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Background: Sleep spindles may be implicated in sensing and regulation of peripheral glucose. Whether spindle density in patients with type 2 diabetes mellitus (T2DM) differs from that of healthy subjects is unknown., Methods: Our retrospective analysis of polysomnography (PSG) studies identified 952 patients with T2DM and 952 sex-, age- and BMI-matched control subjects. We extracted spindles from PSG electroencephalograms and used rank-based statistical methods to test for differences between subjects with and without diabetes. We also explored potential modifiers of spindle density differences. We replicated our analysis on independent data from the Sleep Heart Health Study., Results: We found that patients with T2DM exhibited about half the spindle density during sleep as matched controls (P < 0.0001). The replication dataset showed similar trends. The patient-minus-control paired difference in spindle density for pairs where the patient had major complications were larger than corresponding paired differences in pairs where the patient lacked major complications, despite both patient groups having significantly lower spindle density compared to their respective control subjects. Patients with a prescription for a glucagon-like peptide 1 receptor agonist had significantly higher spindle density than those without one (P ≤ 0.03). Spindle density in patients with T2DM monotonically decreased as their highest recorded HbA1C level increased (P ≤ 0.003)., Conclusions: T2DM patients had significantly lower spindle density than control subjects; the size of that difference was correlated with markers of disease severity (complications and glycemic control). These findings expand our understanding of the relationships between sleep and glucose regulation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Published by Elsevier Ltd.)

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) is one of the legacy separation tools developed by Dr. Andrew Alpert and has been used for developing unique separation methods of hydrophilic compounds, including peptides. In the past it has been studied using designed peptide libraries to elucidate major features of its separation mechanism, while comprehensive peptide retention modeling for ERLIC is still lacking. In this work we employed a proteomics-derived ∼170,000 peptide retention datasets to evaluate major ERLIC retention features using the framework of our Sequence-Specific Retention Calculator model. The separation conditions were adjusted to obtain a wider proteome coverage, particularly for non-modified peptides, resulting in a superior separation orthogonality for a 2D LC combination with reversed-phase C18 LC-MS in the second dimension. The SSRCalc ERLIC model presents a consistent theme with the existing ERLIC retention mechanism, reflecting a dependence on peptide orientation and the position of charged and hydrophilic residues across the peptide backbone. R 2 values of 0.935 and 0.955 accuracy were demonstrated for the standard interpretable SSRCalc model and machine learning algorithm, respectively. The effects of various PTMs on peptide retention were evaluated in this study, covering spontaneous (oxidation, deamidation) and enzymatic (N-terminal acetylation, phosphorylation, glycosylation) modifications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R 2 -value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the amino acids at the terminal positions of the peptide modulate the basicity of the N-terminal amino group or the C-terminal Arg/Lys for tryptic peptides. With respect to the separation orthogonality between HILIC and acidic pH RPLC for two dimensional separations, the orthogonality values were lower at pH 2.7 than operating HILIC at pH 4.5 for the first dimension. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Previous research has demonstrated that children lacking knowledge about genetic disorders may have harmful attitudes toward people with disabilities, but disability awareness can successfully modify these attitudes. We explored adolescents' implicit and explicit attitudes toward peers with genetic conditions to determine whether improved genetics/genomics literacy can mitigate the impact of ableism in this population., Methods: English-speaking adolescents (10-18 years) from British Columbia were invited to complete a Disability Attitudes Implicit Association Test (DA-IAT) and participate in a semi-structured focus group centering on a fictionalized vignette about an adolescent with Down syndrome. We used pragmatism as an analytical paradigm. Descriptive and inferential statistics were used to analyze DA-IAT and sociodemographic data; phronetic iterative analysis with constant comparison as a coding strategy for transcripts; and interpretive description to develop a conceptual model., Results: Twenty-two adolescents completed the DA-IAT and participated in one of four focus groups. Participants had a statistically significant implicit preference for non-disabled people (D-score = 0.72, SD = 0.44; t = 7.18, p < .00001). They demonstrated greater diversity in their explicit attitudes during the focus groups. Although participants articulated a positive attitude toward improved genetics education, results demonstrate their belief that social and personal interactions with disabled peers would be essential to address negative perceptions., Conclusions: This study lays important groundwork to understand, explain, and influence the negative attitudes of adolescents toward individuals with disabilities. Findings will be used to inform the design of interventions that address biased perceptions of people with genetic disorders, with the goal of reducing prejudices and improving social interactions., (© 2024 The Authors. Journal of Adolescence published by Wiley Periodicals LLC on behalf of Foundation for Professionals in Services to Adolescents.)

