DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.

The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

Ambient ionization mass spectrometry applied to new psychoactive substance analysis.

In the past decade a plethora of drugs with similar effects to controlled psychoactive drugs, like cannabis, amfetamine (amphetamine), or lysergic acid diethylamide, have been synthesized. These drugs can collectively be classified under the term new psychoactive substances (NPS) and are used for recreational purposes. The novelty of the substances, alongside the rapid rate of emergence and structural variability, makes their detection as well as their legal control highly challenging, increasing the demand for rapid and easy-to-use analytical techniques for their detection and identification. Therefore, interest in ambient ionization mass spectrometry applied to NPS has grown in recent years, which is largely because it is relatively fast and simple to use and has a low operating cost. This review aims to provide a critique of the suitability of current ambient ionization techniques for the analysis of NPS in the forensic and clinical toxicology fields. Consideration is given to analytical performance and ease of implementation, including ionization efficiency, selectivity, sensitivity, quantification, analyte chemistry, molecular coverage, validation, and practicality.

Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use.

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a "dilute and inject" approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.

Quantification of insulin-like growth factor-I by liquid chromatography-mass spectrometry.

We describe three methods of sample preparation for the liquid chromatography coupled with mass spectrometric measurement of insulin-like growth factor-I concentration in blood. One method involves trypsin digestion, the second involves intact protein quantification and the third method is a combination of the first two. Step-by-step directions are provided for sample collection and handling including transport, storage conditions as well as detailed instructions for preparation for analysis, which can be modified for larger or smaller sample volumes as needed. A fully 15 N-labelled internal standard is used and the merits of a single-point calibrator are discussed. Example instrumental conditions are presented for both intact and digest methods.

Comparison of normal distribution-based and nonparametric decision limits on the GH-2000 score for detecting growth hormone misuse (doping) in sport.

This paper is motivated by the GH-2000 biomarker test, though the discussion is applicable to other diagnostic tests. The GH-2000 biomarker test has been developed as a powerful technique to detect growth hormone misuse by athletes, based on the GH-2000 score. Decision limits on the GH-2000 score have been developed and incorporated into the guidelines of the World Anti-Doping Agency (WADA). These decision limits are constructed, however, under the assumption that the GH-2000 score follows a normal distribution. As it is difficult to affirm the normality of a distribution based on a finite sample, nonparametric decision limits, readily available in the statistical literature, are viable alternatives. In this paper, we compare the normal distribution-based and nonparametric decision limits. We show that the decision limit based on the normal distribution may deviate significantly from the nominal confidence level 1-α or nominal FPR γ when the distribution of the GH-2000 score departs only slightly from the normal distribution. While a nonparametric decision limit does not assume any specific distribution of the GH-2000 score and always guarantees the nominal confidence level and FPR, it requires a much larger sample size than the normal distribution-based decision limit. Due to the stringent FPR of the GH-2000 biomarker test used by WADA, the sample sizes currently available are much too small, and it will take many years of testing to have the minimum sample size required, in order to use the nonparametric decision limits. Large sample theory about the normal distribution-based and nonparametric decision limits is also developed in this paper to help understanding their behaviours when the sample size is large.

Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry.

BACKGROUND: Insulin-like growth factor-I (IGF-1) is measured mainly by immunoassay for the diagnosis and treatment of growth hormone (GH) disorders, and to detect misuse of GH in sport. Immunoassays often have insufficient inter-laboratory agreement, especially between commercial kits. Over the expected range of IGF-1 in blood (∼50-500 ng/mL), in an inter-laboratory study we previously established a measurement imprecision of 11% (%CV) for the digested protein analyzed by LC-MS. Measuring intact IGF-1 by LC-MS should be simpler. However, no inter-laboratory agreement has been published. METHODS: Intact and trypsin-digested IGF-1 in 32 serum samples from healthy volunteers and human growth hormone administration studies were analyzed by LC-MS using different instruments in five laboratories, as well as by immunoassay in a single laboratory. Another 100 samples were analyzed for IGF-1, both intact and after trypsin-digestion, in each laboratory by LC-MS. The statistical relationship between measurements and the imprecision of each assay group was assessed. RESULTS: An intra-laboratory variability of 2-4% CV was obtained. Inter-laboratory variability was greater at 14.5% CV. Orthogonal regression of intact versus trypsin-digestion methods (n = 646) gave a slope of 1.01 and intercept of 2.05 ng/mL. CONCLUSIONS: LC-MS measurements of IGF-1 by intact and trypsin-digestion methods are not statistically different and each is similar to immunoassay. The two LC-MS approaches may be used interchangeably or together to eliminate concerns regarding an immunoassay IGF-1 measurement. Because intact and digested IGF-1 measurements generally agreed within 20% of each other, we propose this as a criterion of assay acceptability.

