A novel multiplex real-time PCR for the identification of mycobacteria associated with zoonotic tuberculosis

Background

Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy.

Methodology/Principal Findings

The aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted.

Conclusions/Significance

The multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the MTC.     

Emission analysis of RE3+ (RE = Sm,Dy):B2O3‐TeO2‐Li2O‐AlF3 glasses

This article reports on the optical properties of 0.5% mol of Sm³⁺, Dy³⁺ ion‐doped B₂O₃‐TeO₂‐Li₂O‐AlF₃ (LiAlFBT) glasses. The glass samples were characterized by optical absorption and emission spectra. Judd‐Ofelt theory was applied to analyze the optical absorption spectra and calculate the intensity parameters and radiative properties of the emission transitions. The emission spectra of Sm³⁺ and Dy³⁺:LiAlFBT glasses showed a bright reddish‐orange emission at 598 nm (⁴G_5/2 → ⁶H_7/2) and an intense yellow emission at 574 nm (⁴F_9/2 → ⁶H_13/2), respectively. Full width at half maximum (FWHM), stimulated emission cross section, gain bandwidth and optical gain values were also calculated to extend the applications of the Sm³⁺ and Dy³⁺:LiAlFBT glasses. Copyright © 2012 John Wiley & Sons, Ltd.     

LED-based interferometric reflectance imaging sensor for quantitative dynamic monitoring of biomolecular interactions

Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the Interferometric Reflectance Imaging Sensor (IRIS) using an inexpensive and durable multi-color LED illumination source to monitor protein-protein and DNA-DNA interactions. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm(2) and DNA single mismatch detection under denaturing conditions in an array format. Our experiments show that this platform has comparable sensitivity to high-end label-free biosensors at a much lower cost with the capability to be translated to field-deployable applications.     

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation

Stimulation of iNKT cells by ??-galactosyl ceramide (??-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells’ response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in ??-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of ??-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.     

Conservation Genetics of Kangaroo Mice, Genus Microdipodops

The two currently recognized species of kangaroo mice, Microdipodops megacephalus and M. pallidus, inhabit sandy soils of the Great Basin Desert in western North America. Given their habitat specificity and the fluctuating climate throughout the Pleistocene, kangaroo mice likely endured a turbulent biogeographic history that resulted in disjunct distributions and isolation of genetic lineages. Recent phylogenetic investigations using mitochondrial data have revealed several mitochondrial clades within this genus that may represent cryptic species. These mitochondrial clades are genetically unique, occupy relatively small distributions, and, as such, may be at an increased risk of extinction due to climate change and extensive recent habitat alteration. Herein, we apply haplotype network, population genetic, and historical demographic analyses to mitochondrial data of each Micropdipodops species and mitochondrial clade to assess conservation genetics within kangaroo mice. Results indicate that each mitochondrial clade is a distinct lineage with little to no gene flow occurring among clades. Additionally, historical demographic analyses support past population expansions and identify locations of past refugium for each distinct lineage. Although mitochondrial data indicate that the clades appear to be in approximate genetic equilibrium and have not suffered any extreme bottlenecks over time, there is still concern for the survival of smaller and more vulnerable Microdipodops subpopulations due to impending habitat threats in the Great Basin Desert.     

Distinct endosomal trafficking requirements for presentation of autoantigens and exogenous lipids by human CD1d molecules

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRalphabeta and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR- APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.     

Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Optimization of mycophenolic acid production in solid state fermentation using response surface methodology

Mycophenolic acid (MPA) can be produced in solid state fermentation. An isolate of Penicillium brevi-compactum ATCC 16024 grown on moist wheat bran produced a titre of 425 mg per kg of wheat bran. Central composite rotatable design and response surface methodology were employed to derive a statistical model for media optimization towards production of mycophenolic acid. Five levels with a five factorial design were adopted. The correlation coefficient was 0.82, ensuring a satisfactory adjustment of the model to the experimental values. This statistical design was very effective in improving the titre of mycophenolic acid up to 3286 mg per kg of wheat bran. Received 24 July 1998/ Accepted in revised form 4 December 1998     

Adenosine metabolism in a rat hippocampal slice preparation: incorporation into S-adenosylhomocysteine

Abstract: The incorporation of [¹⁴C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S -adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of ¹⁴C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S -adenosylhomocysteine, and the free adenosine pool. Labeling of S -adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [¹⁴C]adenosine in the tissue. Addition of l -homocysteine significantly increased the labelling of S -adenosylhomocysteine. The results indicate that S -adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady-state conditions. Further, changes in adenosine concentration, without a concomitant change in l -homocysteine availability, are unlikely to lead to a significant accumulation of S -adenosylhomocysteine. S -Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.