Sleep apnea is a common sleep disorder. The availability of an easy-to-use sleep apnea predictor would provide a public health benefit by promoting early diagnosis and treatment. Our goal was to develop a prediction tool that used commonly available variables and was accessible to the public through a web site. Using data from polysomnography (PSG) studies that measured the apnea-hypopnea index (AHI), we built a machine learning tool to predict the presence of moderate to severe obstructive sleep apnea (OSA) (defined as AHI ≥15). Our tool employs only seven widely available predictor variables: age, sex, weight, height, pulse oxygen saturation, heart rate and respiratory rate. As a preliminary step, we used 16,958 PSG studies to examine eight machine learning algorithms via five-fold cross validation and determined that XGBoost exhibited superior predictive performance. We then refined the XGBoost predictor by randomly partitioning the data into a training and a test set (13,566 and 3392 PSGs, respectively) and repeatedly subsampling from the training set to construct 1000 training subsets. We evaluated each of the resulting 1000 XGBoost models on the single set-aside test set. The resulting classification tool correctly identified 72.5 % of those with moderate to severe OSA as having the condition (sensitivity) and 62.8 % of those without moderate to-severe OSA as not having it (specificity); overall accuracy was 66 %. We developed a user-friendly publicly available website (https://manticore.niehs.nih.gov/OSApredictor). We hope that our easy-to-use tool will serve as a screening vehicle that enables more patients to be clinically diagnosed and treated for OSA., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Published by Elsevier Ltd.)

Purpose: To improve adolescent health measurement, the Global Action for the Measurement of Adolescent health (GAMA) Advisory Group was formed in 2018 and published a draft list of 52 indicators across six adolescent health domains in 2022. We describe the process and results of selecting the adolescent health indicators recommended by GAMA (hereafter, "GAMA-recommended indicators")., Methods: Each indicator in the draft list was assessed using the following inputs: (1) availability of data and stakeholders' perceptions on their relevance, acceptability, and feasibility across selected countries; (2) alignment with current measurement recommendations and practices; and (3) data in global databases. Topic-specific working groups comprised of GAMA experts and representatives of United Nations partner agencies reviewed results and provided preliminary recommendations, which were appraised by all GAMA members and finalized., Results: There are 47 GAMA-recommended indicators (36 core and 11 additional) for adolescent health measurement across six domains: policies, programs, and laws (4 indicators); systems performance and interventions (4); health determinants (7); health behaviors and risks (20); subjective well-being (2); and health outcomes and conditions (10)., Discussion: These indicators are the result of a robust and structured five-year process to identify a priority set of indicators with relevance to adolescent health globally. This inclusive and participatory approach incorporated inputs from a broad range of stakeholders, including adolescents and young people themselves. The GAMA-recommended indicators are now ready to be used to measure adolescent health at the country, regional, and global levels., (Copyright © 2024. Published by Elsevier Inc.)

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mutation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an additional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required., (© 2022 by The American Society of Hematology.)

Optimal health and development from preconception to adulthood are crucial for human flourishing and the formation of human capital. The Nurturing Care Framework, as adapted to age 20 years, conceptualises the major influences during periods of development from preconception, through pregnancy, childhood, and adolescence that affect human capital. In addition to mortality in children younger than 5 years, stillbirths and deaths in 5-19-year-olds are important to consider. The global rate of mortality in individuals younger than 20 years has declined substantially since 2000, yet in 2019 an estimated 8·6 million deaths occurred between 28 weeks of gestation and 20 years of age, with more than half of deaths, including stillbirths, occurring before 28 days of age. The 1000 days from conception to 2 years of age are especially influential for human capital. The prevalence of low birthweight is high in sub-Saharan Africa and even higher in south Asia. Growth faltering, especially from birth to 2 years, occurs in most world regions, whereas overweight increases in many regions from the preprimary school period through adolescence. Analyses of cohort data show that growth trajectories in early years of life are strong determinants of nutritional outcomes in adulthood. The accrual of knowledge and skills is affected by health, nutrition, and home resources in early childhood and by educational opportunities in older children and adolescents. Linear growth in the first 2 years of life better predicts intelligence quotients in adults than increases in height in older children and adolescents. Learning-adjusted years of schooling range from about 4 years in sub-Saharan Africa to about 11 years in high-income countries. Human capital depends on children and adolescents surviving, thriving, and learning until adulthood., Competing Interests: Declaration of interests REB serves on the Board of Directors of Vitamin Angels, a non-profit charitable organisation supporting maternal and child nutrition services in low-income and middle-income countries. ME reports a grant from AstraZeneca for the Young Health Programme, and personal fees from Prudential, outside the submitted work. REB, FV, LH, LL, ADS, DYo, and DYe report grants from the Bill & Melinda Gates Foundation. LH and DYo report grants from USAID, outside the submitted work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Development of a peptide retention prediction model in reversed-phase chromatography is reported for acetylated peptides - both N-terminal (α-) and side chain of Lys (ε-amine) residues. Large-scale proteomic 2D LC-MS analyses of acetylated/non-acetylated tryptic digest of whole human cell lysate have been used to assemble representative retention data sets of 25,000+ modified/non-modified pairs. This allowed elucidating chromatographic behaviour of modified peptides in three different separation modes: high pH reversed-phase, HILIC separation on amide phase (first dimension of 2D) and reversed-phase separation with formic acid as ion-pairing modifier in the second dimension. On average, N-terminal acetylation increases peptide RP retention at acidic pH by 5 Hydrophobicity Index units (% acetonitrile). Acetylation of first lysine adds another 4.1%. The magnitude of the retention shift varies greatly depending on the number of modified amines, peptide length, and N-terminal peptide sequence. Large retention shifts have been observed for peptides with hydrophobic N-termini and specifically peptides carrying sequences characteristic for amphipathic helical structures - all in complete agreement with major sequence-specific features of RP retention mechanism. The utility of the modified Sequence Specific Retention Calculator model has been verified for the in-vivo N-terminally acetylated peptides detected by 2D LC-MS/MS analysis of a yeast tryptic digest. The effect of N-terminal acetylation was also evaluated for six different HILIC columns, strong cation- and strong anion exchange separations using previously acquired 2D LC-MS/MS data., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Peptide retention time prediction models have been developed for zwitter-ionic ZIC-HILIC and ZIC-cHILIC stationary phases (pH 4.5 eluents) using proteomics-derived retention datasets of ~30 thousand tryptic peptides each. Overall, hydrophilicity of these stationary phases was found to be similar to the previously studied Amide HILIC phase, but lower compared to bare silicas. Peptide retention is driven by interactions of all charged (hydrophilic) residues at pH 4.5 (Asp, Glu, Arg, Lys, His), but shows specificity according to orientation of functional groups in zwitter-ionic pair. Thus, ZIC-cHILIC exhibits an increased contribution of negatively charged Asp and Glu due to the distal positioning of positively charged quaternary amines on the stationary phase. These findings confirm that HILIC interactions are driven by both peptide distribution between water layer adsorbed on the stationary phase and by interactions specific to functional groups of the packing material. Sequence-Specific Retention Calculator HILIC models were optimized for these columns showing 0.967-0.976 R 2 -values between experimental and predicted retention values. ZIC-HILIC separations represent a good choice as a first dimension in 2D LC-MS of peptide mixtures with correlations between retention values of ZIC-HILIC against RPLC found at 0.197 (ZIC-HILIC) and 0.137 (ZIC-cHILIC) R 2 -values, confirming a good orthogonality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