In Vitro Phase I Metabolic Profiling of the Synthetic Cannabinoids AM-694, 5F-NNEI, FUB-APINACA, MFUBINAC, and AMB-FUBINACA.

Synthetic cannabinoids (SCs) constitute one of the most rapidly expanding class of new psychoactive substances. SCs pose a health threat to the individual and to the public due to their central (psychoactive) and peripheral effects. Their pharmacology and toxicology are poorly understood, and the substances can be unexpectedly toxic and harmful. The metabolism of SCs is also relevant in clinical and forensic toxicology as SCs are excreted in urine mostly as their metabolites. Thus, SC metabolites are widely used as markers for identifying SC intake. Herein, we used human liver microsome systems to study the in vitro phase I metabolic profiling of five SCs, namely AM-694, 5F-NNEI, FUB-APINACA, MFUBINAC, and AMB-FUBINACA. The metabolites were detected and structurally elucidated by liquid chromatography-high resolution mass spectrometry. The main metabolic pathway of AM-694 (benzoyl-indole SC) is oxidative defluorination; 5F-NNEI (naphthyl-indole carboxamide SC) follows amide hydrolysis and monohydroxylation at the naphthyl moiety. However, indazole carboxamide substituted with an adamantyl group, such as FUB-APINACA, is likely to produce (isomeric) hydroxylation of the adamantyl group as the main metabolite species. For the substrates that contain ester bonds in their structure, like MFUBINAC and AMB-FUBINACA, the ester hydrolysis metabolite is predominant.

Monitoring Methods of Human Body Joints: State-of-the-Art and Research Challenges.

The world's population is aging: the expansion of the older adult population with multiple physical and health issues is now a huge socio-economic concern worldwide. Among these issues, the loss of mobility among older adults due to musculoskeletal disorders is especially serious as it has severe social, mental and physical consequences. Human body joint monitoring and early diagnosis of these disorders will be a strong and effective solution to this problem. A smart joint monitoring system can identify and record important musculoskeletal-related parameters. Such devices can be utilized for continuous monitoring of joint movements during the normal daily activities of older adults and the healing process of joints (hips, knees or ankles) during the post-surgery period. A viable monitoring system can be developed by combining miniaturized, durable, low-cost and compact sensors with the advanced communication technologies and data processing techniques. In this study, we have presented and compared different joint monitoring methods and sensing technologies recently reported. A discussion on sensors' data processing, interpretation, and analysis techniques is also presented. Finally, current research focus, as well as future prospects and development challenges in joint monitoring systems are discussed.

Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry.

RATIONALE: Procollagen III amino-terminal propeptide (P-III-NP) is currently monitored in human doping control as a biomarker for growth hormone administration and also in clinical diagnostics using immunoassays. Drawbacks to this approach have been highlighted and research is ongoing to develop a mass spectrometric method to complement these methods. However, a lack of traceable reference material, the presence of post-translational modifications (PTMs), and small blood concentration complicate the development of targeted analytical methods for P-III-NP quantification. METHODS: Tryptic digest products of P-III-NP were assessed by liquid chromatography/mass spectrometry (LC/MS). In silico digestion was used to predict P-III-NP peptides for MS analysis; however, these excluded PTMs. With a priori knowledge of PTMs, we associated experimental P-III-NP peptides with those derived by in silico digestion. Synthesized P-III-NP peptides, hT1 (human) and T5 (human/bovine), were used to develop sensitive micro- and nano-flow LC/MS methods to analyse P-III-NP originating from human serum semi-quantitatively. RESULTS: P-III-NP peptides, T1 and T5, were identified using high-resolution accurate MS (HRAMS). PTMs modified the mass of observed peptides. N-terminal pyroglutamation (pE) in T1 and several hydroxylated prolines (hP) in T5 (G-X-hP motif) were observed. With PTM, hT1 and T5 were observed in a digest of immuno-captured P-III-NP by LC/MS. Using a semi-quantitative approach, hP-III-NP at basal concentrations of 2 ng/mL (50 pmol) could be estimated from a 200-µL sample volume. CONCLUSIONS: Consideration of PTMs is needed to identify P-III-NP peptides produced by digestion with trypsin. The information presented here now gives the most appropriate peptide sequences for synthesizing suitable reference materials required for quantification of human P-III-NP in blood and evidences methodology that is sufficiently sensitive to develop a quantitative method.

Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy.

γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor (detect, identify, and quantify) exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake.

Rapid Analysis of Anabolic Steroid Metabolites in Urine by Combining Field Asymmetric Waveform Ion Mobility Spectrometry with Liquid Chromatography and Mass Spectrometry.

