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Janick Weberpals,1 Sudha R Raman,2 Pamela A Shaw,3 Hana Lee,4 Massimiliano Russo,1 Bradley G Hammill,2 Sengwee Toh,5 John G Connolly,5 Kimberly J Dandreo,6 Fang Tian,7 Wei Liu,7 Jie Li,7 José J Hernández-Muñoz,7 Robert J Glynn,1 Rishi J Desai1 1Division of Pharmacoepidemiology and Pharmacoeconomics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 2Department of Population Health Sciences, Duke University School of Medicine, Durham, NC, USA; 3Biostatistics Division, Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA; 4Office of Biostatistics, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA; 5Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care Institute, Boston, MA, USA; 6Department of Population Medicine, Harvard Pilgrim Health Care Institute, Boston, MA, USA; 7Office of Surveillance and Epidemiology, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USACorrespondence: Janick Weberpals, Instructor in Medicine, Division of Pharmacoepidemiology and Pharmacoeconomics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 1620 Tremont Street, Suite 3030-R, Boston, MA, 02120, USA, Tel +1 617-278-0932, Fax +1 617-232-8602, Email jweberpals@bwh.harvard.eduObjective: Partially observed confounder data pose challenges to the statistical analysis of electronic health records (EHR) and systematic assessments of potentially underlying missingness mechanisms are lacking. We aimed to provide a principled approach to empirically characterize missing data processes and investigate performance of analytic methods.Methods: Three empirical sub-cohorts of diabetic SGLT2 or DPP4-inhibitor initiators with complete information on HbA1c, BMI and smoking as confounders of interest (COI) formed the basis of data simulation under a plasmode framework. A true null treatment effect, including the COI in the outcome generation model, and four missingness mechanisms for the COI were simulated: completely at random (MCAR), at random (MAR), and two not at random (MNAR) mechanisms, where missingness was dependent on an unmeasured confounder and on the value of the COI itself. We evaluated the ability of three groups of diagnostics to differentiate between mechanisms: 1)-differences in characteristics between patients with or without the observed COI (using averaged standardized mean differences [ASMD]), 2)-predictive ability of the missingness indicator based on observed covariates, and 3)-association of the missingness indicator with the outcome. We then compared analytic methods including “complete case”, inverse probability weighting, single and multiple imputation in their ability to recover true treatment effects.Results: The diagnostics successfully identified characteristic patterns of simulated missingness mechanisms. For MAR, but not MCAR, the patient characteristics showed substantial differences (median ASMD 0.20 vs 0.05) and consequently, discrimination of the prediction models for missingness was also higher (0.59 vs 0.50). For MNAR, but not MAR or MCAR, missingness was significantly associated with the outcome even in models adjusting for other observed covariates. Comparing analytic methods, multiple imputation using a random forest algorithm resulted in the lowest root-mean-squared-error.Conclusion: Principled diagnostics provided reliable insights into missingness mechanisms. When assumptions allow, multiple imputation with nonparametric models could help reduce bias.Keywords: electronic health records, missing data, diagnostics, imputation, analytics

Underreporting of energy consumption by self-report is well-recognized, but previous studies using recovery biomarkers have not been sufficiently large to establish whether participant characteristics predict misreporting. In 2004-2005, 544 participants in the Women's Health Initiative Dietary Modification Trial completed a doubly labeled water protocol (energy biomarker), 24-hour urine collection (protein biomarker), and self-reports of diet (assessed by food frequency questionnaire (FFQ)), exercise, and lifestyle habits; 111 women repeated all procedures after 6 months. Using linear regression, the authors estimated associations of participant characteristics with misreporting, defined as the extent to which the log ratio (self-reported FFQ/nutritional biomarker) was less than zero. Intervention women in the trial underreported energy intake by 32% (vs. 27% in the comparison arm) and protein intake by 15% (vs. 10%). Younger women had more underreporting of energy (p = 0.02) and protein (p = 0.001), while increasing body mass index predicted increased underreporting of energy and overreporting of percentage of energy derived from protein (p = 0.001 and p = 0.004, respectively). Blacks and Hispanics underreported more than did Caucasians. Correlations of initial measures with repeat measures (n = 111) were 0.72, 0.70, 0.46, and 0.64 for biomarker energy, FFQ energy, biomarker protein, and FFQ protein, respectively. Recovery biomarker data were used in regression equations to calibrate self-reports; the potential application of these equations to disease risk modeling is presented. The authors confirm the existence of systematic bias in dietary self-reports and provide methods of correcting for measurement error. [ABSTRACT FROM AUTHOR]

Gaze control requires the coordination of movements of both eyes and head to fixate on a target. Using our biologically constrained architecture for gaze control we show how the relationships between the coupled sensorimotor systems can be learnt autonomously from scratch, allowing for adaptation as the system grows or changes. Infant studies suggest developmental learning strategies, which can be applied to sensorimotor learning in humanoid robots. We examine environmental constraints for the learning of eye and head coupled mappings, and give results from implementations on an iCub robot. The results show the impact of these constraints and how they can be overcome to benefit the development of fast, cumulative, on-line learning of coupled sensorimotor systems.