We report a novel somatic mutation in the kinase domain of JAK2 (R938Q) in a high-risk pediatric case of B-cell acute lymphoblastic leukemia (ALL). The patient developed on-therapy relapse at 12 months, and interestingly, the JAK2 locus acquired loss of heterozygosity during treatment resulting in 100% mutation load. Furthermore, we show that primary ALL mononuclear cells harboring the JAK2 R938Q mutation display reduced sensitivity to the JAK1/2 ATP-competitive inhibitor ruxolitinib in vitro, compared to ALL cells that carry a more common JAK2 pseudokinase domain mutation. Our findings are in line with previous reports that demonstrate that mutations within the kinase domain of JAK2 are associated with resistance to type I JAK inhibitors. Importantly, given the recent inclusion of ruxolitinib in trial protocols for children with JAK pathway alterations, we predict that inter-patient genetic variability may result in suboptimal responses to JAK inhibitor therapy in a subset of cases. The need for alternate targeted and/or combination therapies for patients who display inherent or developed resistance to JAK inhibitor therapy will be warranted, and we propose that kinase-mutants less sensitive to type I JAK inhibitors may present a currently unexplored platform for investigation of improved therapies., (Copyright © 2017. Published by Elsevier Inc.)

A comprehensive cross-platform and cross-assay evaluation using nine technology platforms and four cytokine immunoassays (IL-6, TNFα, IL-17a, IL-2) was performed by comparing assay precision, sensitivity, parallelism and data correlation between platforms. The precision was acceptable for most evaluated assays. In addition to comparing the analytical assay sensitivity using a spiked recombinant analyte in buffer, forty serum samples from both normal controls and multiple sclerosis patients were used to measure the frequency of endogenous analyte detection (FEAD) as a parameter of each assay's ability to detect the endogenous analyte. The highest FEAD measurements were observed on the Simoa™, Erenna®, Milliplex® and Imperacer® platforms. However, only Simoa and Erenna results showed a high correlation across all evaluated cytokine assays, followed by a more moderate correlation of results across platforms for the V-plex™, high sensitivity ELISA and the Ella™ IL-6 and TNFα assays. In contrast, results from the evaluated cytokine assays on the Milliplex, AMMP™ ViBE® and Imperacer platforms did not correlate to each other nor to other evaluated assays. Acceptable parallelism was observed for the Simoa, Erenna, V-plex and Ella assays but not for the Milliplex, AMMP ViBE and Imperacer assays. In conclusion, the Simoa, Erenna,V-plex and Ella platforms performed well in one or more evaluated cytokine assays. Among those, the Simoa and Erenna assays had the highest sensitivity for detection of cytokines present at sub-pg/mL levels in human serum. In addition, the cross-platform and cross-assay comparisons demonstrated that different immunoassays may yield different results, which underscores the importance of performing such comparative evaluations, especially in the absence of reliable reference standards for the quantitative assessments of biomarkers in immunoassays., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Little is known about the use and effectiveness of over-the-counter (OTC) fish oil supplements for triglyceride (TG) lowering., Objectives: To (1) perform a medication-use evaluation (MUE) and (2) assess the efficacy of OTC fish oil., Methods: Retrospective, observational cohort study using electronic medical records and the pharmacy database from Parkland Health and Hospital System in Dallas, Texas. Parkland is a tax-supported county institution that provides patients with single-brand OTC fish oil. Two separate analyses were conducted. Six hundred seventeen patients (prescribed fish oil between July 1, 2012, and August 31, 2012) were included in the MUE analysis and 235 patients (109 fish oil, 72 fenofibrate, and 54 gemfibrozil, prescribed between January 1, 2012, and July 31, 2013) were included in the efficacy analysis. The main outcome measure for the MUE was fish oil prescribing habits including dosages and patient adherence, as defined by medication possession ratio. The main outcome measure for the efficacy analysis was change in lipids measured using the last value before fish oil treatment and the first value after fish oil treatment., Results: MUE: 617 patients received prescriptions for OTC fish oil. Sixty-four percent were prescribed a total daily dose of 2000 mg. Only 25% of patients were adherent. Efficacy analysis: despite being prescribed suboptimal doses, fish oil reduced TGs by 29% (95% confidence interval, 34.3-22.7). Compared with fish oil therapy, fibrate therapy resulted in a greater TG reduction: 48.5% (55.1-41.0) with fenofibrate and 49.8% (57.6-40.5) with gemfibrozil (P < .0001, both medications compared with fish oil)., Conclusions: Health care providers prescribe suboptimal doses of fish oil, and adherence is poor. Even at low doses (2 g/d), though, fish oil lowers TGs by 29%., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screened positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The feasibility of performing diffusion-weighted MRI (DWI) of primary undifferentiated nasopharyngeal carcinoma (NPC) has not been assessed and it is unknown whether the apparent diffusion coefficients (ADC) of primary NPC differs from that of lymphoma or squamous cell carcinoma (SCC) in the head and neck. One hundred patients with newly diagnosed NPC, head and neck lymphoma or SCC underwent echo-planar DWI. ADCs of primary tumours were compared by Kruskal-Wallis test and Mann-Whitney U tests with Bonferroni correction using p<0.05 and p<0.017 respectively to indicate statistical significance. The utility of ADC thresholds for discriminating tumour histology was evaluated by receiver operating characteristic analysis. DWI was successful in 45/65 with NPC, 5/7 with lymphoma and 26/28 with SCC. Mean ADC (+/-SD) of NPC, lymphoma and SCC were 0.98+/-0.161, 0.75+/-0.190, 1.14+/-0.196 (x10(-3)mm(2)/s) respectively which were significantly different (p<0.001-0.003). Optimized ADC thresholds of 0.779, 0.768 and 1.07(x10(-3)mm(2)/s) achieved maximal discriminatory accuracies of 100%, 93% and 70% for SCC/lymphoma, NPC/lymphoma, and SCC/NPC respectively. Echo-planar DWI is a feasible technique for investigating primary NPC although limited in a third of patients due to susceptibility artifacts around the skull base. While the overall ADC value of NPC is significantly different to that of SCC and lymphoma, on a case by case basis overlapping ADCs between these tumours limit its theoretical utility at this site. Nevertheless, DWI may still be useful clinically to distinguish NPC from nasopharyngeal lymphoma in populations with endemic NPC due to the relative rarity of nasopharyngeal SCC., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Currently fielded treatments for nerve agent intoxication promote survival, but do not afford complete protection against either nerve agent-induced motor and cognitive deficits or neuronal pathology. The use of human plasma-derived butyrylcholinesterase (HuBuChE) to neutralize the toxic effects of nerve agents in vivo has been shown to both aid survival and protect against decreased cognitive function after nerve agent exposure. Recently, a commercially produced recombinant form of human butyrylcholinesterase (r-HuBuChE; PharmAthene Inc.) expressed in the milk of transgenic goats has become available. This material is biochemically similar to plasma-derived HuBuChE in in vitro assays. The pharmacokinetic characteristics of a polyethylene glycol coated (pegylated) form of r-HuBuChE were determined in guinea pigs; the enzyme was rapidly bioavailable with a half-life (t(1/2)) and pharmacokinetic profile that resembled that of plasma-derived huBuChE. Guinea pigs were injected with 140mg/kg (i.m.) of pegylated r-HuBuChE 18h prior to exposure (sc) to 5.5xLD(50) VX or soman. VX and soman were administered in a series of three injections of 1.5xLD(50), 2.0xLD(50), and 2.0xLD(50), respectively, with injections separated by 2h. Pretreatment with pegylated r-HuBuChE provided 100% survival against multiple lethal doses of VX and soman. Guinea pigs displayed no signs of nerve agent toxicity following exposure. Assessments of motor activity, coordination, and acquisition of spatial memory were performed for 2 weeks following nerve agent exposure. There were no measurable decreases in motor or cognitive function during this period. In contrast, animals receiving 1.5xLD(50) challenges of soman or VX and treated with standard atropine, 2-PAM, and diazepam therapy showed 50 and 100% survival, respectively, but exhibited marked decrements in motor function and, in the case of GD, impaired spatial memory acquisition. The advances in this field have resulted in the decision to select both the plasma-derived and the recombinant form of BuChE for advanced development and transition to clinical trials. Efforts have now been expanded to identify a catalytic protein capable of not only binding, but also rapidly hydrolyzing the standard threat nerve agents. Recent work has focused on paraoxonase-1 (PON1), a naturally occurring human serum enzyme with the capacity to catalyze the hydrolysis of nerve agents, albeit too slowly to afford dramatic protection. Using rational design, several amino acids involved in substrate binding have been identified and site-directed mutations have revealed that residue H115 plays an important role in binding. In addition, the stereospecificity of PON1 for the catalytic hydrolysis of soman has been examined. The enzyme exhibits a slight stereospecificity for the C+P+ isomer of soman, which is due more to preferential binding than to selective hydrolysis of this isomer. The results suggest that it may be possible to engineer a mutant form of PON1 with enhanced activity and stereospecificity for the most toxic nerve agent isoforms.

Flavonoids are polyphenolic compounds that are widespread in the plant kingdom, and structure-activity relationships (SAR) for vascular relaxation effects were examined for 17 of them using porcine coronary arteries. Density functional theory was employed to calculate the chemical parameters of these compounds. The order of potency for vascular relaxation was as follows: flavones (apigenin and luteolin) >or= flavonols (kaempferol and quercetin)>isoflavones (genistein and daidzein)>flavanon(ol)es (naringenin)>chalcones (phloretin)>anthocyanidins (pelargonidin)>flavan(ol)es ((+)-catechin and (-)-epicatechin). SAR analysis revealed that for good relaxation activity, the 5-OH, 7-OH, 4'-OH, C2=C3 and C4=O functionalities were essential. Comparison of rutin with quercetin, genistin with genistein, and puerarin with daidzein demonstrated that the presence of a glycosylation group greatly reduced relaxation effect. Total energy and molecular volume were also predictive of their relaxation activities. Our findings indicated that the most effective relaxing agents are apigenin, luteolin, kaempferol and genistein. These flavonoids possess the key chemical structures demonstrated in our SAR analysis.

Background: Priming with autologous tumor vaccine followed by ex vivo expansion of activated T cells is a feasible experimental strategy. This paper describes the application of this cellular therapy to treat patients with late-stage non-small-cell lung cancer (NSCLC)., Methods: Twenty-one patients with Stages III and IV NSCLC were treated. Tumor samples were obtained surgically (five patients) or by using aco-axial biopsy needle (16 patients). Each course of vaccination consisted of irradiated tumor cells, mixed with GM-CSF and injected intradermally on Day 1, followed by GM-CSF only on Days 2- 5. The course was repeated 10-14 days later. Lymphocytes were collected 10-14 day after the second course and ex vivo expanded using IL-2 and anti-CD3 Ab. The expansion products were then re-infused into the patients., Results: Twelve out of 16 biopsies resulted in optimal cell numbers for vaccine preparation. Nineteen out of 21 patients achieved a delayed type hypersensitivity (DTH response after two courses of vaccination. In 18/21 patients, the ex vivo expansion products contained > 1.6 x 10(10) cells. Subset analysis showed 77.0-97.2% T cells with a CD4:CD8 ratio of 0.65-4.0; natural killer cells were 2.0-18.6%. There were no significant toxicities. The median survival of all 21 patients was 18.6 months, with a 1-year survival of 51.6%., Conclusion: Autologous tumor cell-vaccination may be combined with ex vivo expansion of lymphocytes as adoptive cellular immunotherapy for advanced NSCLC. Overall survival in this cohort of poor prognosis patients compared favorably with results reported in the literature.

The heptapeptide toxins produced by the blue-green alga (cyanobacterium) Microcystis aeruginosa are selectively hepatotoxic in mammals. The characteristic post-mortem pathology of the liver is extensive lobular disruption due to sinusoidal breakdown, leakage of blood into the tissue and hepatocyte disintegration. Isolated hepatocytes incubated with toxin show severe structural deformity and surface blebbing. This paper demonstrates the effects of Microcystis toxins on the contraction and aggregation of actin microfilaments, and on the relocation and breakdown of cytokeratin intermediate filaments, in cultured hepatocytes. Earlier work did not show changes in the assembly/disassembly of actin; however, this paper demonstrates the change in cytokeratin from intermediate filaments to distributed granules in the cytoplasm of toxin-affected cells. Acrylamide gel electrophoresis of cytoskeletal fractions from hepatocytes did not show changes in total cytokeratins; however, marked changes in the immunogenicity of cytokeratins at 52 and 58 kDa were seen on toxin exposure of cells. Measurement of 32P-phosphorylation of proteins in toxin-affected cells incubated with [32P]orthophosphate showed a dramatic increase compared to control incubations. This is in agreement with research elsewhere describing phosphatase inhibition in vitro by Microcystis toxins. The data indicate that phosphorylated cytokeratin is a major component of cytoplasmic fraction phosphorylated protein after toxin exposure to hepatocytes. It is concluded that the mechanism of Microcystis toxicity to the hepatocyte is through cytoskeletal damage leading to loss of cell morphology, cell to cell adhesion and finally cellular necrosis. The underlying biochemical lesion is likely to be phosphatase inhibition causing hyperphosphorylation of a number of hepatocyte proteins, including those cytokeratins responsible for microfilament orientation and intermediate filament integrity.

