X-ray Source Heights in a Solar Flare: Thick-target versus Thermal Conduction Front Heating

Observations of solar flares with RHESSI have shown X-ray sources traveling along flaring loops, from the corona down to the chromosphere and back up. The 28 November 2002 C1.1 flare, first observed with RHESSI by Sui et al. 2006 and quantitatively analyzed by O'Flannagain et al. 2013, very clearly shows this behavior. By employing numerical experiments, we use these observations of X-ray source height motions as a constraint to distinguish between heating due to a non-thermal electron beam and in situ energy deposition in the corona. We find that both heating scenarios can reproduce the observed light curves, but our results favor non-thermal heating. In situ heating is inconsistent with the observed X-ray source morphology and always gives a height dispersion with photon energy opposite to what is observed.Comment: Accepted to Ap

Evaluation of dimethyl sulfoxide (DMSO) as a mobile phase additive during top 3 label-free quantitative proteomics

AbstractDimethyl sulfoxide (DMSO) has been advocated as a beneficial additive to electrospray solvents for peptide analysis due to the improved ionisation efficiency conferred. Previous reports have shown that the resultant improvements in peptide ion signal intensities are non-uniform. As a result, it was hypothesised that inclusion of DMSO in electrospray solvents could be detrimental to the outcome of intensity-based label-free absolute quantification approaches, specifically the top 3 method. The effect of DMSO as a mobile phase additive in top 3 label-free quantification was therefore evaluated. We show that inclusion of DMSO enhances data quality, improving the precision and number of proteins quantified, with no significant change to the quantification values observed in its absence

The use of selected reaction monitoring in quantitative proteomics

Selected reaction monitoring (SRM) has a long history of use in the area of quantitative MS. In recent years, the approach has seen increased application to quantitative proteomics, facilitating multiplexed relative and absolute quantification studies in a variety of organisms. This article discusses SRM, after introducing the context of quantitative proteomics (specifically primarily absolute quantification) where it finds most application, and considers topics such as the theory and advantages of SRM, the selection of peptide surrogates for protein quantification, the design of optimal SRM co-ordinates and the handling of SRM data. A number of published studies are also discussed to demonstrate the impact that SRM has had on the field of quantitative proteomics. </jats:p

Peptide scrambling during collision-induced dissociation is influenced by N-terminal residue basicity

‘Bottom up’ proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H2O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID

RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies

This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (<b>Re</b>versed-<b>p</b>hase <b>li</b>quid chromatography <b>cal</b>ibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses. RePLiCal has been benchmarked against three commercially available peptide RT standard kits and outperforms all in terms of LC gradient coverage. RePLiCal also provides a higher number of calibrant points for chromatographic retention time standardization and normalization. The standard provides stable RTs over long analysis times and can be readily transferred between different LC gradients and nUHPLC instruments. Moreover, RePLiCal can be used to predict RTs for other peptides in a timely manner. Furthermore, it is shown that RePLiCal can be used effectively to evaluate trapping column performance for nUHPLC instruments using trap-elute configurations, to optimize gradients to maximize peptide and protein identification rates, and to recalibrate the <i>m</i>/<i>z</i> scale of mass spectrometry data post-acquisition

Predicting death over 8 years in a prospective cohort of HIV-infected women: the Women's Interagency HIV Study.

ObjectivesPredicting mortality in middle-aged HIV-infected (HIV+) women on antiretroviral therapies (ART) is important for understanding the impact of HIV infection. Several health indices have been used to predict mortality in women with HIV infection. We evaluated: (1) an HIV biological index, Veterans Aging Cohort Study (VACS); (2) a physical index, Fried Frailty Index (FFI); and (3) a mental health index, Center for Epidemiologic Studies-Depression (CES-D). Proportional hazards regression analyses were used to predict death and included relevant covariates.DesignProspective, observational cohort.SettingMulticentre, across six sites in the USA.Participants1385 multirace/ethnic ART-experienced HIV+ women in 2005.Primary and secondary outcomesAll deaths, AIDS deaths and non-AIDS deaths up to ~8 years from baseline.ResultsIncluded together in one model, VACS Index was the dominant, significant independent predictor of all deaths within 3 years (HR=2.20, 95% CI 1.83, 2.65, χ2=69.04, p&lt;0.0001), and later than 3 years (HR=1.55, 95% CI 1.30, 1.84, χ2=23.88, p&lt;0.0001); followed by FFI within 3 years (HR=2.06, 95% CI 1.19, 3.57, χ2=6.73, p=0.01) and later than 3 years (HR=2.43, 95% CI 1.58, 3.75, χ2=16.18, p=0.0001). CES-D score was not independently associated with mortality.Conclusions and relevanceThis is the first simultaneous evaluation of three common health indices in HIV+ adults. Indices reflecting physical and biological ageing were associated with death

Are lower fasting plasma glucose levels at diagnosis of type 2 diabetes associated with improved outcomes? U.K. Prospective Diabetes Study 61

WSTĘP. Cukrzyca typu 2 może się rozwijać przez wiele lat przed postawieniem diagnozy. W tym czasie u wielu osób dochodzi już do rozwoju jej powikłań. Wczesne wykrycie i leczenie cukrzycy może temu zapobiec, ale jak dotąd brakuje dowodów na poparcie tej tezy. MATERIAŁ I METODY. U 5088 spośród 5102 uczestników badania UKDPS (United Kingdom Prospective Diabetes Study) dokonano porównania kontroli glikemii oraz klinicznych i pośrednich kryteriów badania w zależności od glikemii na czczo (FPG, fasting plasma glucose) w momencie rozpoznania choroby. Grupy porównywane cechowały się niską ( 140 do < 180 mg/dl [7,8 do < 10,0 mmol/l]) lub wysoką (ł 180 mg/dl [ł 10,0 mmol/l]) glikemią. Porównano również grupy pacjentów różniące się objawami klinicznymi cukrzycy. WYNIKI. W grupie pacjentów z niższymi stężeniami glukozy na czczo stwierdzono rzadsze występowanie retinopatii cukrzycowej, nieprawidłowych wyników biotezjometrii oraz deklarowanych zaburzeń erekcji. Stopień wzrostu FPG i HbA1c podczas badania był identyczny we wszystkich trzech grupach, chociaż obserwowano przetrwanie pierwotnych różnic. U osób mieszczących się w grupie z najniższą glikemią na czczo ryzyko wystąpienia każdego z określonych wcześniej klinicznych kryteriów badania, wyłączywszy udar mózgu, było istotnie niższe. W grupie o średnich wartościach glikemii ryzyko wszystkich kryteriów badania, poza udarem i zawałem serca, było istotnie zmniejszone. Grupy o niskiej i średniej FPG charakteryzowało istotnie zmniejszone ryzyko progresji retinopatii, zmniejszenia czucia wibracji i rozwoju mikroalbuminurii. WNIOSKI. Chorzy, u których rozpoznaje się cukrzycę typu 2 z niższymi wartościami glikemii na czczo, znajdują się na wczesnym etapie rozwoju choroby i ryzyko rozwoju klinicznych kryteriów badania jest u nich mniejsze mimo progresji cukrzycy. Ponieważ większość pacjentów w momencie rozpoznania nie ma wyraźnych objawów klinicznych, do ich identyfikacji pożądane jest wprowadzenie programów aktywnego wykrywania cukrzycyINTRODUCTION. Type 2 diabetes may be present for several years before diagnosis, by which time many patients have already developed diabetic complications. Earlier detection and treatment may reduce this burden, but evidence to support this approach is lacking. MATERIAL AND METHODS. Glycemic control and clinical and surrogate outcomes were compared for 5,088 of 5,102 U.K. Diabetes Prospective Study participants according to whether they had low (< 140 mg/dl [< 7.8 mmol/l]), intermediate (140 to < 180 mg/dl [7.8 to < 10.0 mmol/l]), or high (&#8805; 180 mg/dl [&#8805; 10 mmol/l]) fasting plasma glucose (FPG) levels at diagnosis. Individuals who presented with and without diabetic symptoms were also compared. RESULTS. Fewer people with FPG in the lowest category had retinopathy, abnormal biothesiometer measurements, or reported erectile dysfunction. The rate of increase in FPG and HbA1c during the study was identical in all three groups, although absolute differences persisted. Individuals in the low FPG group had a significantly reduced risk for each predefined clinical outcome except stroke, whereas those in the intermediate group had significantly reduced risk for each outcome except stroke and myocardial infarction. The low and intermediate FPG groups had a significantly reduced risk for progression of retinopathy, reduction in vibration sensory threshold, or development of microalbuminuria. CONCLUSIONS. People presenting with type 2 diabetes with lower initial glycemia who may be earlier in the course of their disease had fewer adverse clinical outcomes despite similar glycemic progression. Since most such people are asymptomatic at diagnosis, active case detection programs would be required to identify them

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. Reliable and accurate quantification of the proteins present in a cell or tissue remains a major challenge for post-genome scientists. Proteins are the primary functional molecules in biological systems and knowledge of their abundance and dynamics is an important prerequisite to a complete understanding of natural physiological processes, or dysfunction in disease. Accordingly, much effort has been spent in the development of reliable, accurate and sensitive techniques to quantify the cellular proteome, the complement of proteins expressed at a given time under defined conditions (1). Moreover, the ability to model a biological system and thus characterize it in kinetic terms, requires that protein concentrations be defined in absolute numbers (2, 3). Given the high demand for accurate quantitative proteome data sets, there has been a continual drive to develop methodology to accomplish this, typically using mass spectrometry (MS) as the analytical platform. Many recent studies have highlighted the capabilities of MS to provide good coverage of the proteome at high sensitivity often using yeast as a demonstrator system (4⇓⇓⇓⇓⇓–10), suggesting that quantitative proteomics has now “come of age” (1). However, given that MS is not inherently quantitative, most of the approaches produce relative quantitation and do not typically measure the absolute concentrations of individual molecular species by direct means. For the yeast proteome, epitope tagging studies using green fluorescent protein or tandem affinity purification tags provides an alternative to MS. Here, collections of modified strains are generated that incorporate a detectable, and therefore quantifiable, tag that supports immunoblotting or fluorescence techniques (11, 12). However, such strategies for copies per cell (cpc) quantification rely on genetic manipulation of the host organism and hence do not quantify endogenous, unmodified protein. Similarly, the tagging can alter protein levels - in some instances hindering protein expression completely (11). Even so, epitope tagging methods have been of value to the community, yielding high coverage quantitative data sets for the majority of the yeast proteome (11, 12). MS-based methods do not rely on such nonendogenous labels, and can reach genome-wide levels of coverage. Accurate estimation of absolute concentrations i.e. protein copy number per cell, also usually necessitates the use of (one or more) external or internal standards from which to derive absolute abundance (4). Examples include a comprehensive quantification of the Leptospira interrogans proteome that used a 19 protein subset quantified using selected reaction monitoring (SRM)1 to calibrate their label-free data (8, 13). It is worth noting that epitope tagging methods, although also absolute, rely on a very limited set of standards for the quantitative western blots and necessitate incorporation of a suitable immunogenic tag (11). Other recent, innovative approaches exploiting total ion signal and internal scaling to estimate protein cellular abundance (10, 14), avoid the use of internal standards, though they do rely on targeted proteomic data to validate their approach. The use of targeted SRM strategies to derive proteomic calibration standards highlights its advantages in comparison to label-free in terms of accuracy, precision, dynamic range and limit of detection and has gained currency for its reliability and sensitivity (3, 15⇓–17). Indeed, SRM is often referred to as the “gold standard proteomic quantification method,” being particularly well-suited when the proteins to be quantified are known, when appropriate surrogate peptides for protein quantification can be selected a priori, and matched with stable isotope-labeled (SIL) standards (18⇓–20). In combination with SIL peptide standards that can be generated through a variety of means (3, 15), SRM can be used to quantify low copy number proteins, reaching down to ∼50 cpc in yeast (5). However, although SRM methodology has been used extensively for S. cerevisiae protein quantification by us and others (19, 21, 22), it has not been used for large protein cohorts because of the requirement to generate the large numbers of attendant SIL peptide standards; the largest published data set is only for a few tens of proteins. It remains a challenge therefore to robustly quantify an entire eukaryotic proteome in absolute terms by direct means using targeted MS and this is the focus of our present study, the Census Of the Proteome of Yeast (CoPY). We present here direct and absolute quantification of nearly 2000 endogenous proteins from S. cerevisiae grown in steady state in a chemostat culture, using the SRM-based QconCAT approach. Although arguably not quantification of the entire proteome, this represents an accurate and rigorous collection of direct yeast protein quantifications, providing a gold-standard data set of endogenous protein levels for future reference and comparative studies. The highly reproducible SIL-SRM MS data, with robust CVs typically less than 20%, is compared with other extant data sets that were obtained via alternative analytical strategies. We also report a matched high quality transcriptome from the same cells using RNA-seq, which supports additional calculations including a refined estimate of the total protein content in yeast cells, and a simple linear model of translation explaining 70% of the variance between RNA and protein levels in yeast chemostat cultures. These analyses confirm the validity of our data and approach, which we believe represents a state-of-the-art absolute quantification compendium of a significant proportion of a model eukaryotic proteome