Abstract Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Abstract Background Motor neurone disease (MND) is a devastating illness which leads to muscle weakness and death, usually within 2-3 years of symptom onset. Respiratory insufficiency is a common cause of morbidity, particularly in later stages of MND and respiratory complications are the leading cause of mortality in MND patients. Non Invasive Ventilation (NIV) is the current standard therapy to manage respiratory insufficiency. Some MND patients however do not tolerate NIV due to a number of issues including mask interface problems and claustrophobia. In those that do tolerate NIV, eventually respiratory muscle weakness will progress to a point at which intermittent/overnight NIV is ineffective. The NeuRx RA/4 Diaphragm Pacing System was originally developed for patients with respiratory insufficiency and diaphragm paralysis secondary to stable high spinal cord injuries. The DiPALS study will assess the effect of diaphragm pacing (DP) when used to treat patients with MND and respiratory insufficiency. Method/Design 108 patients will be recruited to the study at 5 sites in the UK. Patients will be randomised to either receive NIV (current standard care) or receive DP in addition to NIV. Study participants will be required to complete outcome measures at 5 follow up time points (2, 3, 6, 9 and 12 months) plus an additional surgery and 1 week post operative visit for those in the DP group. 12 patients (and their carers) from the DP group will also be asked to complete 2 qualitative interviews. Discussion The primary objective of this trial will be to evaluate the effect of Diaphragm Pacing (DP) on survival over the study duration in patients with MND with respiratory muscle weakness. The project is funded by the National Institute for Health Research, Health Technology Assessment (HTA) Programme (project number 09/55/33) and the Motor Neurone Disease Association and the Henry Smith Charity. Trial Registration: Current controlled trials ISRCTN53817913. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health.

Abstract Chinese medicinal materials may be authenticated by molecular identification. As a definitive approach to molecular identification of medicinal materials, forensically informative nucleotide sequencing (FINS) comprises four steps, namely (1) DNA extraction from biological samples, (2) selection and amplification of a specific DNA fragment, (3) determination of the sequence of the amplified DNA fragment and (4) cladistic analysis of the sample DNA sequence against a DNA database. Success of the FINS identification depends on the selection of DNA region and reference species. This article describes the techniques and applications of FINS for authenticating Chinese medicinal materials.

Abstract Background The objective of this research was to develop a disease-specific measure for fatigue in patients with motor neurone disease (MND) by generating data that would fit the Rasch measurement model. Fatigue was defined as reversible motor weakness and whole-body tiredness that was predominantly brought on by muscular exertion and was partially relieved by rest. Methods Qualitative interviews were undertaken to confirm the suitability of a previously identified set of 52 neurological fatigue items as relevant to patients with MND. Patients were recruited from five U.K. MND clinics. Questionnaires were administered during clinic or by post. A sub-sample of patients completed the questionnaire again after 2-4 weeks to assess test-retest validity. Exploratory factor analyses and Rasch analysis were conducted on the item set. Results Qualitative interviews with ten MND patients confirmed the suitability of 52 previously identified neurological fatigue items as relevant to patients with MND. 298 patients consented to completing the initial questionnaire including this item set, with an additional 78 patients completing the questionnaire a second time after 4-6 weeks. Exploratory Factor Analysis identified five potential subscales that could be conceptualised as representing: 'Energy', 'Reversible muscular weakness' (shortened to 'Weakness'), 'Concentration', 'Effects of heat' and 'Rest'. Of the original five factors, two factors 'Energy' and 'Weakness' met the expectations of the Rasch model. A higher order fatigue summary scale, consisting of items from the 'Energy' and 'Weakness' subscales, was found to fit the Rasch model and have acceptable unidimensionality. The two scales and the higher order summary scale were shown to fulfil model expectations, including assumptions of unidimensionality, local independency and an absence of differential item functioning. Conclusions The Neurological Fatigue Index for MND (NFI-MND) is a simple, easy-to-administer fatigue scale. It consists of an 8-item fatigue summary scale in addition to separate scales for measuring fatigue experienced as reversible muscular weakness and fatigue expressed as feelings of low energy and whole body tiredness. The underlying two factor structure supports the patient concept of fatigue derived from qualitative interviews in this population. All three scales were shown to be reliable and capable of interval level measurement.

