A liquid chromatography tandem mass spectrometry method for plasma glucagon.

Glucagon is a peptide involved in controlling the body's blood glucose levels. The majority of analytical methods used for its quantitation are based on immunoassays that suffer from cross-reactivity with other peptides. For accurate routine analysis a liquid chromatography tandem mass spectrometry (LC-MSMS) was developed. Glucagon was extracted from plasma samples by a combination of protein precipitation using ethanol and mixed anion exchange solid phase extraction. Linearity for glucagon was above 0.99 (r2) up to a concentration range of 771 ng/L with a lower limit of quantification established at 19 ng/L. Precision of the method was below 9% (coefficient of variation). Recovery was 93%. Correlations with the existing immunoassay displayed a significant negative bias.

Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma.

Asymmetric dimethylguanidino valeric acid (ADGV), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are three arginine metabolites which have utility in the assessment of cardiovascular disease, renal disease and non-alcoholic fatty liver disease (NAFLD). Translation of these research metabolomic markers into routine clinical use requires the development of robust assays with appropriately assessed preanalytical variables and traceable clinical reference intervals. A hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADGV, ADMA and SDMA was developed. Sample stability and collection conditions were scrutinised to determine any preanalytical factors that could affect quantification under routine laboratory conditions. Patient samples from 120 males and 120 females were used to derive preliminary reference intervals. All three analytes were quantifiable in human plasma using unique MS/MS transitions. The analytes were stable for up to a week once separated from red cells, though reduced stability was observed upon extraction of the analytes from plasma. The assay was linear for concentration of ADGV between 1.6 nmol/L and 200 nmol/L and for ADMA and SDMA between 0.1 µmol/L and 4.0 µmol/L. The accuracy for all analytes was 97-103% and interday and intraday imprecisions (coefficients of variation) were less than 10%. ADGV concentrations were noted to be lower in the female reference population when compared to males. The analytical method shows excellent performance and is sufficiently robust to be used in the clinical investigation of cardiovascular disease and NAFLD.

Novel insights from a multiomics dissection of the Hayflick limit.

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

Fully Phased Sequence of a Diploid Human Genome Determined de Novo from the DNA of a Single Individual.

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood.

BACKGROUND AND AIMS: The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection. METHODS: Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76). RESULTS: Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method. CONCLUSIONS: Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

eIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.

Variations in measurement of sexual activity based on EHR definitions.

OBJECTIVE: The goal of this study was to compare the performance of 4 operational definitions of sexual activity by using data electronically abstracted from electronic health records (EHRs) and examine how documentation of Chlamydia screening and positivity vary according to definition of sexual activity. METHODS: Extracts were created from EHRs of adolescent females 12 to 19 years old who had ≥1 visit to a primary care practice during 2011 at 4 US pediatric health care organizations. We created 4 definitions of sexual activity derived from electronically abstracted indicator variables. Percent sexually active, documentation of Chlamydia screening, and rate of positive Chlamydia test results per 1000 adolescent females according to the sexual activity definition were calculated. RESULTS: The most commonly documented individual indicator of sexual activity was "patient report of being sexually active" (mean across 4 sites: 19.2%). The percentage of adolescent females classified as sexually active varied by site and increased as more indicator variables were included. As the definition of sexual activity expanded, the percentage of sexually active females who received at least 1 Chlamydia test decreased. Using a broader definition of sexual activity resulted in improved identification of adolescent females with Chlamydia infection. For each sexual activity definition and performance item, the difference was statistically significant (P < .0001). CONCLUSIONS: Information about sexual activity may be gathered from a variety of data sources, and changing the configurations of these indicators results in differences in the percentage of adolescent females classified as sexually active, screened for Chlamydia infection, and Chlamydia infection rates.

An electronic diary is shown to be more reliable than a paper diary: results from a randomized crossover study in patients with persistent asthma.