The combination of field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of glucuronide and sulfate metabolites of seven anabolic-androgenic steroids in urine. Separation by FAIMS-MS was investigated in positive ion mode for selected cationic adducts (H+, NH4+, Na+, K+, and Cs+). LC-FAIMS-MS analysis of the doubly sodiated adducts ([M + 2Na - H]+) of isobaric and coeluting steroid metabolites allowed their rapid (8 min) qualitative and quantitative determination in spiked urine using hydrophilic interaction liquid chromatography prior to FAIMS-MS separation, with discrimination >95% achieved between the steroids investigated. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection in the range 1-6 ng mL-1, limits of quantification in the range 3-20 ng mL-1, with reproducibility (%RSD < 10%; n = 6) and linearity (R2 > 0.99). The LC-FAIMS-MS method demonstrates increases in signal-to-noise ratios for the doubly sodiated steroid metabolites in unspiked urine (>250%) by the reduction of isobaric interferences from the matrix. An alternative or additional tool for identification of the steroid metabolites is based on the observations of different patterns of sodium acetate clusters that are characteristic for each metabolite.

Identification of potent, nonabsorbable agonists of the calcium-sensing receptor for GI-specific administration.

Modulation of gastrointestinal nutrient sensing pathways provides a promising a new approach for the treatment of metabolic diseases including diabetes and obesity. The calcium-sensing receptor has been identified as a key receptor involved in mineral and amino acid nutrient sensing and thus is an attractive target for modulation in the intestine. Herein we describe the optimization of gastrointestinally restricted calcium-sensing receptor agonists starting from a 3-aminopyrrolidine-containing template leading to the identification of GI-restricted agonist 19 (GSK3004774).

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist.

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.

Epidemiology of Major Depressive Disorder Disability in the US Military: FY 2007-2012.

This study assesses the incidence of major depressive disorder (MDD) disability discharge and retirement in the Army, Navy, Marine Corps and Air Force and describes MDD comorbidity. Service members with a disability discharge for either MDD (n = 2,882) or any nonpsychiatric disability (n = 56,145), between fiscal years 2007 and 2012, were included in the study population. Those with MDD disability at first evaluation but not at last evaluation were excluded. The incidence of MDD disability discharge increased significantly in the Army and Air Force between fiscal years 2007 and 2012. MDD disability retirement significantly increased in the Army, Navy, and Air Force. Females, and those who experienced at least one deployment, had higher incidence rates of MDD disability discharge. All services included spinal diseases and posttraumatic stress disorder in their top five comorbid categories. Given the association between trauma and MDD, further research into the role of both combat exposure and injury on MDD is merited.

Preenlistment and Early Service Risk Factors for Traumatic Brain Injury in the Army and Marine Corps: FY 2002-2010.

OBJECTIVE: To determine the preenlistment and early service risk factors for traumatic brain injury (TBI)-related disability in Army and Marine Corps service members. DESIGN: Matched case-control design. MAIN OUTCOME: TBI disability discharges. SUBJECTS: Army and Marine Corps service members with an enlistment record and disability discharge for TBI were included as cases. Controls were selected from the enlisted population with no disability evaluation record and were matched on fiscal year of enlistment, sex, and service at a ratio of 5:1. RESULTS: Older age at enlistment resulted in a significantly increased risk for TBI disability in the crude and adjusted models (adjusted odds ratio [aOR] = 1.49; 95% confidence interval [CI], 1.16-1.91). An enlistment military occupational specialty (MOS) with a combat arms designation resulted in an almost 3-fold increased odds of TBI disability compared with other MOS categories (aOR = 2.75; 95% CI, 2.46-3.09). This remained a significant risk factor for TBI disability in the multivariate model (aOR = 2.74; 95% CI, 2.45-3.08). CONCLUSION: Results from this study help inform the existing body of military TBI research by highlighting the preenlistment demographic and early service risk factors for TBI disability. Further research into the role of age on TBI disability in the military is merited.

LC-MS-Based Metabolomics Discovers Purine Endogenous Associations with Low-Dose Salbutamol in Urine Collected for Antidoping Tests.