The daily sodium content in the diets of infants was determined. Milk and milk products were the main contributor of sodium during early infancy. Commercial baby foods contributed little sodium. As table foods were introduced, the daily sodium intake of the infants rose dramatically. The main sources of sodium in table foods were salt-containing canned foods and added table salt.

In recent years, bottle feeding and the early introduction of solid foods in infants' diets have been proposed as factors contributing to overweight in infancy that might continue into childhood. These hypotheses were examined in a longitudinal survey of 403 infants from birth to 18 months of age. Results from this survey do not support either (a) a relationship between type of milk feeding or time of introduction of solid food and fatness at 6 months of age or (b) the hypothesis that fat infants remain fat.

Fasting blood samples were taken from 13 college students who had never been on oral contraceptives in two menstrual cycles. During the first cycle, the control cycle, each girl donated three blood samples; the first sample was given between days 1 and 5, the second sample between days 13 and 17, and the third sample between days 22 and 26 of the menstrual cycle. In the second menstrual cycle, the experimental cycle, nine girls were given Micronor, a progestogen type oral contraceptive and four girls were given Ortho-Novum SQ, a sequential type oral contraceptive. Four blood samples were obtained from each of the subjects: the first three samples were obtained in the three periods corresponding to those in the control cycle, and the fourth was taken 2 days after the subjects had stopped taking the oral contraceptive. Results showed that estrogen significantly raised plasma vitamin A and triglycerides. The progestogen, at low concentration, had little or no effect on these two lipid materials. At a higher concentration the progestogen enhanced the effect of the estrogen on plasma vitamin A and triglycerides. These effects were extended to at least 2 days after subjects had ceased taking the oral contraceptive. Plasma vitamin E levels in the subjects given Ortho-Novum SQ, were consistently but not statistically higher in the experimental cycle than in the control cycle. The correlation coefficient between vitamin E and triglycerides was statistically significant while those between the vitamin A and vitamin E and between vitamin A and triglycerides were not. Cholesterol was not affected by either Micronor or Ortho-Novum SQ.

Between 1977 and 1979, the H.J. Heinz Company of Canada Ltd conducted a longitudinal survey of infant nutrition from birth to 18 months. In 1980, 189 of the original 403 infants were recruited in a follow-up study. The age of the children in the follow-up study was 3.5 to 4 years. This article describes the food and nutrient intakes of the preschool children. Food intake information was obtained by the 4-day dietary record method. The mean energy intake of the children was adequate to support normal growth. The mean nutrient intakes were above the recommended daily intakes of the Canadian Dietary Standard. All children ate three regular meals and at least one in-between meal snack. Milk was the primary source of energy, protein, fat, calcium, phosphorus, sodium, potassium, and riboflavin. Meat contributed a substantial amount of protein, fat, and niacin. Cereal and cereal products were the primary source of iron and were good sources of carbohydrates, sodium, and the B vitamins. Fruit and fruit juices were the primary sources of carbohydrate and vitamin C. Vitamin supplements provided large amounts of vitamin A and the B vitamins. Since foods alone included sufficient nutrients to provide the recommended daily nutrient intakes, vitamin supplements were largely unnecessary.

Plasma vitamins A, E, and C, plasma triglycerides and cholesterol, and leukocyte vitamin C were examined in young healthy adult females who were cigarette and/or oral contraceptive users. It was found that cigarette smoking slightly increased the levels of vitamin A, triglycerides, and cholesterol while oral contraceptives significantly increased these plasma lipids. The effects of cigarette smoking and oral contraceptives on these substances were additive. Neither cigarette smoking nor oral contraceptives had any significant effect on plasma vitamins E and C. Oral contraceptives slightly decreased the level of leukocyte vitamin C in the cigarette smokers. Cigarette smoking did not impart and acute effect on these parameters.