Abstract Background Overexpression of mutant copper/zinc superoxide dismutase (SOD1) in rodents has provided useful models for studying the pathogenesis of amyotrophic lateral sclerosis (ALS). Microglia have been shown to contribute to ALS disease progression in these models, although the mechanism of this contribution remains to be elucidated. Here, we present the first evidence of the effects of overexpression of mutant (TG G93A) and wild type (TG WT) human SOD1 transgenes on a set of functional properties of microglia relevant to ALS progression, including expression of integrin β-1, spreading and migration, phagocytosis of apoptotic neuronal cell debris, and intracellular calcium changes in response to an inflammatory stimulus. Results TG SOD1 G93A but not TG SOD1 WT microglia had lower expression levels of the cell adhesion molecule subunit integrin β-1 than their NTG control cells [NTG (G93A) and NTG (WT), respectively, 92.8 ± 2.8% on TG G93A, 92.0 ± 6.6% on TG WT, 100.0 ± 1.6% on NTG (G93A), and 100.0 ± 2.7% on NTG (WT) cells], resulting in decreased spreading ability, with no effect on ability to migrate. Both TG G93A and TG WT microglia had reduced capacity to phagocytose apoptotic neuronal cell debris (13.0 ± 1.3% for TG G93A, 16.5 ± 1.9% for TG WT, 28.6 ± 1.8% for NTG (G93A), and 26.9 ± 2.8% for NTG (WT) cells). Extracellular stimulation of microglia with ATP resulted in smaller increase in intracellular free calcium in TG G93A and TG WT microglia relative to NTG controls (0.28 ± 0.02 μM for TG G93A, 0.24 ± 0.03 μM for TG WT, 0.39 ± 0.03 μM for NTG (G93A), and 0.37 ± 0.05 μM for NTG (WT) microglia). Conclusions These findings indicate that, under resting conditions, microglia from mutant SOD1 transgenic mice have a reduced capacity to elicit physiological responses following tissue disturbances and that higher levels of stimulatory signals, and/or prolonged stimulation may be necessary to initiate these responses. Overall, resting mutant SOD1-overexpressing microglia may have reduced capacity to function as sensors of disturbed tissue/cellular homeostasis in the CNS and thus have reduced neuroprotective function.

Abstract Background The Hospital Anxiety and Depression Scale (HADS) is commonly used to assess symptoms of anxiety and depression in motor neurone disease (MND). The measure has never been specifically validated for use within this population, despite questions raised about the scale's validity. This study seeks to analyse the construct validity of the HADS in MND by fitting its data to the Rasch model. Methods The scale was administered to 298 patients with MND. Scale assessment included model fit, differential item functioning (DIF), unidimensionality, local dependency and category threshold analysis. Results Rasch analyses were carried out on the HADS total score as well as depression and anxiety subscales (HADS-T, D and A respectively). After removing one item from both of the seven item scales, it was possible to produce modified HADS-A and HADS-D scales which fit the Rasch model. An 11-item higher-order HADS-T total scale was found to fit the Rasch model following the removal of one further item. Conclusion Our results suggest that a modified HADS-A and HADS-D are unidimensional, free of DIF and have good fit to the Rasch model in this population. As such they are suitable for use in MND clinics or research. The use of the modified HADS-T as a higher-order measure of psychological distress was supported by our data. Revised cut-off points are given for the modified HADS-A and HADS-D subscales.

Abstract Background Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disorder characterised by loss of motor neurons leading to severe weakness and death from respiratory failure within 3-5 years. Riluzole prolongs survival in ALS. A published report has suggested a dramatic effect of lithium carbonate on survival. 44 patients were studied, with 16 randomly selected to take LiCO3 and riluzole and 28 allocated to take riluzole alone. In the group treated with lithium, no patients had died (i.e., 100% survival) at the end of the study (15 months from entry), compared to 71% surviving in the riluzole-only group. Although the trial can be criticised on several grounds, there is a substantial rationale from other laboratory studies that lithium is worth investigating therapeutically in amyotrophic lateral sclerosis. Methods/Design LiCALS is a multi-centre double-blind randomised parallel group controlled trial of the efficacy, safety, and tolerability of lithium carbonate (LiCO3) at doses to achieve stable 'therapeutic' plasma levels (0.4-0.8 mmol/L), plus standard treatment, versus matched placebo plus standard treatment, in patients with amyotrophic lateral sclerosis. The study will be based in the UK, in partnership with the MND Association and DeNDRoN (the Dementias and Neurodegnerative Diseases Clinical Research Network). 220 patients will be recruited. All patients will be on the standard treatment for ALS of riluzole 100 mg daily. The primary outcome measure will be death from any cause at 18 months defined from the date of randomisation. Secondary outcome measures will be changes in three functional rating scales, the ALS Functional Rating Scale-Revised, The EuroQOL (EQ-5D), and the Hospital Anxiety and Depression Scale. Eligible patients will have El Escorial Possible, Laboratory-supported Probable, Probable or Definite amyotrophic lateral sclerosis with disease duration between 6 months and 36 months (inclusive), vital capacity ≥ 60% of predicted within 1 month prior to randomisation and age at least18 years. Discussion Patient recruitment began in June 2009 and the last patient is expected to complete the trial protocol in November 2011. Trial registration Current controlled trials ISRCTN83178718

