Addressing the needs of traumatic brain injury with clinical proteomics.

BackgroundNeurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.ContentWe discuss the challenges of discovering new and validating identified neurotrauma marker candidates using proteomics-based strategies, including targeting, selection strategies and the application of mass spectrometry (MS) technologies and their potential impact to the neurotrauma field.SummaryMany studies use TBI marker candidates based on literature reports, yet progress in genomics and proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from such 'long lists' is still pending, as only few can be taken through the process of preclinical verification and large scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and protection of concussion victims from repeated exposures that could result in lasting neurological deficits

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes.

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation

A deep, multi-epoch Chandra HETG study of the ionized outflow from NGC 4051

Actively accreting supermassive black holes significantly impact the evolution of their host galaxies, truncating further star formation by expelling large fractions of gas with wide-angle outflows. The X-ray band is key to understanding how these black hole winds affect their environment, as the outflows have high temperatures ($\sim$10$^{5-8}$K). We have developed a Bayesian framework for characterizing Active Galactic Nuclei (AGN) outflows with an improved ability to explore parameter space and perform robust model selection. We applied this framework to a new 700 ks and an archival 315 ks Chandra High Energy Transmission Gratings observation of the Seyfert galaxy NGC 4051. We have detected six absorbers intrinsic to NGC 4051. These wind components span velocities from 400 km s$^{-1}$ to 30,000 km s$^{-1}$. We have determined that the most statistically significant wind component is purely collisionally ionized, which is the first detection of such an absorber. This wind has $T\approx10^7$ K and $v\approx880$ km s$^{-1}$ and remains remarkably stable between the two epochs. Other slow components also remain stable across time. Fast outflow components change their properties between 2008 and 2016, suggesting either physical changes or clouds moving in and out of the line of sight. For one of the fast components we obtain one of the tightest wind density measurements to date, log $n/$[cm$^{-3}$]=13.0$^{+0.01}_{-0.02}$, and determine that it is located at $\sim$240 gravitational radii. The estimated total outflow power surpasses 5% of the bolometric luminosity (albeit with large uncertainties) making it important in the context of galaxy-black hole interactions.Comment: 27 pages, 17 figures, 10 tables, accepted for publication in MNRA

Photoionization Models for High-density Gas

Relativistically broadened and redshifted 6.4–6.9 keV iron K lines are observed from many accretion powered objects, including X-ray binaries and active galactic nuclei. The existence of gas close to the central engine implies large radiation intensities and correspondingly large gas densities if the gas is to remain partially ionized. Simple estimates indicate that high gas densities are needed to allow for the survival of iron against ionization. These are high enough that rates for many atomic processes are affected by mechanisms related to interactions with nearby ions and electrons. Radiation intensities are high enough that stimulated processes can be important. Most models currently in use for interpreting relativistic lines use atomic rate coefficients designed for use at low densities and neglect stimulated processes. In our work so far we have presented atomic structure calculations with the goal of providing physically appropriate models at densities consistent with line-emitting gas near compact objects. In this paper we apply these rates to photoionization calculations, and produce ionization balance curves and X-ray emissivities and opacities that are appropriate for high densities and high radiation intensities. The final step in our program will be presented in a subsequent paper in which model atmosphere calculations will incorporate these rates into synthetic spectra

Identification of the Major Expressed S-Layer and Cell Surface-Layer-Related Proteins in the Model Methanogenic Archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A

Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UnlabelledSyntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.ImportanceBacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA

Improved measurements of turbulence in the hot gaseous atmospheres of nearby giant elliptical galaxies

We present significantly improved measurements of turbulent velocities in the hot gaseous haloes of nearby giant elliptical galaxies. Using deep XMM-Newton Reflection Grating Spectrometer (RGS) observations and a combination of resonance scattering and direct line broadening methods, we obtain well bounded constraints for 13 galaxies. Assuming that the turbulence is isotropic, we obtain a best-fitting mean 1D turbulent velocity of similar to 110 km s(-1). This implies a typical 3D Mach number similar to 0.45 and a typical non-thermal pressure contribution of similar to 6 per cent in the cores of nearby massive galaxies. The intrinsic scatter around these values is modest-consistent with zero, albeit with large statistical uncertainty-hinting at a common and quasi-continuous mechanism sourcing the velocity structure in these objects. Using conservative estimates of the spatial scales associated with the observed turbulent motions, we find that turbulent heating can be sufficient to offset radiative cooling in the inner regions of these galaxies (< 10 kpc, typically 2-3 kpc). The full potential of our analysis methods will be enabled by future X-ray micro-calorimeter observations

Improved measurements of turbulence in the hot gaseous atmospheres of nearby giant elliptical galaxies

We present significantly improved measurements of turbulent velocities in the hot gaseous haloes of nearby giant elliptical galaxies. Using deep XMM-Newton Reflection Grating Spectrometer (RGS) observations and a combination of resonance scattering and direct line broadening methods, we obtain well bounded constraints for 13 galaxies. Assuming that the turbulence is isotropic, we obtain a best-fitting mean 1D turbulent velocity of similar to 110 km s(-1). This implies a typical 3D Mach number similar to 0.45 and a typical non-thermal pressure contribution of similar to 6 per cent in the cores of nearby massive galaxies. The intrinsic scatter around these values is modest-consistent with zero, albeit with large statistical uncertainty-hinting at a common and quasi-continuous mechanism sourcing the velocity structure in these objects. Using conservative estimates of the spatial scales associated with the observed turbulent motions, we find that turbulent heating can be sufficient to offset radiative cooling in the inner regions of these galaxies (< 10 kpc, typically 2-3 kpc). The full potential of our analysis methods will be enabled by future X-ray micro-calorimeter observations

Insights into the location and dynamics of the coolest X-ray emitting gas in clusters of galaxies

We extend our previous study of the cool gas responsible for the emission of O VII X-ray lines in the cores of clusters and groups of galaxies. This is the coolest X-ray emitting phase and connects the 10 000 K H alpha emitting gas to the million degree phase, providing a useful tool to understand cooling in these objects. We study the location of the O VII gas and its connection to the intermediate Fe XVII and hotter O VIII phases. We use high-resolution X-ray grating spectra of elliptical galaxies with strong Fe XVII line emission and detect O VII in 11 of 24 objects. Comparing the O VII detection level and resonant scattering, which is sensitive to turbulence and temperature, suggests that O VII is preferably found in cooler objects, where the Fe XVII resonant line is suppressed due to resonant scattering, indicating subsonic turbulence. Although a larger sample of sources and further observations is needed to distinguish between effects from temperature and turbulence, our results are consistent with cooling being suppressed at high turbulence as predicted by models of active galactic nuclei feedback, gas sloshing and galactic mergers. In some objects, the O VII resonant-to-forbidden line ratio is decreased by either resonant scattering or charge exchange boosting the forbidden line, as we show for NGC 4636. Charge exchange indicates interaction between neutral and ionized gas phases. The Perseus cluster also shows a high Fe XVII forbidden-to-resonance line ratio, which can be explained with resonant scattering by low-turbulence cool gas in the line of sight.Peer reviewe