Current antidoping analytical methods are tailored mainly to the targeting of known drugs and endogenous molecules. This causes difficulties in rapidly reacting to emerging threats, such as designer drugs, biological therapeutic agents, and technologies. Biomarkers are considered as a promising approach for the fight against these threats to sport. The main purpose of this study was to find surrogate biomarkers induced by the intake of small amounts of the model compound salbutamol and explore a sensitive approach to help screen for possible drug misuse. Urine samples (91) from athletes with detectable salbutamol (30) and negative samples (61) were analyzed using a UHPLC-MS. A third group (30) was created by spiking salbutamol into negative samples to eliminate confounding effects. Data were then analyzed in XCMS to extract metabolic features. Orthogonal partial least squares-discriminant analysis was performed to select features correlated with detectable salbutamol (p(corr) > 0.5) and ROC analysis was performed to measure the predictive potential of the markers. Univariate analysis including Mann-Whitney U test and Spearman's correlation was conducted on selected markers. A total of 7000 metabolic features were parsed, one feature identified as hypoxanthine increased with salbutamol (p < 0.001). The ROC curve of hypoxanthine returned an AUC of 0.79 (p < 0.001). Correlation with salbutamol (r = 0.415, p < 0.01, Spearman's correlation) showed hypoxanthine and purine metabolism have association with salbutamol administration. This surrogate discovery approach needs further PK studies but in the meantime can be used as an intelligence-based complementary approach for targeting of athletes to be further tested.

Towards a blood-based diagnostic panel for bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a costly, devastating and life shortening mental disorder that is often misdiagnosed, especially on initial presentation. Misdiagnosis frequently results in ineffective treatment. We investigated the utility of a biomarker panel as a diagnostic test for BD. METHODS AND FINDINGS: We performed a meta-analysis of eight case-control studies to define a diagnostic biomarker panel for BD. After validating the panel on established BD patients, we applied it to undiagnosed BD patients. We analysed 249 BD, 122 pre-diagnostic BD, 75 pre-diagnostic schizophrenia and 90 first onset major depression disorder (MDD) patients and 371 controls. The biomarker panel was identified using ten-fold cross-validation with lasso regression applied to the 87 analytes available across the meta-analysis studies. We identified 20 protein analytes with excellent predictive performance [area under the curve (AUC)⩾0.90]. Importantly, the panel had a good predictive performance (AUC 0.84) to differentiate 12 misdiagnosed BD patients from 90 first onset MDD patients, and a fair to good predictive performance (AUC 0.79) to differentiate between 110 pre-diagnostic BD patients and 184 controls. We also demonstrated the disease specificity of the panel. CONCLUSIONS: An early and accurate diagnosis has the potential to delay or even prevent the onset of BD. This study demonstrates the potential utility of a biomarker panel as a diagnostic test for BD.

Statistical methodology for age-adjustment of the GH-2000 score detecting growth hormone misuse.

BACKGROUND: The GH-2000 score has been developed as a powerful and unique technique for the detection of growth hormone misuse by sportsmen and women. The score depends upon the measurement of two growth hormone (GH) sensitive markers, insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). With the collection and establishment of an increasingly large database it has become apparent that the score shows a positive age effect in the male athlete population, which could potentially place older male athletes at a disadvantage. METHODS: We have used results from residual analysis of the general linear model to show that the residual of the GH-2000 score when regressed on the mean-age centred age is an appropriate way to proceed to correct this bias. As six GH-2000 scores are possible depending on the assays used for determining IGF-I and P-III-NP, methodology had to be explored for including six different age effects into a unique residual. Meta-analytic techniques have been utilized to find a summary age effect. RESULTS: The age-adjusted GH-2000 score, a form of residual, has similar mean and variance as the original GH-2000 score and, hence, the developed decision limits show negligible change when compared to the decision limits based on the original score. We also show that any further scale-transformation will not change the adjusted score. Hence the suggested adjustment is optimal for the given data. The summary age effect is homogeneous across the six scores, and so the generic adjustment of the GH-2000 score formula is justified. CONCLUSIONS: A final revised GH-2000 score formula is provided which is independent of the age of the athlete under consideration.

Epidemiology of asthma-related disability in the U.S. Armed Forces: 2007-2012.

OBJECTIVE: To characterize the demographic, disability and deployment characteristics of U.S. Armed Forces personnel with an asthma-related disability discharge, which includes separation (without benefits) and retirement (with disability benefits). METHODS: Incidence rates for personnel evaluated for disability discharge and/or disability retired due to asthma and due to all other causes of disability discharge were calculated per 100,000 active duty enlisted service members by year. Multivariate logistical regression was used to examine the associations between disability retirement and several demographic and disability characteristics of service members evaluated for asthma-related disability discharge versus those evaluated for any other non-respiratory condition for each branch of military service. RESULTS: Service members evaluated for disability discharge related to asthma most often do not have comorbidity and are disability retired rather than separated, with rates of disability retirement increasing over time. Groups with a significantly higher incidence of evaluation for asthma-related disability include females, individuals who entered the military prior to the age of 20, non-Whites, and those with a history of deployment to Iraq or Afghanistan. The characteristic most associated with the odds of disability retirement was a history of deployment. CONCLUSIONS: New-onset asthma occurring after military entry often causes occupational impairment in service members, especially in those that have been deployed to Iraq or Afghanistan.