Rigorous Determination of the Stoichiometry of Protein Phosphorylation Using Mass Spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment

From large-scale loops to the sites of dense flaring loops: Preferential conditions for long-period pulsations in solar flares

Copyright © 2010 American Astronomical Society / IOP PublishingLong-period quasi-periodic pulsations (QPPs) of solar flares are a class apart from shorter period events. By involving an external resonator, the mechanism they call upon differs from traditional QPP models, but has wider applications. We present a multi-wavelength analysis of spatially resolved QPPs, with periods around 10 minutes, observed in the X-ray spectrum primarily at energies between 3 and 25 keV. Complementary observations obtained in Hα and radio emission in the kHz to GHz frequency range, together with an analysis of the X-ray plasma properties provide a comprehensive picture that is consistent with a dense flaring loop subject to periodic energization and thermalization. The QPPs obtained in Hα and type III radio bursts, with similar periods as the QPPs in soft X-rays, have the longest periods ever reported for those types of data sets. We also report 1-2 GHz radio emission, concurrent with but unrestricted to the QPP time intervals, which is multi-structured at regularly separated narrowband frequencies and modulated with ~18 minute periods. This radio emission can be attributed to the presence of multiple "quiet" large-scale loops in the background corona. Large scale but shorter inner loops below may act as preferential resonators for the QPPs. The observations support interpretations consistent with both inner and outer loops subject to fast kink magnetohydrodynamic waves. Finally, X-ray imaging indicates the presence of double coronal sources in the flaring sites, which could be the particular signatures of the magnetically linked inner loops. We discuss the preferential conditions and the driving mechanisms causing the repeated flaring

Mechanistic insights into the activation of the IKK kinase complex by the Kaposi’s Sarcoma Herpes virus oncoprotein vFLIP

Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi’s Sarcoma Herpes virus (KSHV) pathogenesis where it is essential for the survival of primary effusion lymphoma (PEL). Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) kinase complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilised, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilised by endogenous cytokines and cellular FLIP homologues

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated

Implications of the Actin Cytoskeleton on the Multi-Step Process of [ PSI+] Prion Formation

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell

Isotopic evidence for dominant secondary production of HONO in near-ground wildfire plumes

Nitrous acid (HONO) is an important precursor to hydroxyl radical (OH) that determines atmospheric oxidative capacity and thus impacts climate and air quality. Wildfire is not only a major direct source of HONO, it also results in highly polluted conditions that favor the heterogeneous formation of HONO from nitrogen oxides (NOx= NO + NO2) and nitrate on both ground and particle surfaces. However, these processes remain poorly constrained. To quantitatively constrain the HONO budget under various fire and/or smoke conditions, we combine a unique dataset of field concentrations and isotopic ratios (15N / 14N and 18O / 16O) of NOx and HONO with an isotopic box model. Here we report the first isotopic evidence of secondary HONO production in near-ground wildfire plumes (over a sample integration time of hours) and the subsequent quantification of the relative importance of each pathway to total HONO production. Most importantly, our results reveal that nitrate photolysis plays a minor role (\u3c5 %) in HONO formation in daytime aged smoke, while NO2-to-HONO heterogeneous conversion contributes 85 %–95 % to total HONO production, followed by OH + NO (5 %–15 %). At nighttime, heterogeneous reduction of NO2 catalyzed by redox active species (e.g., iron oxide and/or quinone) is essential (≥ 75 %) for HONO production in addition to surface NO2 hydrolysis. Additionally, the 18O / 16O of HONO is used for the first time to constrain the NO-to-NO2 oxidation branching ratio between ozone and peroxy radicals. Our approach provides a new and critical way to mechanistically constrain atmospheric chemistry and/or air quality models on a diurnal timescale

Ecological drivers of global gradients in avian dispersal inferred from wing morphology

An organism’s ability to disperse influences many fundamental processes, from speciation and geographical range expansion to community assembly. However, the patterns and underlying drivers of variation in dispersal across species remain unclear, partly because standardised estimates of dispersal ability are rarely available. Here we present a global dataset of avian hand-wing index (HWI), an estimate of wing shape widely adopted as a proxy for dispersal ability in birds. We show that HWI is correlated with geography and ecology across 10,338 (>99%) species, increasing at higher latitudes and with migration, and decreasing with territoriality. After controlling for these effects, the strongest predictor of HWI is temperature variability (seasonality), with secondary effects of diet and habitat type. Finally, we also show that HWI is a strong predictor of geographical range size. Our analyses reveal a prominent latitudinal gradient in HWI shaped by a combination of environmental and behavioural factors, and also provide a global index of avian dispersal ability for use in community ecology, macroecology, and macroevolution

Short-Term Impact of Point-Source Enrichment on the Behavior of Gestating Sows Housed in Groups

Environmental enrichment is an important strategy to improve the welfare of farm animals. However, relatively little is known about enrichment for gestating sows, especially those raised on farms with slatted floors and for which provision of straw may be difficult. The objective of this study was therefore to investigate the short-term (4 d) impact of a point-source enrichment object on the behavior of gestating sows housed in group pens. Four pens of gestating sows on a university research farm were randomly allocated to receive either enrichment or no enrichment (control) in a 2 by 2 crossover design. Time budgets were established by video recording focal sows' behaviors (n = 10 focals per pen) every 15 min between 0800 and 1500 every day. Enrichment use was further characterized by continuous behavior sampling for a 1 h interval between 0830 and 0930 each day. The impact of parity, lameness and presence of stereotypical behavior such as sham chewing on enrichment use was evaluated. Over the course of the study, focal sows spent approximately 73% of observations inactive [either lying down (70%), standing (2%), or sitting (1%)]. Within the remaining observations, sows were most commonly observed sham-chewing (16%), followed by 3% exploring, 2% feeding, 2% walking and 1% interacting with the enrichment when it was available. Low-parity sows, moderately-lame sows, and sows observed sham chewing at baseline displayed more consistent enrichment use over the course of the study (p = 0.02, p < 0.01, p = 0.04, respectively). While no adverse behavioral effects (increased agonism or sham chewing) due to provision or removal of the enrichment object were observed and while 85% of sows were observed to interact with enrichment at least once, interest declined sharply after the first day. We conclude that further research is needed to identify effective and sustainable enrichment strategies for gestating sows

Characteristics of Nontuberculous Mycobacterial Infections at a Midwestern Tertiary Hospital: A Retrospective Study of 365 Patients.

Background: The prevalence of infections due to nontuberculous mycobacteria (NTM) is increasing worldwide, yet little is known about the epidemiology and pathophysiology of these ubiquitous environmental organisms. Pulmonary disease due to Mycobacterium avium complex is most prevalent, but many other NTM species can cause disease in virtually any organ system. As NTM becomes an increasingly common cause of morbidity and mortality, more information is needed about the epidemiology of NTM disease. Methods: We conducted a retrospective chart review of all patients with cultures that grew NTM at a Midwestern tertiary hospital from 1996 to 2017. Information on demographics, medical history, clinical findings, treatment, and outcome was obtained from medical records of all NTM isolates. American Thoracic Society/Infectious Diseases Society of America criteria were used to define pulmonary NTM infections. Results: We identified 1064 NTM isolates, 365 of which met criteria for NTM infection. Pulmonary cases predominated (185 of 365; 50.7%), followed by skin/soft tissue (56 of 365; 15.3%), disseminated (40 of 365; 11%), and lymphatic (28 of 365; 7.7%) disease. Conclusions: This large cohort provides information on the demographics, risk factors, and disease course of patients with pulmonary and extrapulmonary NTM infections. Most patients had medical comorbidities that resulted in anatomic, genetic, or immunologic risk factors for NTM infection. Further population-based studies and increased disease surveillance are warranted to further characterize NTM infection prevalence and trends