OBJECTIVES: Test-retest reliability of an asthma paper diary versus an electronic diary (e-diary) with an integrated peak flow meter was investigated. The equivalence of the two modes was also evaluated. METHODS: Prospective, randomized crossover study design in adolescents (12-17 years) and adults (≥18 years). Key inclusion criteria were persistent asthma, Asthma Control Test (ACT) scores ≥16, use of inhaled corticosteroid with or without long-acting beta-agonist for ≥12 weeks, nocturnal awakenings <2 times in the past week, and activity limitations <1 per week. Participants were randomized to either paper then e-diary or e-diary then paper, to be completed for 14 days each. RESULTS: Forty-seven participants completed all study visits. Weekly percentage of symptom-free days (SFDs) and rescue-free days (RFDs) were calculated. Intraclass correlation coefficients (ICCs) of Week 1 mean SFD and RFD (test) and Week 2 mean SFD and RFD (retest), respectively, were estimated in three groups defined as stable: (i) minimal changes in asthma symptoms, as measured by the global patient reported symptom change question, (ii) less than 15% change (absolute value) in 1 second FEV(1) at adjacent study visits, and (iii) changes in ACT scores less than three points for each mode. SFD demonstrated acceptable ICC (≥0.70) using all three definitions of asthma stability for both modes. CONCLUSION: Acceptable reproducibility of the percentage of RFD (ICC = 0.78) was only observed for the e-diary using the FEV(1) stability criterion. The ICCs for SFD and RFD were acceptable, 0.84 and 0.70, respectively, suggesting better reliability for the e-diary.

Moving towards a comprehensive assessment of lower urinary tract symptoms (LUTS).

AIMS: To evaluate the utility of the International Prostate Symptom Score (IPSS) and the LUTS Tool when assessing lower urinary tract symptoms (LUTS). Secondary objectives were to examine associations of LUTS and treatment seeking. METHODS: Analyses were performed using EpiLUTS data, a population-based, cross-sectional, Internet survey of men and women (aged ≥40) in Sweden, UK, and US with a sample of 30,000 participants. Participants completed the IPSS and the LUTS Tool. Prevalence rates of symptoms captured by the LUTS Tool were compared using IPSS summary scores: 0, 1-7, 8-19, and ≥20. LUTS Tool subscale scores were calculated. Pearson correlations between the LUTS Tool symptoms and subscales and IPSS symptoms were performed. Logistic regressions evaluated the associations of IPSS scores and LUTS Tool subscales with treatment seeking for LUTS. RESULTS: The IPSS did not assess some symptoms (i.e., incontinence) for which there was a high prevalence among participants. Correlations between the 7 symptoms assessed by the IPSS and LUTS Tool were moderate to high ranging between 0.37 (nocturia women) and 0.77 (weak stream men), indicating concordance. While the LUTS Tool subscales and the total IPSS score were all significantly associated with treatment seeking, the LUTS Tool OAB subscale in men and Voiding subscale in women most strongly predicted treatment seeking. CONCLUSION: LUTS that are excluded from the IPSS, most notably incontinence, were prevalent even among mildly symptomatic participants. Since storage symptoms appear to drive treatment seeking, identifying, and treating these symptoms is essential when caring for patients with LUTS.

Urinary incontinence and its relationship to mental health and health-related quality of life in men and women in Sweden, the United Kingdom, and the United States.

BACKGROUND: Differences in health burden associated with urinary incontinence (UI) subtypes have been previously described, but the majority of studies are in women. Additional research is needed to examine the prevalence and burden of UI subtype including postmicturition incontinence, nocturnal enuresis, coital incontinence, and incontinence for unspecified reasons. OBJECTIVE: Examine the burden of UI in men and women in Sweden, the United Kingdom, and the United States. DESIGN, SETTING, AND PARTICIPANTS: Secondary analyses of the Epidemiology of Lower Urinary Tract Symptoms (EpiLUTS), a cross-sectional Internet survey, were performed. Participants who reported UI were categorized as (1) urgency urinary incontinence (UUI) only, (2) stress urinary incontinence (SUI) only, (3) mixed urinary incontinence (MUI), (4) UUI plus other incontinence (OI), (5) SUI plus OI, or (6) OI. Differences in health outcomes across UI groups were explored by gender using descriptive statistics and general linear models. MEASUREMENTS: Outcomes included treatment seeking for urinary symptoms, perception of bladder condition, depression, anxiety, and health-related quality of life (HRQL). RESULTS AND LIMITATIONS: Of 14 140 men and 15 860 women, 6479 men (45.8%) and 10 717 women (67.6%) reported UI. The most prevalent UI subgroups were OI in men and SUI in women. MUI and SUI plus OI had the greatest treatment seeking among men, whereas MUI and UUI plus OI had the greatest treatment seeking among women. Men with MUI had the highest rates of anxiety, followed by those with UUI plus OI and SUI plus OI, and OI with a similar trend observed for depression. Anxiety and depression were highest in SUI plus OI and MUI women. MUI and UUI plus OI men and women had significantly lower HRQL compared with other UI groups. CONCLUSIONS: UI is common in men and women aged >40. Individuals with UUI combined with SUI or OI bear a greater mental health burden and report poorer HRQL.

Validation of the AQLQ12+ among adolescents and adults with persistent asthma.

