Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways

Effects of Sperm Conjugation and Dissociation on Sperm Viability In Vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability

Human arachnoid granulations Part I: a technique for quantifying area and distribution on the superior surface of the cerebral cortex

<p>Abstract</p> <p>Background</p> <p>The arachnoid granulations (AGs) are herniations of the arachnoid membrane into the dural venous sinuses on the surface of the brain. Previous morphological studies of AGs have been limited in scope and only one has mentioned surface area measurements. The purpose of this study was to investigate the topographic distribution of AGs on the superior surface of the cerebral cortex.</p> <p>Methods</p> <p><it>En face </it>images were taken of the superior surface of 35 formalin-fixed human brains. AGs were manually identified using Adobe Photoshop, with a pixel location containing an AG defined as 'positive'. A set of 25 standard fiducial points was marked on each hemisphere for a total of 50 points on each image. The points were connected on each hemisphere to create a segmented image. A standard template was created for each hemisphere by calculating the average position of the 25 fiducial points from all brains. Each segmented image was mapped to the standard template using a linear transformation. A topographic distribution map was produced by calculating the proportion of AG positive images at each pixel in the standard template. The AG surface area was calculated for each hemisphere and for the total brain superior surface. To adjust for different brain sizes, the proportional involvement of AGs was calculated by dividing the AG area by the total area.</p> <p>Results</p> <p>The total brain average surface area of AGs was 78.53 ± 13.13 mm²(n = 35) and average AG proportional involvement was 57.71 × 10^-4± 7.65 × 10^-4. Regression analysis confirmed the reproducibility of AG identification between independent researchers with r²= 0.97. The surface AGs were localized in the parasagittal planes that coincide with the region of the lateral lacunae.</p> <p>Conclusion</p> <p>The data obtained on the spatial distribution and <it>en face </it>surface area of AGs will be used in an <it>in vitro </it>model of CSF outflow. With an increase in the number of samples, this analysis technique can be used to study the relationship between AG surface area and variables such as age, race and gender.</p

Bayesian Methods for Exoplanet Science

Exoplanet research is carried out at the limits of the capabilities of current telescopes and instruments. The studied signals are weak, and often embedded in complex systematics from instrumental, telluric, and astrophysical sources. Combining repeated observations of periodic events, simultaneous observations with multiple telescopes, different observation techniques, and existing information from theory and prior research can help to disentangle the systematics from the planetary signals, and offers synergistic advantages over analysing observations separately. Bayesian inference provides a self-consistent statistical framework that addresses both the necessity for complex systematics models, and the need to combine prior information and heterogeneous observations. This chapter offers a brief introduction to Bayesian inference in the context of exoplanet research, with focus on time series analysis, and finishes with an overview of a set of freely available programming libraries.Comment: Invited revie

DOSCATs: Double standards for protein quantification

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB

Integrating modes of policy analysis and strategic management practice : requisite elements and dilemmas

There is a need to bring methods to bear on public problems that are inclusive, analytic, and quick. This paper describes the efforts of three pairs of academics working from three different though complementary theoretical foundations and intervention backgrounds (i.e., ways of working) who set out together to meet this challenge. Each of the three pairs had conducted dozens of interventions that had been regarded as successful or very successful by the client groups in dealing with complex policy and strategic problems. One approach focused on leadership issues and stakeholders, another on negotiating competitive strategic intent with attention to stakeholder responses, and the third on analysis of feedback ramifications in developing policies. This paper describes the 10 year longitudinal research project designed to address the above challenge. The important outcomes are reported: the requisite elements of a general integrated approach and the enduring puzzles and tensions that arose from seeking to design a wide-ranging multi-method approach

Transit Timing and Duration Variations for the Discovery and Characterization of Exoplanets

Transiting exoplanets in multi-planet systems have non-Keplerian orbits which can cause the times and durations of transits to vary. The theory and observations of transit timing variations (TTV) and transit duration variations (TDV) are reviewed. Since the last review, the Kepler spacecraft has detected several hundred perturbed planets. In a few cases, these data have been used to discover additional planets, similar to the historical discovery of Neptune in our own Solar System. However, the more impactful aspect of TTV and TDV studies has been characterization of planetary systems in which multiple planets transit. After addressing the equations of motion and parameter scalings, the main dynamical mechanisms for TTV and TDV are described, with citations to the observational literature for real examples. We describe parameter constraints, particularly the origin of the mass/eccentricity degeneracy and how it is overcome by the high-frequency component of the signal. On the observational side, derivation of timing precision and introduction to the timing diagram are given. Science results are reviewed, with an emphasis on mass measurements of transiting sub-Neptunes and super-Earths, from which bulk compositions may be inferred.Comment: Revised version. Invited review submitted to 'Handbook of Exoplanets,' Exoplanet Discovery Methods section, Springer Reference Works, Juan Antonio Belmonte and Hans Deeg, Eds. TeX and figures may be found at https://github.com/ericagol/TTV_revie

An adult cystic fibrosis patient presenting with persistent dyspnea: case report

BACKGROUND: Persistent dyspnea is a common finding in the cystic fibrosis patient that typically leads to further work up of an alternative pulmonary etiology. Adult cystic fibrosis patients; however, are growing in numbers and they are living into the ages in which coronary artery disease becomes prevalent. Coronary disease should be included in the consideration of diagnostic possibilities. CASE PRESENTATION: A 52-year-old white male with cystic fibrosis was evaluated for exertional dyspnea associated with vague chest discomfort. Diagnostic testing revealed normal white blood cell, hemoglobin and platelet count, basic metabolic panel, fasting lipid profile, HbA1c, with chest radiograph confirming chronic cystic findings unchanged from prior radiographs and an electrocardiogram that revealed sinus rhythm with left anterior fascicular block. Stress thallium testing demonstrated a reversible anteroseptal perfusion defect with a 55% left ventricular ejection fraction. Heart catheterization found a 99% occlusion of the left anterior descending artery extending into the two diagonal branches, with 100% obstruction of the left anterior descending artery at the trifurcation and 70% lesion affecting the first posterior lateral branch of the circumflex artery. CONCLUSION: This case report represents the first description in the medical literature of a cystic fibrosis patient diagnosed with symptomatic coronary artery disease. Applying a standard clinical practice guide proved useful toward evaluating a differential diagnosis for a cystic fibrosis patient presenting with dyspnea and chest discomfort

The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence