorophore (1) (Fig. 1). Molecular beacons are extremely specific, and can clearly discriminate between targets differing only by a single nucleotide (2,3). When present in a PCR reaction where their target is the amplification product, molecular beacons can form a stable hybrid with the amplicon during the annealing step. The intensity of fluorescence at the annealing step in each amplification cycle is a direct measure of amplicon concentration (2,4) (Fig. 2). Another interesting feature of molecular beacons is that they can be coupled to a variety of differently colored fluorophores. This allows multiplex PCR reactions where different DNA fragments can be amplified and detected simultaneously in the same tube (2,3). Fig. 1. Operation of molecular beacons. On their own, these molecules are nonfluorescent, because the stem hybrid keeps the fluorophore () close to the quencher (). When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence (1). Fig. 2. Real time measurement of amplicon synthesis during PCR using molecular beacons. (A) Four PCR reactions were initiated with a different number of template molecules (indicated). The concentration of amplicons present after each cycle of amplification was determined by measuring fluorescence during the last few seconds of the annealing step. (B) Inverted relationship between the threshold cycle (the cycle at which the fluorescent signal becomes detectable above the background) and the logarithm of the initial number of template molecules. In this example, the target is M. tuberculosis H37Rv chromosomal DNA. The primers-molecular beacon set used in the reaction was specific for sigA (reprinted from ref. 4).
PubMed | The Public Health Research Institute
Type: Journal Article | Journal: Clinical microbiology reviews | Year: 2011
Infection with Mycobacterium tuberculosis causes a variety of clinical conditions ranging from life-long asymptomatic infection to overt disease with increasingly severe tissue damage and a heavy bacillary burden. Immune biomarkers should follow the evolution of infection and disease because the host immune response is at the core of protection against disease and tissue damage in M. tuberculosis infection. Moreover, levels of immune markers are often affected by the antigen load. We review how the clinical spectrum of M. tuberculosis infection correlates with the evolution of granulomatous lesions and how granuloma structural changes are reflected in the peripheral circulation. We also discuss how antigen-specific, peripheral immune responses change during infection and how these changes are associated with the physiology of the tubercle bacillus. We propose that a dynamic approach to immune biomarker research should overcome the challenges of identifying those asymptomatic and symptomatic stages of infection that require antituberculosis treatment. Implementation of such a view requires longitudinal studies and a systems immunology approach leading to multianalyte assays.