Development of specific PCR assays for the detection of Cryptocaryon irritans

Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the “white spot” disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems

Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and “FeMoco”

NifEN plays a key role in the biosynthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ΔnifB NifEN, contains only two permanent [Fe4S4]-like clusters. The second, designated NifENPrecursor, contains the permanent clusters and a precursor form of FeMoco. The third, designated NifEN“FeMoco”, contains the permanent [Fe4S4]-like clusters and a fully complemented, “FeMoco”-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe4S4]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe4S4]+ clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and “FeMoco” have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the “FeMoco” in NifEN“FeMoco” exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and “FeMoco” in NifEN

Can a standard dose of eicosapentaenoic acid (EPA) supplementation reduce the symptoms of delayed onset of muscle soreness?

Unaccustomed exercise can result in delayed onset of muscle soreness (DOMS) which can affect athletic performance. Although DOMS is a useful tool to identify muscle damage and remodelling, prolonged symptoms of DOMS may be associated with the over-training syndrome. In order to reduce the symptoms of DOMS numerous management strategies have been attempted with no significant effect on DOMS-associated cytokines surge. The present study aimed to investigate the acute and chronic effects of a 2x180 mg per day dose of eicosapentaenoic acid (EPA) on interleukin-6 (IL-6) mediated inflammatory response and symptoms associated with DOMS. Methods: Seventeen healthy non-smoking females (age 20.4 +/- 2.1 years, height 161.2 +/- 8.3cm and mass 61.48 +/- 7.4kg) were randomly assigned to either placebo (N = 10) or EPA (N = 7). Serum IL-6, isometric and isokinetic (concentric and eccentric) strength, and rating of perceived exertion (RPE) were recorded on four occasions: i-prior to supplementation, ii-immediately after three weeks of supplementation (basal effects), iii-48 hours following a single bout of resistance exercise (acute training response effects), and iv-48 hours following the last of a series of three bouts of resistance exercise (chronic training response effects). Results: There was only a group difference in the degree of change in circulating IL-6 levels. In fact, relative to the first baseline, by the third bout of eccentric workout, the EPA group had 103 +/- 60% increment in IL-6 levels whereas the placebo group only had 80 +/- 26% incremented IL-6 levels (P = 0.020). We also describe a stable multiple linear regression model which included measures of strength and not IL-6 as predictors of RPE scale. Conclusion: The present study suggests that in doubling the standard recommended dose of EPA, whilst this may still not be beneficial at ameliorating the symptoms of DOMS, it counter intuitively appears to enhance the cytokine response to exercise. In a context where previous in vitro work has shown EPA to decrease the effects of inflammatory cytokines, it may in fact be that the doses required in vivo is much larger than current recommended amounts. An attempt to dampen the exercise-induced cytokine flux in fact results in an over-compensatory response of this system

A Nationwide Population-Based Cohort Study: Will Anxiety Disorders Increase Subsequent Cancer Risk?

BACKGROUND: The aim of this study was to evaluate a possible association between malignancy and anxiety disorders (AD) in Taiwan. METHODS: We employed data from the National Health Insurance system of Taiwan. The AD cohort contained 24,066 patients with each patient randomly frequency matched according to age and sex with 4 individuals from the general population without AD. Cox's proportional hazard regression analysis was conducted to estimate the influence of AD on the risk of cancer. RESULTS: Among patients with AD, the overall risk of developing cancer was only 1% higher than among subjects without AD, and the difference was not significant (hazard ratio [HR] = 1.01, 95% confidence interval [95% CI] = 0.95-1.07). With regard to individual types of cancer, the risk of developing prostate cancer among male patients with AD was significantly higher (HR = 1.32, 95% CI = 1.02-1.71). On the other hand, the risk of cervical cancer among female patients with AD was marginally significantly lower than among female subjects without AD (HR = 0.72, 95% CI = 0.51-1.03). LIMITATIONS: One major limitation is the lack of information regarding the life style or behavior of patients in the NHI database, such as smoking and alcohol consumption. CONCLUSIONS: Despite the failure to identify a relationship between AD and the overall risk of cancer, we found that Taiwanese patients with AD had a higher risk of developing prostate cancer and a lower risk of developing cervical cancer

Symbiotic Legume Nodules Employ Both Rhizobial Exo- and Endo-Hydrogenases to Recycle Hydrogen Produced by Nitrogen Fixation

BACKGROUND: In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N(2), an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H(2)). While in most legume nodules this H(2) is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H(2). Rather, endogenous H(2) is efficiently respired at the expense of O(2), driving oxidative phosphorylation, recouping ATP used for H(2) production, and increasing the efficiency of symbiotic nodule N(2) fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity. METHODOLOGY/PRINCIPAL FINDINGS: Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H(2) and thus are fully competent for endogenous H(2) recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H(2) quantitatively and thus suffer complete loss of H(2) recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H(2) may carry both exo- and endo-hydrogenase gene-sets. CONCLUSIONS/SIGNIFICANCE: In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H(2) produced by N(2) fixation. Thus, H(2) recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases

Genetic and Proteomic Approaches to Identify Cancer Drug Targets

While target-based small-molecule discovery has taken centre-stage in the pharmaceutical industry, there are many cancer-promoting proteins not easily addressed with a traditional target-based screening approach. In order to address this problem, as well as to identify modulators of biological states in the absence of knowing the protein target of the state switch, alternative phenotypic screening approaches, such as gene expression-based and high-content imaging, have been developed. With this renewed interest in phenotypic screening, however, comes the challenge of identifying the binding protein target(s) of small-molecule hits. Emerging technologies have the potential to improve the process of target identification. In this review, we discuss the application of genomic (gene expression-based), genetic (short hairpin RNA and open reading frame screening), and proteomic approaches to protein target identification

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration