An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: Rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (<1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is =2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins

Origins of Diamond Surface Noise Probed by Correlating Single-Spin Measurements with Surface Spectroscopy

The nitrogen vacancy (NV) center in diamond exhibits spin-dependent fluorescence and long spin coherence times under ambient conditions, enabling applications in quantum information processing and sensing. NV centers near the surface can have strong interactions with external materials and spins, enabling new forms of nanoscale spectroscopy. However, NV spin coherence degrades within 100 nanometers of the surface, suggesting that diamond surfaces are plagued with ubiquitous defects. Prior work on characterizing near-surface noise has primarily relied on using NV centers themselves as probes; while this has the advantage of exquisite sensitivity, it provides only indirect information about the origin of the noise. Here we demonstrate that surface spectroscopy methods and single spin measurements can be used as complementary diagnostics to understand sources of noise. We find that surface morphology is crucial for realizing reproducible chemical termination, and use these insights to achieve a highly ordered, oxygen-terminated surface with suppressed noise. We observe NV centers within 10 nm of the surface with coherence times extended by an order of magnitude

Elevated Hepcidin Is Part of a Complex Relation That Links Mortality with Iron Homeostasis and Anemia in Men and Women with HIV Infection.

BACKGROUND: Early and chronic inflammation is a hallmark of HIV infection, and inflammation is known to increase hepcidin expression. Consequently, hepcidin may be a key determinant of the iron homeostasis and anemia associated with poorer HIV prognoses. OBJECTIVE: The objective of this study was to understand how hepcidin is related to anemia, iron homeostasis, and inflammation at HIV diagnosis and to investigate associations between hepcidin and all-cause mortality in HIV infection. METHODS: In a retrospective cohort, baseline plasma hepcidin was measured by competitive enzyme immunoassay within 3 mo of HIV diagnosis in 196 antiretroviral-naive Gambians. Iron homeostasis [hemoglobin, plasma transferrin, ferritin, iron, soluble transferrin receptor (sTfR)] and inflammation [α1-antichymotrypsin (ACT)] from the same plasma sample were available, as were absolute CD4 cell counts, age, gender, body mass index (BMI), and HIV type. RESULTS: Anemia was common across the spectrum of immunosuppression [CD4 cell counts (prevalence of anemia): >500 cells/μL (68%), 200-500 cells/μL (73%), and <200 cells/μL (89%); P = 0.032] and in men (81%) and women (76%). Increasing hepcidin was associated with iron homeostasis biomarkers (higher ferritin and lower transferrin, hemoglobin, and sTfR), inflammation (higher ACT), and key health indicators (lower CD4 or BMI, advancing age, and male gender; P < 0.001 except for hemoglobin, P = 0.021). Elevated hepcidin was associated with greater all-cause mortality in a dose-dependent manner [intermediate vs. lowest tertile: unadjusted HR (95% CI), 1.95 (1.22, 3.10); upper vs. lowest tertile: 3.02 (1.91, 4.78)]. Principal components analysis identified 2 patterns composed of hepcidin-ferritin-transferrin, with or without ACT, and iron-sTfR-hemoglobin that may distinguish inflammation and erythropoiesis iron functions. CONCLUSIONS: Elevated hepcidin is independently associated with greater mortality in men and women with HIV infection, and hepcidin is also part of a complex relation linking iron homeostasis, anemia, and HIV. Understanding the mechanisms and role of hepcidin modulation may further guide evidence-based interventions needed to counter detrimental iron homeostasis and anemia in HIV infection

Mining the LIPG Allelic Spectrum Reveals the Contribution of Rare and Common Regulatory Variants to HDL Cholesterol

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5′ UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5′ UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci

Ultrafast dynamics in microemulsions: Optical Kerr effect study of the dispersed oil phase in a carbon disulride-dodecyltrimethylammonium bromide-water microemulsion

The ultrafast, optically-heterodyne-detected optical Kerr effect has been used to investigate the dynamics of the dispersed oil phase of a novel oil-in-water microemulsion. The carbon disulfide-dodecyltrimethylammonium bromide-water (CS2-DTAB-H2O) system has been characterized by viscosity and conductivity measurements. The results of these measurements are discussed in terms of possible shape changes and interparticle interactions. Ultrafast measurements are reported for several different compositions of the microemulsion. The results are compared and contrasted with the dynamics of neat CS2 and CS2 dissolved in n-alkanes. The dynamics of CS2 dispersed in the microemulsion do not approach those of the neat liquid in any case, even at the highest possible loading, implying that the dynamics in the oil phase are strongly perturbed by the surfactant. The slow (picosecond) diffusive reorientation of the CS2 does not reflect the macroscopic viscosity of the microemulsion

Ultrafast dynamics in complex fluids observed through the ultrafast optically-heterodyne-detected optical-Kerr-effect (OHD-OKE)

The ultrafast molecular dynamics of complex fluids have been recorded using the optically-heterodyne-detected optical-Kerr-effect (OHD-OKE). The OHD-OKE method is reviewed and some recent refinements to the method are described. Applications to a range of complex fluids, including microemulsions, polymer melts and solutions, liquid crystal and ionic liquids are surveyed. The level of detail attainable with the OHD-OKE method in these complex fluids is discussed. The prospects for future experiments are discussed

Polyfunctional T cell responses are a hallmark of HIV-2 infection

HIV-2 is distinguished clinically and immunologically from HIV-1 infection by delayed disease progression and maintenance of HIV-specific CD4(+) T cell help in most infected subjects. Thus, HIV-2 provides a unique natural human model in which to investigate correlates of immune protection against HIV disease progression. Here, we report a detailed assessment of the HIV-2-specific CD4(+) and CD8(+) T cell response compared to HIV-1, using polychromatic flow cytometry to assess the quality of the HIV-specific T cell response by measuring IFN-gamma, IL-2, TNF-alpha, MIP-1beta, and CD107a mobilization (degranulation) simultaneously following Gag peptide stimulation. We find that HIV-2-specific CD4(+) and CD8(+) T cells are more polyfunctional that those specific for HIV-1 and that polyfunctional HIV-2-specific T cells produce more IFN-gamma and TNF-alpha on a per-cell basis than monofunctional T cells. Polyfunctional HIV-2-specific CD4(+) T cells were generally more differentiated and expressed CD57, while there was no association between function and phenotype in the CD8(+) T cell fraction. Polyfunctional HIV-specific T cell responses are a hallmark of non-progressive HIV-2 infection and may be related to good clinical outcome in this setting

High-density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin-1

Background and PurposeMonocyte-derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High-density lipoproteins (HDLs) have many cardio-protective properties including potent anti-inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur