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Thomas Leth Jensen,1 Andreas Brønden,1 Kristian Karstoft,1,2 David Peick Sonne,1,2 Mikkel Bring Christensen1– 3 1Department of Clinical Pharmacology, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, DK-2400, Denmark; 2Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DK-2200, Denmark; 3Center for Translational Research, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, DK-2400, DenmarkCorrespondence: Mikkel Bring Christensen, Email Mikkel.bring.christensen@regionh.dkAbstract: Obesity is becoming more frequent and has several negative health impacts. Recent advances in weight management strategies have primarily resided in pharmaceutical treatments, and the glucagon-like peptide-1 (GLP-1) receptor agonists have shown great potential in terms of body weight reduction in addition to improving glycemic control in patients with type 2 diabetes (T2D). Recently, the dual GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist tirzepatide has been developed. Tirzepatide has shown strong effects on glycated hemoglobin (HbA1C) levels in several clinical trials including participants with T2D (SURPASS program). In addition to its lowering effect on HbA1C, tirzepatide leads to substantial reductions in body weight, and a series of clinical trials (SURMOUNT program) have investigated the effects on body weight as the primary outcome. In these two trial programs, tirzepatide in doses of 5 mg to 15 mg administered subcutaneously once weekly resulted in body weight reduction of up to 15% in participants with T2D and up to 21% in participants without T2D, despite comparable baseline bodyweight. Across the two trial programs, adverse effects were mainly gastrointestinal (nausea, diarrhea, and vomiting) occurring with similar incidences of vomiting and lower incidences of diarrhea and nausea in trial participants with T2D compared to trials participants without T2D. Overall, discontinuation due to adverse events occurred in 3– 7% of participants with no major differences between individuals with and without T2D. The higher weight-reducing efficacy of tirzepatide in trial participants without T2D is currently unexplained and may be partly reflected in dissimilarities in frequencies of gastrointestinal adverse events. The weight reducing effects of tirzepatide hold great promise for weight management in obese patients regardless of the presence of T2D.Keywords: tirzepatide, weight loss, type 2 diabetes, GLP-1, GIP, dual agonists, safety

Purpose: The Danish National Hospital Medicine Register (SMR) was established in 2018 to centralize and standardize medication use data across Danish hospitals. This task was previously managed by individual hospital registration systems across the five regions. This initiative addresses the need for a unified, detailed understanding of hospital medication use to monitor healthcare delivery, improve patient outcomes, and support research., Methods: The SMR has comprehensive coverage of medication use in hospitals in Denmark, offering a national overview that was previously missing. It features high-quality data, with efforts to ensure completeness and accuracy. The data collected encompasses key categories such as types of medications, doses, administration times and practices, and specific treatment indications. The SMR facilitates collaboration among Danish regional health authorities and national health agencies, enhancing decision-making and planning across the regions., Results: The establishment of the SMR has provided a centralized and standardized database for medication use across Danish hospitals. This unification replaces the previously fragmented systems, allowing for better monitoring of healthcare delivery and supporting improvements in patient outcomes and research., Conclusion: The upcoming version of the SMR will include data on dispensed outpatient medications, covering most medication use across all hospitals. This expansion will further enhance the register's utility for health authorities, clinicians, and researchers by providing a more comprehensive understanding of medication use in Denmark., (© 2024 The Author(s). Pharmacoepidemiology and Drug Safety published by John Wiley & Sons Ltd.)

Background: Avian influenza viruses pose significant risk to human health. Vaccines targeting the hemagglutinin of these viruses are poorly immunogenic without the use of adjuvants., Methods: Twenty healthy men and women (18-49 years of age) were randomized to receive 2 doses of inactivated influenza A/H5N1 vaccine alone (IIV) or with AS03 adjuvant (IIV-AS03) 1 month apart. Urine and serum samples were collected on day 0 and on days 1, 3, and 7 following first vaccination and subjected to metabolomics analyses to identify metabolites, metabolic pathways, and metabolite clusters associated with immunization., Results: Seventy-three differentially abundant (DA) serum and 88 urine metabolites were identified for any postvaccination day comparison. Pathway analysis revealed enrichment of tryptophan, tyrosine, and nicotinate metabolism in urine and serum among IIV-AS03 recipients. Increased urine abundance of 4-vinylphenol sulfate on day 1 was associated with serologic response based on hemagglutination inhibition responses. In addition, 9 DA urine metabolites were identified in participants with malaise compared to those without., Conclusions: Our findings suggest that tryptophan, tyrosine, and nicotinate metabolism are upregulated among IIV-AS03 recipients compared with IIV alone. Metabolites within these pathways may serve as measures of immunogenicity and may provide mechanistic insights for adjuvanted vaccines., Clinical Trials Registration: NCT01573312., Competing Interests: Potential conflicts of interest. C. B. C. reports consulting fees from GSK, Pfizer, TD Cowen, Moderna, CommenseBio, and AstraZeneca for unrelated work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This paper presents quasiexperimental evidence of Covid-19 transmission through casual contact between customers in retail stores. For a large sample of individuals in Denmark, we match card payment data, indicating exactly where and when each individual made purchases, with Covid-19 test data, indicating when each individual was tested and whether the test was positive. The resulting dataset identifies more than 100,000 instances where an infected individual made a purchase in a store and, in each instance, allows us to track the infection dynamics of other individuals who made purchases in the same store around the same time. We estimate transmissions by comparing the infection rate of exposed customers, who made a purchase within 5 min of an infected individual, and nonexposed customers, who made a purchase in the same store 16 to 30 min before. We find that exposure to an infected individual in a store increases the infection rate by around 0.12 percentage points ( P < 0.001) between day 3 and day 7 after exposure. The estimates imply that transmissions in stores contributed around 0.04 to the reproduction number for the average infected individual and significantly more in the period where Omicron was the dominant variant., Competing Interests: Competing interests statement:The authors declare no competing interest.

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34 + hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies., Competing Interests: R.T.G., T.W., and A.V. are inventors on a patent that has been filed by the Technical University of Denmark (DTU) and has been licensed to Artisan Bio (patent #WO2023137233A2, published 2023-07-20, based on the U.S. Provisional Patent Application No. 63/300,244, filed January 17, 2022). R.T.G., T.W., A.B., and R.F.B. have financial interests in Artisan Bio. L.K.N. is a member of the advisory board of Artisan Bio., (© 2023.)

Objective: Poisonings contribute significantly to morbidity and mortality of patients. Some patients have numerous contacts to a poison information center, indicating repeated poisoning exposures. Information on the involved substances is necessary to explore methods to prevent self-harm and reduce mortality. The objective of this study was to characterize the patient population with repeated poison exposures in Denmark and identify the substances involved., Methods: This study was a retrospective cohort study of enquiries to the nationwide Danish Poison Information Centre and the Danish National Patient Registry. The databases were used to identify patients with more than five individual poisoning episodes within a 12-month-period between 1 January 2013, and 31 December 2017., Results: One hundred and thirty-seven patients and 995 patients met the inclusion criteria in the Danish Poison Information Centre and the Danish National Patient Registry, respectively. The majority were women (82.5% and 66.3% for the Danish Poison Information Centre and the Danish National Patient Registry cohorts, respectively). The mean age was 24.7 and 29.5 years. Psychiatric comorbidities were frequent with 74.5% and 67.0% suffering from personality disorders and 70.1% and 54.5% from affective disorders in the Danish Poison Information Centre and the Danish National Patient Registry cohorts, respectively. One thousand seven hundred and fifty-two poisoning episodes were identified in the Danish Poison Information Centre database, and the most common types of substance were 'pharmaceuticals' (1,420 episodes). The most common medications ingested were quetiapine, paracetamol and cyclizine. Median number of contacts to the Danish Poison Information Centre was 10. Patients with one or more poisoning episodes involving cyclizine had on average 11.4 poisoning episodes involving cyclizine. In the Danish National Patient Registry cohort 80.9% were alive after 10 years compared to 97.7% in the background population., Conclusion and Implications: Most poisonings were intentional and occurred among younger women. Psychiatric comorbidity was frequent. Most often, pharmaceuticals were the toxic substance, mainly quetiapine, paracetamol and cyclizine. Changing the status of cyclizine from over the counter to prescription only medication, and implementing stricter rules for prescribing quetiapine, could limit future poisoning incidences.

Managing critically ill children is a rare and challenging event requiring training to ensure adequate and timely quality of care. Thus, health professionals train for pediatric emergencies in a simulated setting. Virtual reality (VR) is a promising modality for simulation, and current evidence highlights the potential of VR for simulating pediatric emergencies. However, more studies are needed to determine the aspects of VR design and implementation that support transfer of learning.

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial., Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths., Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies., Competing Interests: JG, TLJ, TG, CEG, KJS were employed by The Emmes Company. EA has consulted for Pfizer, Sanofi Pasteur, GSK, Janssen, Moderna, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc. and Sanofi Pasteur. He serves on a data adjudication board for WCG and ACI Clinical. His institution has also received funding from NIH to conduct clinical trials of COVID-19 vaccines. MM reported potential competing interests: laboratory and clinical trial contract funding for vaccines or MAB vs SARS-CoV-2 with Lilly, Pfizer, and Sanofi; personal fees for Scientific Advisory Board service from Merck, Meissa Vaccines, Inc., and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MG declared a shared affiliation with the author RH to the handling editor at the time of review., (Copyright © 2023 Goll, Bosinger, Jensen, Walum, Grimes, Tharp, Natrajan, Blazevic, Head, Gelber, Steenbergen, Patel, Sanz, Rouphael, Anderson, Mulligan and Hoft.)

Background: Parent of origin-specific allelic expression of imprinted genes is epigenetically controlled. In cancer, imprinted genes undergo both genomic and epigenomic alterations, including frequent copy number changes. We investigated whether copy number loss or gain of imprinted genes in cancer cell lines is associated with response to chemotherapy treatment., Results: We analyzed 198 human imprinted genes including protein-coding genes and noncoding RNA genes using data from tumor cell lines from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We examined whether copy number of the imprinted genes in 35 different genome locations was associated with response to cancer drug treatment. We also analyzed associations of pretreatment expression and DNA methylation of imprinted genes with drug response. Higher copy number of BLCAP, GNAS, NNAT, GNAS-AS1, HM13, MIR296, MIR298, and PSIMCT-1 in the chromosomal region 20q11-q13.32 was associated with resistance to multiple antitumor agents. Increased expression of BLCAP and HM13 was also associated with drug resistance, whereas higher methylation of gene regions of BLCAP, NNAT, SGK2, and GNAS was associated with drug sensitivity. While expression and methylation of imprinted genes in several other chromosomal regions was also associated with drug response and many imprinted genes in different chromosomal locations showed a considerable copy number variation, only imprinted genes at 20q11-q13.32 had a consistent association of their copy number with drug response. Copy number values among the imprinted genes in the 20q11-q13.32 region were strongly correlated. They were also correlated with the copy number of cancer-related non-imprinted genes MYBL2, AURKA, and ZNF217 in that chromosomal region. Expression of genes at 20q11-q13.32 was associated with ex vivo drug response in primary tumor samples from the Beat AML 1.0 acute myeloid leukemia patient cohort. Association of the increased copy number of the 20q11-q13.32 region with drug resistance may be complex and could involve multiple genes., Conclusions: Copy number of imprinted and non-imprinted genes in the chromosomal region 20q11-q13.32 was associated with cancer drug resistance. The genes in this chromosomal region may have a modulating effect on tumor response to chemotherapy., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers., (© 2022. The Author(s).)

Ribosomopathies constitute a range of disorders associated with defective protein synthesis mainly affecting hematopoietic stem cells (HSCs) and erythroid development. Here, we demonstrate that deletion of poly-pyrimidine-tract-binding protein 1 (PTBP1) in the hematopoietic compartment leads to the development of a ribosomopathy-like condition. Specifically, loss of PTBP1 is associated with decreases in HSC self-renewal, erythroid differentiation, and protein synthesis. Consistent with its function as a splicing regulator, PTBP1 deficiency results in splicing defects in hundreds of genes, and we demonstrate that the up-regulation of a specific isoform of CDC42 partly mimics the protein-synthesis defect associated with loss of PTBP1. Furthermore, PTBP1 deficiency is associated with a marked defect in ribosome biogenesis and a selective reduction in the translation of mRNAs encoding ribosomal proteins. Collectively, this work identifies PTBP1 as a key integrator of ribosomal functions and highlights the broad functional repertoire of RNA-binding proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

To handle energetic materials safely, it is important to have knowledge about their sensitivity. Density functional theory (DFT) has proven a valuable tool in the study of energetic materials, and in the current work, DFT is employed to study the thermal unimolecular decomposition of 2,4,6-trinitrophenol (picric acid, PA), 3-methyl-2,4,6-trinitrophenol (methyl picric acid, mPA), and 3,5-dimethyl-2,4,6-trinitrophenol (dimethyl picric acid, dmPA). These compounds have similar molecular structures, but according to the literature, mPA is far less sensitive to impact than the other two compounds. Three pathways believed important for the initiation reactions are investigated at 0 and 298.15 K. We compare the computed energetics of the reaction pathways with the objective of rationalizing the unexpected sensitivity behavior. Our results reveal a few if any significant differences in the energetics of the three molecules, and thus do not reflect the sensitivity deviations observed in experiments. These findings point toward the potential importance of crystal structure, crystal morphology, bimolecular reactions, or combinations thereof on the impact sensitivity of nitroaromatics.

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His 6 segment. This tag promoted acylation of the N-terminal N α -amine resulting in stable conjugates. Herein, we report the peptide sequences His n -Lys-His m , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys N ϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method., (© 2022 Wiley-VCH GmbH.)

The U2AF1 gene is a core part of mRNA splicing machinery and frequently contains somatic mutations that contribute to oncogenesis in myelodysplastic syndrome, acute myeloid leukemia, and other cancers. A change introduced in the GRCh38 version of the human reference build prevents detection of mutations in this gene, and others, by variant calling pipelines. This study describes the problem in detail and shows that a modified GRCh38 reference build with unchanged coordinates can be used to ameliorate the issue., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)

Background and Aims: To estimate during pregnancy correlations between frequency of self-reported use of marijuana and quantified marijuana metabolite in biospecimens including urine, sera and umbilical cord homogenate., Design: Prospective cohort., Setting: Two urban hospitals in Colorado with legal recreational and medicinal marijuana., Participants: Pregnant women (<16 weeks gestation) self-reporting marijuana use., Measurements: Participants completed a written self-report survey and provided biospecimens at <16 weeks gestation (n = 46), 18 to 22 weeks gestation (n = 43), 32 to 36 weeks gestation (n = 39), and delivery (n = 37). Self-reported marijuana use frequency was calculated based on past-month days of use multiplied by number of daily uses. Maternal urine and sera were tested for presence (>5 ng/mL) of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH). Liquid chromatography tandem mass spectrometry quantified THC-COOH in umbilical cord homogenate (ng/g). Last marijuana use by any measure was recorded to evaluate the time frame over which THC-COOH remains detectable (>0.10 ng/g) in cord., Findings: From December 2017 through May 2019, 51 pregnant women enrolled, and 46 were included in analyses (2 withdrew and 3 had a spontaneous abortion). The majority were normal weight, White or Black race, and insured by Medicaid. At the time of enrollment between 7 to 15 weeks' gestation, 87% had ongoing use by self-report, or positive urine or serum. The majority (33 [66%]) stopped using before delivery. Sera and urine results were strongly correlated with self-reported use frequency (Spearman correlation coefficient [r] range 0.70-0.87 across visits, P < 0.001), and with each other. There was only one positive cord result when use stopped before 22 weeks. Frequency of self-reported marijuana use at delivery had strong correlation with quantified cord THC-COOH (r = 0.80, 95% CI = 0.62-0.89)., Conclusions: Quantified umbilical cord THC-COOH appears to strongly correlate with frequency of maternal marijuana use in the last month of pregnancy. Earlier use can be measured by either quantitative urine or serum assay., (© 2021 Society for the Study of Addiction.)

Background: There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine., Methods: A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results., Results: MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses., Conclusions: MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development., Clinical Trials Registration: NCT00914732., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

In recent years, gene therapy has been raising hopes toward viable treatment strategies for rare genetic diseases for which there has been almost exclusively supportive treatment. We here review this progress at the pre-clinical and clinical trial levels as well as market approvals within diseases that specifically affect the brain and spinal cord, including degenerative, developmental, lysosomal storage, and metabolic disorders. The field reached an unprecedented milestone when Zolgensma® (onasemnogene abeparvovec) was approved by the FDA and EMA for in vivo adeno-associated virus-mediated gene replacement therapy for spinal muscular atrophy. Shortly after EMA approved Libmeldy®, an ex vivo gene therapy with lentivirus vector-transduced autologous CD34-positive stem cells, for treatment of metachromatic leukodystrophy. These successes could be the first of many more new gene therapies in development that mostly target loss-of-function mutation diseases with gene replacement (e.g., Batten disease, mucopolysaccharidoses, gangliosidoses) or, less frequently, gain-of-toxic-function mutation diseases by gene therapeutic silencing of pathologic genes (e.g., amyotrophic lateral sclerosis, Huntington's disease). In addition, the use of genome editing as a gene therapy is being explored for some diseases, but this has so far only reached clinical testing in the treatment of mucopolysaccharidoses. Based on the large number of planned, ongoing, and completed clinical trials for rare genetic central nervous system diseases, it can be expected that several novel gene therapies will be approved and become available within the near future. Essential for this to happen is the in depth characterization of short- and long-term effects, safety aspects, and pharmacodynamics of the applied gene therapy platforms., Competing Interests: DW is co-founder and consultant of CombiGene AB (Lund, Sweden) and CG is employed by UCB Nordic A/S (Copenhagen, Denmark). The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jensen, Gøtzsche and Woldbye.)

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user's own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3))., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Jensen TL et al.)

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC-MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/β signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1β, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host-pathogen interactions.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis . To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Francisella tularensis ( F. tularensis ) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

In order to predict the impact sensitivity of high explosives, we designed and evaluated several models based on the trigger linkage hypothesis and the Arrhenius equation. To this effect, we calculated the heat of detonation, temperature of detonation, and bond dissociation energy for 70 energetic molecules. The bond dissociation energy divided by the temperature of detonation proved to be a good predictor of the impact sensitivity of nitroaromatics, with a coefficient of determination (R 2 ) of 0.81. A separate Bayesian analysis gave similar results, taking model complexity into account. For nitramines, there was no relationship between the impact sensitivity and the bond dissociation energy. None of the models studied gave good predictions for the impact sensitivity of liquid nitrate esters. For solid nitrate esters, the bond dissociation energy divided by the temperature of detonation showed promising results (R 2 = 0.85), but since this regression was based on only a few data points, it was discredited when model complexity was accounted for by our Bayesian analysis. Since the temperature of detonation correlated with the impact sensitivity for nitroaromatics, nitramines, and nitrate esters, we consider it to be one of the leading predictive factors of impact sensitivity for energetic materials.

Background: Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial., Methods: CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects., Results: At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects., Conclusions: Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background : Vaccinia is known to induce antibody and cellular responses. Plasmablast, circulating follicular helper T (cT FH ) cells, cytokine-expressing CD4 T cells, and memory B cells were compared between subcutaneous (SC) and needle-free jet injection (JI) recipients of non-replicating modified vaccinia Ankara (MVA) vaccine. Methods : Vaccinia-naïve adults received MVA SC or by JI on Days 1 and 29. Vaccinia-specific antibodies were quantified by plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay. Plasmablast, cT FH , and cytokine-expressing CD4 T cells were assessed on Days 1, 8, 15, 29, 36, 43 (cT FH and CD4+ only) and 57. Memory B cells were measured on Days 1 and 57. Results : Of the 36 enrolled subjects, only 22 received both vaccinations and had evaluable specimens after the second vaccine. Plasmablasts peaked one week after each vaccine. Day 15 plasmablasts correlated with peak PRNT titers. cT FH peaked on Days 8 and 36 and correlated with Day 36 plasmablasts. CD4+ peaked at Day 29 and one-third produced ≥2 cytokines. Day 57 memory B cells ranged from 0.1% to 0.17% of IgG-secreting B cells. Conclusions : This study provides insights into the cellular responses to non-replicating MVA, currently used as a vector for a variety of novel vaccines., Competing Interests: E.J.A. has received research funding unrelated to this paper from Novavax, Micron, PaxVax, Pfizer, Merck, Sanofi-Pasteur, Regeneron, and Medimmune and has also consulted for AbbVie and Pfizer unrelated to this study. N.R has received research funding unrelated to this paper from Sanofi-Pasteur, Pfizer, and Merck. All other authors declare no conflict of interest.

Background: Tularemia is a potential biological weapon due to its high infectivity and ease of dissemination. This study aimed to characterize the innate and adaptive responses induced by two different lots of a live attenuated tularemia vaccine and compare them to other well-characterized viral vaccine immune responses. Methods: Microarray analyses were performed on human peripheral blood mononuclear cells (PBMCs) to determine changes in transcriptional activity that correlated with changes detected by cellular phenotyping, cytokine signaling, and serological assays. Transcriptional profiles after tularemia vaccination were compared with yellow fever [YF-17D], inactivated [TIV], and live attenuated [LAIV] influenza. Results: Tularemia vaccine lots produced strong innate immune responses by Day 2 after vaccination, with an increase in monocytes, NK cells, and cytokine signaling. T cell responses peaked at Day 14. Changes in gene expression, including upregulation of STAT1 , GBP1 , and IFIT2 , predicted tularemia-specific antibody responses. Changes in CCL20 expression positively correlated with peak CD8+ T cell responses, but negatively correlated with peak CD4+ T cell activation. Tularemia vaccines elicited gene expression signatures similar to other replicating vaccines, inducing early upregulation of interferon-inducible genes. Conclusions: A systems vaccinology approach identified that tularemia vaccines induce a strong innate immune response early after vaccination, similar to the response seen after well-studied viral vaccines, and produce unique transcriptional signatures that are strongly correlated to the induction of T cell and antibody responses.

Fasting of mice is a common procedure, which can affect the outcome of the study as well as animal welfare. In this study, we assess the effects of fasting, fasting duration and fasting initiation time in relation to light schedule and present suggestions for optimization of fasting. Male C57BL/6NCrl mice were fasted for 0, 3, 6, 12, 18 and 24 hours initiated either in the light period (photophase) or the dark period (scotophase). Body weight, gastric content, body temperature, corticosterone and 19 routine clinical chemistry parameters were evaluated. Fasting caused significant changes in most of the measured parameters. Increasing duration of fasting resulted in increasing physiological changes. Fasting initiated in the scotophase caused more significant changes than fasting initiated in the photophase. To cause the least physiological changes in mice and increase animal welfare, mice should preferably be fasted in the photophase and for the shortest possible period allowed by the experimental purpose of fasting.

Background: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood., Methods: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types., Results: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs., Conclusions: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Understanding levels of in utero drug exposure is important to properly customize the immediate, as well as ongoing, medical and social management needs of affected newborns. Here, we present the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of 4 cannabinoid analytes in two neonatal matrices. The analytes targeted were Δ 9 -tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN). The matrices analyzed were umbilical cord tissue and meconium. A fifth analyte, cannabidiol (CBD), was also detected uniquely in meconium. Extracts were analyzed by LC-MS/MS in negative electrospray ionization mode. Paired meconium and umbilical cord samples (i.e., one specimen from each matrix collected from each single birth, n = 46 pairs) were tested to evaluate concentration and metabolite profiles. THCA was detected in all positive (containing one or more analytes) meconium samples (n = 32). CBN, THC, 11-OH-THC, and CBD were present in 57% (n = 26), 39% (n = 18), 24% (n = 11), and 20% (n = 9), respectively. Concentrations were lower in the umbilical cord samples for all analytes (i.e., 0.27-537 ng/g for meconium and 0.1-9 ng/g for umbilical cord). In umbilical cord THCA was also detected in all positive samples (n = 19) while THC, CBN, and 11-OH-THC were present in 24% (n = 11), 17% (n = 8), and 11% (n = 5), respectively. Testing neonatal matrices for cannabinoids could be used to support studies designed to detect newborns exposed to cannabis in utero , as well as provide data that could be examined for correlations with clinical and social outcomes., (© 2019 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V. All rights reserved.)

In utero exposure to cannabis may cause various short- and long-term health problems in newborns, such as low birth weight and neonatal withdrawal syndrome. Drug testing with umbilical cord tissue can be used to identify in utero exposure to cannabis. Here, we described a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that simultaneously quantifies four cannabinoids in umbilical cord tissue, including tetrahydrocannabinol (THC), 11-nor-Δ9-carboxy-THC (THC-COOH), cannabinol (CBN), and 11-hydroxy-THC (11-OH-THC). Umbilical cord specimens are weighed and homogenized, and cannabinoids are extracted using anion exchange solid-phase extraction columns (AX-SPE). Liquid chromatography separation is performed, and quantitative results are obtained using LC-MS/MS.

Objective: To compare self-reported maternal marijuana use with quantitative biological sampling for a marijuana metabolite, 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, in umbilical cord homogenate in a state with legalized marijuana., Methods: We conducted a cross-sectional study of women approached at the time of admission for delivery with live, singleton pregnancies at 24 weeks of gestation or greater at two urban medical centers in Colorado. Maternal marijuana use was estimated by 1) report to a health care provider on admission history and physical, 2) survey of self-reported use, and 3) liquid chromatography-tandem mass spectrometry analysis of umbilical cord homogenate for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid. Women were categorized by survey-reported last use (30 days ago or less, 30 days to 1 year, more than 1 year, never) and proportion of women with cord results above the limit of detection and limit of quantification for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid was reported for each group. Comparisons between groups were made using contingency tables. Correlation between survey-reported frequency of use and quantitative 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid cord homogenate results was evaluated., Results: We included 116 women with self-report surveys linked to cord assay results. Six percent (95% CI 2.5-12.0%) of participants reported use in the past 30 days on survey and 2.6% (95% CI 0.5-7.4%) of participants reported marijuana use to health care providers. On umbilical cord assay, 22.4% (95% CI 15.2-31.1%) had detectable 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid. The proportion of women with detectable 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid increased with more recent self-reported use. Survey-reported frequency of use in the past 30 days had moderate correlation with quantified umbilical cord 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (correlation coefficient 0.44, 95% CI 0.28-0.58, P<.001)., Conclusion: Umbilical cord sampling results in higher estimates of prenatal marijuana use than self-report even in the setting of legalization. Umbilical cord assays for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid demonstrate promise for quantifying use. Future studies should examine how the use of biological sampling informs the association between marijuana use and perinatal outcomes.

Background: Reflection through shared storytelling is an established and well-documented way of learning in clinical placement studies in nursing education. Digital development has provided storytelling activities with increased possibilities., Aim: The aim of this study was to explore nursing students' experiences with student-created digital storytelling as a tool for reflection during clinical placement studies., Methods: The study employed an explorative qualitative design. Five semi-structured focus group interviews with students (=37) from two different nursing education institutions in Norway were conducted during fall 2016 and spring 2017., Findings: The findings showed that the use of the student-created digital storytelling was a trigger for engagement, promoted feelings of ownership of the reflection, and resulted in a deeper understanding. However, the study also revealed that the storyteller might be placed in a vulnerable position., Conclusion and Implications for Practice: Student-created digital storytelling seems to be valuable for processes of reflection in clinical placement studies in nursing education. However, one should be aware of aspects concerning the vulnerable role of the storyteller as sharing multimedia technology messages with peer students seems to make students feel more exposed., (Copyright © 2018. Published by Elsevier Ltd.)

Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA-Seq is increasingly being used to measure human RNA expression on a genome-wide scale. Expression profiles can be interrogated to identify and functionally characterize treatment-responsive genes. Ultimately, such controlled studies promise to reveal insights into molecular mechanisms of treatment effects, identify biomarkers, and realize personalized medicine. RNA-Seq Reports (RSEQREP) is a new open-source cloud-enabled framework that allows users to execute start-to-end gene-level RNA-Seq analysis on a preconfigured RSEQREP Amazon Virtual Machine Image (AMI) hosted by AWS or on their own Ubuntu Linux machine. The framework works with unstranded, stranded, and paired-end sequence FASTQ files stored locally, on Amazon Simple Storage Service (S3), or at the Sequence Read Archive (SRA). RSEQREP automatically executes a series of customizable steps including reference alignment, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially expressed genes, heatmaps, co-expressed gene clusters, enriched pathways, and a series of custom visualizations. The framework outputs a file collection that includes a dynamically generated PDF report using R, knitr, and LaTeX, as well as publication-ready table and figure files. A user-friendly configuration file handles sample metadata entry, processing, analysis, and reporting options. The configuration supports time series RNA-Seq experimental designs with at least one pre- and one post-treatment sample for each subject, as well as multiple treatment groups and specimen types. All RSEQREP analyses components are built using open-source R code and R/Bioconductor packages allowing for further customization. As a use case, we provide RSEQREP results for a trivalent influenza vaccine (TIV) RNA-Seq study that collected 1 pre-TIV and 10 post-TIV vaccination samples (days 1-10) for 5 subjects and two specimen types (peripheral blood mononuclear cells and B-cells)., Competing Interests: No competing interests were disclosed.

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates. IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine., (Copyright © 2017 American Society for Microbiology.)

Adjuvants enhance immunity elicited by vaccines through mechanisms that are poorly understood. Using a systems biology approach, we investigated temporal protein expression changes in five primary human immune cell populations: neutrophils, monocytes, natural killer cells, T cells, and B cells after administration of either an Adjuvant System 03 adjuvanted or unadjuvanted split-virus H5N1 influenza vaccine. Monocytes demonstrated the strongest differential signal between vaccine groups. On day 3 post-vaccination, several antigen presentation-related pathways, including MHC class I-mediated antigen processing and presentation, were enriched in monocytes and neutrophils and expression of HLA class I proteins was increased in the Adjuvant System 03 group. We identified several protein families whose proteomic responses predicted seroprotective antibody responses (>1:40 hemagglutination inhibition titer), including inflammation and oxidative stress proteins at day 1 as well as immunoproteasome subunit (PSME1 and PSME2) and HLA class I proteins at day 3 in monocytes. While comparison between temporal proteomic and transcriptomic results showed little overlap overall, enrichment of the MHC class I antigen processing and presentation pathway in monocytes and neutrophils was confirmed by both approaches., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood., Objective and Methods: We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18-49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination., Results: Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination., Conclusions: Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed., Trial Registration: ClinicalTrials.gov NCT01573312., Competing Interests: KME has received grant funding for group B streptococcal vaccine research from Novartis and has participated as a member on a Novartis Data Safety Monitoring Board on influenza vaccines. CBC has received institutional grant support for staphylococcal vaccine research from Pfizer and Novartis Vaccines. JBG, TJL, and HH are employed by The Emmes Corporation. The Emmes Corporation is an independent contract research organization NIH-NIAID-DMID contracted with for collecting and analyzing data for clinical trials and studies to evaluate the safety and efficacy of vaccines, devices, and therapeutics for infectious diseases. The Emmes Corporation does not have any patents, products in development or marketed products to declare. These affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials; however, we have not released raw sequencing data to maintain accordance with subject confidentiality requirements as outlined in the study’s Informed Consent Document, as approved by the Vanderbilt University Medical Center Institutional Review Board. The other authors declare that they have no conflicts of interest.

A new application of utilizing ultra-wideband (UWB) technology to sense wind turbine blade deflections is introduced in this paper for wind energy cost reduction. The lower UWB band of 3.1-5.3 GHz is applied. On each blade, there will be one UWB blade deflection sensing system, which consists of two UWB antennas at the blade root and one UWB antenna at the blade tip. The detailed topology and challenges of this deflection sensing system are addressed. Due to the complexity of the problem, this paper will first realize the on-blade UWB radio link in the simplest case, where the tip antenna is situated outside (and on the surface of) a blade tip. To investigate this case, full-blade time-domain measurements are designed and conducted under different deflections. The detailed measurement setups and results are provided. If the root and tip antenna locations are properly selected, the first pulse is always of sufficient quality for accurate estimations under different deflections. The measured results reveal that the blade tip-root distance and blade deflection can be accurately estimated in the complicated and lossy wireless channels around a wind turbine blade. Some future research topics on this application are listed finally.

Background: The medial hamstring muscle has the potential to prevent excessive dynamic valgus and external rotation of the knee joint during sports. Thus, specific training targeting the medial hamstring muscle seems important to avoid knee injuries., Objective: The aim was to investigate the medial and lateral hamstring muscle activation balance during 14 selected therapeutic exercises., Study Design: The study design involved single-occasion repeated measures in a randomised manner. Sixteen female elite handball and soccer players with a mean (SD) age of 23 (3) years and no previous history of knee injury participated in the present study. Electromyographic (EMG) activity of the lateral (biceps femoris - BF) and medial (semitendinosus - ST) hamstring muscle was measured during selected strengthening and balance/coordination exercises, and normalised to EMG during isometric maximal voluntary contraction (MVC). A two-way analysis of variance was performed using the mixed procedure to determine whether differences existed in normalised EMG between exercises and muscles., Results: Kettlebell swing and Romanian deadlift targeted specifically ST over BF (Δ17-22%, p<0.05) at very high levels of normalised EMG (73-115% of MVC). In contrast, the supine leg curl and hip extension specifically targeted the BF over the ST (Δ 20-23%, p<0.05) at very high levels of normalised EMG (75-87% of MVC)., Conclusion: Specific therapeutic exercises targeting the hamstrings can be divided into ST dominant or BF dominant hamstring exercises. Due to distinct functions of the medial and lateral hamstring muscles, this is an important knowledge in respect to prophylactic training and physical therapist practice.

The relationship between incarceration and recidivism was investigated in a sample of 627 adult male sexual offenders. Incarceration for the index offense was unrelated to sexual or violent recidivism. This was the case whether incarceration was examined as a dichotomous variable (incarceration vs. community sentence) or as a continuous variable (length of incarceration). Risk for sexual recidivism was assessed with a modified version of the Rapid Risk Assessment for Sexual Offense Recidivism. There was no evidence that the relationship between incarceration and recidivism was confounded or moderated by risk or that length of incarceration and recidivism were non-linearly associated. Sentencing sexual offenders to terms of incarceration appears to have little, if any, impact on sexual and violent recidivism following release.

Introduction: The aim of this study was to evaluate the registration of childhood CNS tumours in the Danish Cancer Registry (CR) from 1980 to 1996, based on completeness and validity., Materials and Methods: The completeness of the CR was estimated by examination of the Danish National Hospital Register and an independent hospital registry. To determine the validity of the CR, 640 cases of childhood CNS tumours identified in the CR were compared with the corresponding medical record, with regard to cancer morphology and date of diagnosis., Results: The completeness of the CR was 98-99%. For 82% of all cases, the morphology registered in the CR was identical with that in the medical record. The validity of the morphological diagnosis was 84% for astrocytomas, 88% for ependymomas, 95% for medulloblastomas, 47% for other gliomas and 76% for miscellaneous intracranial and intraspinal neoplasms. In a further 7% of all cases, the morphological diagnosis in the CR was incorrect with respect to details but correct within these main diagnostic groups, 6% were tumours of another main diagnostic group than that registered in the CR and 5% were not tumours, were tumours outside the study criteria or were registered in the hospital journal as suspected tumours. The date of diagnosis in the CR and that in the medical records were concordant in 88% of the evaluated cases., Conclusion: Registration of childhood CNS tumours in the CR was highly complete, while the validity of the morphological diagnosis differed among the main diagnostic groups. The relatively low validity might be explained by the anatomic location of CNS tumours, which makes tissue sampling for histological verification and therefore diagnosis difficult.

Down syndrome children with acute megakaryocytic leukemia (AMkL) have higher cure rates than non-Down syndrome acute myeloid leukemia (AML) patients treated with cytosine arabinoside (ara-C). Megakaryoblasts from Down syndrome AML patients are more sensitive in vitro to ara-C than cells from non-Down syndrome AML patients. Somatic mutations in the GATA1 transcription factor have been detected exclusively and almost uniformly in Down syndrome AMkL patients, suggesting a potential linkage to the chemotherapy sensitivity of Down syndrome megakaryoblasts. Stable transfection of wild-type GATA1 cDNA into the Down syndrome AMkL cell line CMK resulted in decreased (8- to 17-fold) ara-C sensitivity and a threefold-lower generation of the active ara-C metabolite ara-CTP compared with that for mock-transfected CMK cells. High intracellular levels of uridine arabinoside (ara-U) (an inactive ara-C catabolite generated by cytidine deaminase) and cytidine deaminase transcripts were detected in GATA1-transfected CMK sublines, whereas no ara-U was detected in mock-transfected cells. Cytidine deaminase transcripts were a median 5.1-fold (P = .002) lower in Down syndrome megakaryoblasts (n = 16) than in blast cells from non-Down syndrome patients (n = 56). These results suggest that GATA1 transcriptionally upregulates cytidine deaminase and that the presence or absence of GATA1 mutations in AML blasts likely confers differences in ara-C sensitivities due to effects on cytidine deaminase gene expression, which, in turn, contributes to the high cure rate of Down syndrome AMkL patients.

Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5'-Deletion and site-directed mutagenesis of the dCK upstream region (positions -464 to -27) confirmed the importance of two GC-boxes (positions -317 to -309 and -213 to -206) and two E-boxes (positions -302 to -297 and -278 to -273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp- and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp- and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21-localized gene, cystathionine-beta-synthase (CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5'-cDNA ends (5'-RACE) analysis demonstrated exclusive use of the CBS -1b promoter in the cell lines, and transient transfections with the full-length CBS -1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS -1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f- or GT-d-binding site resulted in an approximately 90% decrease of the CBS -1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to the CBS -1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns of CBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.

Cystathionine-beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously described essential transactivating roles for specificity protein 1 (Sp1), Sp3, nuclear factor Y (NF-Y), and USF-1 in the regulation of the CBS-1b promoter. Differential binding of Sp1/Sp3 to the CBS-1b promoter due to differences in Sp1/Sp3 phosphorylation, and Sp1/Sp3 synergism with NF-Y might, in part, explain cell-specific patterns of CBS expression. In this report, the roles of various NF-YA isoforms in influencing cell-specific differences in CBS gene expression were determined in HT1080 and HepG2 cells. Seven unique NF-YA isoforms were detected in HT1080 by reverse transcriptase-PCR (RT-PCR) and DNA sequencing, characterized by deletions in the glutamine-rich and/or serine/threonine-rich domains. Only four of the seven NF-YA isoforms were found in HepG2 cells. The six alternatively spliced NF-YA isoforms all showed significantly less synergistic transactivation of the CBS-1b promoter with Sp1 than wild-type NF-YA, as determined by cotransfections in Drosophila SL2 cells with NF-YB and NF-YC. Further, all six alternatively spliced NF-YA isoforms inhibited the synergistic transactivation of the CBS-1b promoter by wild-type NF-Y and Sp1. Thus, the cellular distributions of these alternatively spliced NF-YA isoforms could impart an important cell-specific component to CBS transcriptional regulation, by virtue of their abilities to directly synergize with Sp1/Sp3 and interfere with transactivation of the CBS-1b promoter by wild-type NF-Y. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down's syndrome (DS).

Controlled release nitrogen (N) fertilizers have been commonly used in horticultural applications such as turf grasses and container-grown woody perennials. Agrium, a major N manufacturer in North and South America, is developing a low-cost controlled release urea (CRU) product for use in field crops such as grain corn, canola, wheat, and other small grain cereals. From 1998 to 2000, 11 field trials were conducted across western Canada to determine if seed-placed CRU could maintain crop yields and increase grain N and N use efficiency when compared to the practice of side-banding of urea N fertilizer. CRU was designed to release timely and adequate, but not excessive, amounts of N to the crop. Crop uptake of N from seed-placed CRU was sufficient to provide yields similar to those of side-banded urea N. Grain N concentrations of the CRU treatments were higher, on average, than those from side-banded urea, resulting in 4.2% higher N use efficiency across the entire N application range from 25 to 100 kg ha(-1). Higher levels of removal of N in grain from CRU compared to side-banded urea can result in less residual N remaining in the soil, and limit the possibility of N losses due to denitrification and leaching.