A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species

MPA in Labor: Securing the Pearl Cays of Nicaragua

Implementation of Marine Protected Areas (MPAs) has always a step-zero, i.e., an initial phase when the idea is incepted, communicated and negotiated among stakeholders. What happens during this phase is likely to have an impact later on. If not done right, the management of the MPA may encounter problems at later stage that will be difficult to correct. Inspired by this working theory, this article describes the effort to establish the Pearl Cays off the Caribbean coast of Nicaragua as a protected area. This case-study illustrates the critical actions to be taken during step-zero, i.e., what needs to be considered and done before an MPA is formally declared. The area investigated consists of a number of small islands (cays) and coral reefs, fishing grounds and marine turtle nesting areas. Throughout history, the cays have played an important role in sustaining livelihoods of nearby communities. Although the idea of an MPA was originally conservation, the communities saw it as an opportunity to regain ownership and control of the cays. By Nicaraguan law, in order to establish protected areas, consultation and approval from local people is required. In the case of the Pearl Cays, this has proved difficult. The article demonstrates how MPA initiatives must sometimes relate to already ongoing complex social processes in the area where they are to be instigated

Acetate supplementation modulates brain histone acetylation and decreases interleukin-1β expression in a rat model of neuroinflammation

<p>Abstract</p> <p>Background</p> <p>Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression.</p> <p>Methods</p> <p>In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1β expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively.</p> <p>Results</p> <p>We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9), histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1β protein and mRNA levels were increased by 1.3- and 10-fold, respectively, and acetate supplementation reduced this expression to control levels.</p> <p>Conclusion</p> <p>Based on these results, we conclude that dietary acetate supplementation attenuates neuroglial activation by effectively reducing pro-inflammatory cytokine expression by a mechanism that may involve a distinct site-specific pattern of histone acetylation and histone deacetylase expression in the brain.</p

The N-terminal region of the PA subunit of the RNA polymerase of influenza A/HongKong/156/97 (H5N1) influences promoter binding.

BACKGROUND: The RNA polymerase of influenza virus is a heterotrimeric complex of PB1, PB2 and PA subunits which cooperate in the transcription and replication of the viral genome. Previous research has shown that the N-terminal region of the PA subunit of influenza A/WSN/33 (H1N1) virus is involved in promoter binding. METHODOLOGY/PRINCIPAL FINDINGS: Here we extend our studies of the influenza RNA polymerase to that of influenza strains A/HongKong/156/97 (H5N1) and A/Vietnam/1194/04 (H5N1). Both H5N1 strains, originally isolated from patients in 1997 and 2004, showed significantly higher polymerase activity compared with two classical human strains, A/WSN/33 (H1N1) and A/NT/60/68 (H3N2) in vitro. This increased polymerase activity correlated with enhanced promoter binding. The N-terminal region of the PA subunit was the major determinant of this enhanced promoter activity. CONCLUSIONS/SIGNIFICANCE: Overall we suggest that the N-terminal region of the PA subunit of two recent H5N1 strains can influence promoter binding and we speculate this may be a factor in their virulence

A search for rare B → Dμ+μ− decays

A search for rare B→Dμ+μ− decays is performed using proton-proton collision data collected by the LHCb experiment, corresponding to an integrated luminosity of 9 fb−1. No significant signals are observed in the non-resonant μ+μ− modes, and upper limits of B(B0→D ̄ ̄ ̄ ̄0μ+μ−)&lt;5.1×10−8, B(B+→D+sμ+μ−)&lt;3.2×10−8, B(B0s→D ̄ ̄ ̄ ̄0μ+μ−)&lt;1.6×10−7 and fc/fu⋅B(B+c→D+sμ+μ−)&lt;9.6×10−8 are set at the 95\% confidence level, where fc and fu are the fragmentation fractions of a B meson with a c and u quark respectively in proton-proton collisions. Each result is either the first such measurement or an improvement by three orders of magnitude on an existing limit. Separate upper limits are calculated when the muon pair originates from a J/ψ→μ+μ− decay. The branching fraction of B+c→D+sJ/ψ multiplied by the fragmentation-fraction ratio is measured to be fc/fu⋅B(B+c→D+sJ/ψ)=(1.63±0.15±0.13)×10−5, where the first uncertainty is statistical and the second systematic

Observation of Two New Excited Ξb0 States Decaying to Λb0 K-π+

Two narrow resonant states are observed in the Λb0K-π+ mass spectrum using a data sample of proton-proton collisions at a center-of-mass energy of 13 TeV, collected by the LHCb experiment and corresponding to an integrated luminosity of 6 fb-1. The minimal quark content of the Λb0K-π+ system indicates that these are excited Ξb0 baryons. The masses of the Ξb(6327)0 and Ξb(6333)0 states are m[Ξb(6327)0]=6327.28-0.21+0.23±0.12±0.24 and m[Ξb(6333)0]=6332.69-0.18+0.17±0.03±0.22 MeV, respectively, with a mass splitting of Δm=5.41-0.27+0.26±0.12 MeV, where the uncertainties are statistical, systematic, and due to the Λb0 mass measurement. The measured natural widths of these states are consistent with zero, with upper limits of Γ[Ξb(6327)0]&lt;2.20(2.56) and Γ[Ξb(6333)0]&lt;1.60(1.92) MeV at a 90% (95%) credibility level. The significance of the two-peak hypothesis is larger than nine (five) Gaussian standard deviations compared to the no-peak (one-peak) hypothesis. The masses, widths, and resonant structure of the new states are in good agreement with the expectations for a doublet of 1D Ξb0 resonances

Design of the ECCE Detector for the Electron Ion Collider

Preprint submitted to Nuclear Instruments and Methods A. The file archived on this institutional repository has not been certified by peer review.32 pages, 29 figures, 9 tablesThe EIC Comprehensive Chromodynamics Experiment (ECCE) detector has been designed to address the full scope of the proposed Electron Ion Collider (EIC) physics program as presented by the National Academy of Science and provide a deeper understanding of the quark-gluon structure of matter. To accomplish this, the ECCE detector offers nearly acceptance and energy coverage along with excellent tracking and particle identification. The ECCE detector was designed to be built within the budget envelope set out by the EIC project while simultaneously managing cost and schedule risks. This detector concept has been selected to be the basis for the EIC project detector.Office of Science in the Department of Energy, the National Science Foundation, and the Los Alamos National Laboratory Laboratory Directed Research and Development (LDRD) 20200022DR; This research used resources of the Compute and Data Environment for Science (CADES) at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05- 00OR22725. The work of AANL group are supported by the Science Committee of RA, in the frames of the research project # 21AG-1C028. And we gratefully acknowledge that support of Brookhaven National Lab and the Thomas Jefferson National Accelerator Facility which are operated under contracts DESC0012704 and DE-AC05-06OR23177 respectivel