BACKGROUND: To date, there is limited psychometric evidence on the Asthma Quality of Life Questionnaire (AQLQ12 +) among populations that include adolescents and adults. OBJECTIVE: To provide evidence of the psychometric properties of the AQLQ12+ as a measure of asthma-specific quality of life (QOL) in patients with persistent asthma treated with a combination of inhaled glucocorticoid and long-acting beta2-agonist, as well as explore the predictors of at least a minimally important AQLQ12+ improvement. METHODS: The psychometric properties of the AQLQ12+ were assessed through post hoc analysis of two large (n = 740 and 778) Phase III, randomized, double-blinded, placebo-controlled efficacy studies of mometasone furoate/formoterol fumarate (MF/F) combination compared with monotherapy in subjects with persistent asthma previously treated with either low-dose or medium-dose inhaled glucocorticoids. RESULTS: With 15% and 8% participation from 12- to 17-year olds, blinded trial data demonstrated excellent reproducibility (ICC range: 0.76-0.85) and moderate-to-strong construct validity with other measures of asthma health at baseline and over time for the AQLQ12 +. A greater percentage of the MF/F treatment group (44%) achieved an important change at 26 weeks on the AQLQ12+ compared with formoterol fumarate (F, 23%) and placebo (18%) treatment groups in the low-dose study (P < 0.001) and the medium-dose study (50% (MF/F) versus 34% (F) and 23% (placebo); P < 0.001). Pre-randomization nighttime awakenings and rescue medications use were significant predictors of AQLQ12+ improvement. CONCLUSIONS: These findings provide strong support for the measurement properties of the AQLQ12+ among patients with persistent asthma and confidence in the AQLQ12+ improvements demonstrated by the MF/F treatment group.

An assessment of change in asthma control among adolescents and adults with persistent asthma in mometasone furoate/formoterol fumarate clinical trials.

OBJECTIVE: This article provides evidence on the psychometric properties of the Asthma Control Questionnaire (ACQ) in adolescent and adult patients with persistent asthma treated with a combination of inhaled glucocorticoid and long-acting beta2-agonist (LABAs), and explores the factors associated with important improvements in asthma control. METHODS: Data from patients in two large (n = 737 and 772) Phase III, randomized, double-blind, parallel-group, multi-center, placebo-controlled studies of mometasone furoate/formoterol fumarate (MF/F) combination formulation compared with monotherapies in subjects with persistent asthma previously treated with either low- or medium-dose inhaled glucocorticoids were used to evaluate the ACQ psychometric properties and predictors of important ACQ improvement, defined as an ACQ score decline from baseline of 0.5 or more at the end of treatment. RESULTS: With 15% and 8% participation from adolescents in the low- and medium-dose studies, the ACQ yielded acceptable reliability (intraclass correlation coefficient ≥ 0.75), and baseline and change scores demonstrated moderate to strong correlations with other baseline measures and change scores in other measures of asthma-related health, including the Asthma Quality of Life Questionnaire (AQLQ12+) domains and total scores. More MF/F treatment group patients (48%) achieved an important ACQ change at 26 weeks compared with MF (32%), F (26%), and placebo (18%) treatment groups (p < .001). Use of rescue medications before randomization was a significant predictor of important ACQ improvement in both studies. CONCLUSIONS: These findings support the psychometric properties of the ACQ to measure asthma control among persistent asthma patients and provide confidence in the significant improvements in asthma control demonstrated by the MF/F treatment group.

Natural variation in Drosophila stressed locomotion meets or exceeds variation caused by hsp70 mutation: analysis of behavior and performance.

Thermotolerance involves more than life or death. Investigating the complexity of this trait will aid identification of its genetic contributors. We examined variation in thermally stressed walking behavior and performance in natural Drosophila melanogaster strains and strains mutant for the heat shock protein Hsp70, to determine which aspects of locomotion are affected by heat shock and genotype. We developed software for the large-scale capture, analysis, and visualization of locomotion, and determined: (1) Heat shock and thermal pretreatment significantly and differentially impact fly locomotor behavior and performance. (2) Stressed locomotion traits vary extensively among natural strains. (3) Interactions among treatments, strains, and traits are substantial and often counterintuitive. (4) Hsp70 overexpressing flies are faster and more basally thermoprotected in performance than Hsp70 null flies, but null flies are more unidirectional. (5) Natural variation in most stressed locomotion traits exceeds that caused by Hsp70 mutation, reveals uncoupling between thermoprotection of behavior and performance, and suggests significant genetic variation for trait-specific modifiers of thermotolerance.