Philip D. Charles

The Mass of the Neutron Star in Cygnus X-2 (V1341 Cygni)

Cygnus X-2 is one of the brightest and longest known X-ray sources. We present high-resolution optical spectroscopy of Cyg X-2 obtained over 4 yr, which gives an improved mass function of 0.69 ± 0.03 M☉ (1 σ). In addition, we resolve the rotationally broadened absorption features of the secondary star for the first time, deriving a rotation speed of v sin i=34.2±2.5 km s-1 (1 σ), which leads to a mass ratio of q=Mc/M X = 0.34 ± 0.04 (1 σ, assuming a tidally locked and Roche lobe-filling secondary). Hence, with the lack of X-ray eclipses (i.e., i73°) we can set firm 95% confidence lower limits to the neutron star mass of MX > 1.27 M☉ and to the companion star mass of Mc > 0.39 M☉. However, by additionally requiring that the companion must exceed 0.75 M☉ (as required theoretically to produce a steady low-mass X-ray binary), then MX > 1.88 M☉ and i < 61° (95% confidence lower and upper limit, respectively), thereby making Cyg X-2 the highest mass neutron star measured to date. If confirmed, this would set significant constraints on the equation of state of nuclear matter.

Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High‐throughput definition of HLA class I‐associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T‐cell responses against pathogens such as HIV‐1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo‐assisted database searching to define the HLA class I‐associated immunopeptidome of HIV‐1‐infected human cells. We here report for the first time the identification of 75 HIV‐1‐derived peptides bound to HLA class I complexes that were purified directly from HIV‐1‐infected human primary CD4+ T cells and the C8166 human T‐cell line. Importantly, one‐third of eluted HIV‐1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T‐cell responses have previously been reported but for which the precise HLA class I‐binding sequences have not yet been defined. These results validate and expand the current knowledge of virus‐specific antigenic peptide presentation during HIV‐1 infection and provide novel targets for T‐cell vaccine development.

A new soft X-ray mode in the AM Herculis object E2003 + 225

Exosat observations of the AM Her object E2003 + 225 made in June and September 1985, are presented together with optical and ultraviolet observations. The averages of soft X-ray count rate doubled between October 1983 and June 1985; the simultaneously measured V band intensity fell by a factor of 3 and the UV flux by 20 percent. The medium-energy X-ray flux did not increase significantly. In September 1985, E2003 + 225 was observed to have a radically different soft X-ray light curve from that seen previously, but an average intensity similar to that of June 1985. The new soft X-ray light curve showed two similar peaks and two unequal minima separated by half a period. The optical light curve observed 2 days earlier showed no major change in shape. These observations provide a serious challenge to the standard model of AM Her objects. 18 references.

Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells

ABSTRACT Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance. IMPORTANCE The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

Expanding the yeast protein arginine methylome.

Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono‐methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA‐binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg‐Gly‐Gly motifs or Asn‐rich contexts.

Cerebrospinal fluid macrophage biomarkers in amyotrophic lateral sclerosis.

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is a heterogeneous clinical syndrome involving multiple molecular pathways. The development of biomarkers for use in therapeutic trials is a priority. We sought to use a high‐throughput proteomic method to identify novel biomarkers in individual cerebrospinal fluid (CSF) samples.

Perturbed cholesterol and vesicular trafficking associated with dengue blocking in Wolbachia -infected Aedes aegypti cells

Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders.Wolbachia infection of mosquitoes can block dengue virus infection and is tested in field trials, but the mechanism of action is unclear. Using proteomics, Geoghegan et al. here identify effects of Wolbachia on cholesterol homeostasis and dengue virus replication in Aedes aegypti.

Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes.

Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn’s disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation.

Optimizing 2D gas chromatography mass spectrometry for robust tissue, serum and urine metabolite profiling

Two-dimensional gas chromatography mass spectrometry (GCxGC-MS) is utilized to an increasing extent in biomedical metabolomics. Here, we established and adapted metabolite extraction and derivatization protocols for cell/tissue biopsy, serum and urine samples according to their individual properties. GCxGC-MS analysis revealed detection of ~600 molecular features from which 165 were characterized representing different classes such as amino acids, fatty acids, lipids, carbohydrates, nucleotides and small polar components of glycolysis and the Krebs cycle using electron impact (EI) spectrum matching and validation using external standard compounds. Advantages of two-dimensional gas chromatography based resolution were demonstrated by optimizing gradient length and separation through modulation between the first and second column, leading to a marked increase in metabolite identification due to improved separation as exemplified for lactate versus pyruvate, talopyranose versus methyl palmitate and inosine versus docosahexaenoic acid. Our results demonstrate that GCxGC-MS represents a robust metabolomics platform for discovery and targeted studies that can be used with samples derived from the clinic.

ING photometry and spectroscopy of the optical counterpart of the x‐ray transient GRO J0422+32

We present new photometric observations of the GRO transient J0422+32 which show a two‐hour and a five‐hour variation of only 0.1 magnitude in amplitude. Spectroscopic observations during the late decline of the August 1992 outburst and during the August 1993 outburst show variability in the double‐peaked Balmer and He ii profiles on a timescale of hours and reveal red‐shifted emission components at two epochs.