Possible detection of two giant extrasolar planets orbiting the eclipsing polar UZ Fornacis

We present new high-speed, multi-observatory, multi-instrument photometry of the eclipsing polar UZ For in order to measure precise mid-eclipse times with the aim of detecting any orbital period variations. When combined with published eclipse times and archival data spanning ~27 years, we detect departures from a linear and quadratic trend of ~60 s. The departures are strongly suggestive of two cyclic variations of 16(3) and 5.25(25) years. The two favoured mechanisms to drive the periodicities are either two giant extrasolar planets as companions to the binary (with minimum masses of 6.3(1.5)M(Jupiter) and 7.7(1.2)M(Jupiter)) or a magnetic cycle mechanism (e.g. Applegate's mechanism) of the secondary star. Applegate's mechanism would require the entire radiant energy output of the secondary and would therefore seem to be the least likely of the two, barring any further refinements in the effect of magnetic fieilds (e.g. those of Lanza et al.). The two planet model can provide realistic solutions but it does not quite capture all of the eclipse times measurements. A highly eccentric orbit for the outer planet would fit the data nicely, but we find that such a solution would be unstable. It is also possible that the periodicities are driven by some combination of both mechanisms. Further observations of this system are encouraged.Comment: 10 pages, 4 figures, 2 table

A First Comparison of Kepler Planet Candidates in Single and Multiple Systems

In this letter we present an overview of the rich population of systems with multiple candidate transiting planets found in the first four months of Kepler data. The census of multiples includes 115 targets that show 2 candidate planets, 45 with 3, 8 with 4, and 1 each with 5 and 6, for a total of 170 systems with 408 candidates. When compared to the 827 systems with only one candidate, the multiples account for 17 percent of the total number of systems, and a third of all the planet candidates. We compare the characteristics of candidates found in multiples with those found in singles. False positives due to eclipsing binaries are much less common for the multiples, as expected. Singles and multiples are both dominated by planets smaller than Neptune; 69 +2/-3 percent for singles and 86 +2/-5 percent for multiples. This result, that systems with multiple transiting planets are less likely to include a transiting giant planet, suggests that close-in giant planets tend to disrupt the orbital inclinations of small planets in flat systems, or maybe even to prevent the formation of such systems in the first place.Comment: 13 pages, 13 figures, submitted to ApJ Letter