Abstract Background Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Results Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Conclusions Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

Abstract Background Thousands of plants and animals possess pharmacological properties and there is an increased interest in using these materials for therapy and health maintenance. Efficacies of the application is critically dependent on the use of genuine materials. For time to time, life-threatening poisoning is found because toxic adulterant or substitute is administered. DNA barcoding provides a definitive means of authentication and for conducting molecular systematics studies. Owing to the reduced cost in DNA authentication, the volume of the DNA barcodes produced for medicinal materials is on the rise and necessitates the development of an integrated DNA database. Description We have developed an integrated DNA barcode multimedia information platform- Medicinal Materials DNA Barcode Database (MMDBD) for data retrieval and similarity search. MMDBD contains over 1000 species of medicinal materials listed in the Chinese Pharmacopoeia and American Herbal Pharmacopoeia. MMDBD also contains useful information of the medicinal material, including resources, adulterant information, medical parts, photographs, primers used for obtaining the barcodes and key references. MMDBD can be accessed at http://www.cuhk.edu.hk/icm/mmdbd.htm. Conclusions This work provides a centralized medicinal materials DNA barcode database and bioinformatics tools for data storage, analysis and exchange for promoting the identification of medicinal materials. MMDBD has the largest collection of DNA barcodes of medicinal materials and is a useful resource for researchers in conservation, systematic study, forensic and herbal industry.

Abstract Background Lifetime physical activity energy expenditure (PAEE) is an important determinant of risk for many chronic diseases but remains challenging to measure. Previously reported historical physical activity (PA) questionnaires appear to be reliable, but their validity is less well established. Methods We sought to design and validate an historical adulthood PA questionnaire (HAPAQ) against objective PA measurements from the same individuals. We recruited from a population-based cohort in Cambridgeshire, UK, (Medical Research Council Ely Study) in whom PA measurements, using individually calibrated heart rate monitoring, had been obtained in the past, once between 1994 and 1996 and once between 2000 and 2002. 100 individuals from this cohort attended for interview. Historical PA within the domains of home, work, transport, sport and exercise was recalled using the questionnaire by asking closed questions repeated for several discrete time periods from the age of 20 years old to their current age. The average PAEE from the 2 periods of objective measurements was compared to the self-reported data from the corresponding time periods in the questionnaire. Results Significant correlations were observed between HAPAQ-derived and objectively measured total PAEE for both time periods (Spearman r = 0.44; P < 0.001). Similarly, self-reported time spent in vigorous PA was significantly correlated with objective measurements of vigorous PA (Spearman r = 0.40; P < 0.001). Conclusions HAPAQ demonstrates convergent validity for total PAEE and vigorous PA. This instrument will be useful for ranking individuals according to their past PA in studies of chronic disease aetiology, where activity may be an important underlying factor contributing to disease pathogenesis.

Abstract Background Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFδ/δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFδ/δ mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFδ/δ mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFδ/δ mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFδ/δ mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Abstract Background The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus. Results 3020 Pisum germplasm samples from the John Innes Pisum germplasm collection were genotyped for 45 retrotransposon based insertion polymorphism (RBIP) markers by the Tagged Array Marker (TAM) method. The data set was stored in a purpose-built Germinate relational database and analysed by both principal coordinate analysis and a nested application of the Structure program which yielded substantially similar but complementary views of the diversity of the genus Pisum. Structure revealed three Groups (1-3) corresponding approximately to landrace, cultivar and wild Pisum respectively, which were resolved by nested Structure analysis into 14 Sub-Groups, many of which correlate with taxonomic sub-divisions of Pisum, domestication related phenotypic traits and/or restricted geographical locations. Genetic distances calculated between these Sub-Groups are broadly supported by principal coordinate analysis and these, together with the trait and geographical data, were used to infer a detailed model for the domestication of Pisum. Conclusions These data provide a clear picture of the major distinct gene pools into which the genus Pisum is partitioned and their geographical distribution. The data strongly support the model of independent domestications for P. sativum ssp abyssinicum and P. sativum. The relationships between these two cultivated germplasms and the various sub-divisions of wild Pisum have been clarified and the most likely ancestral wild gene pools for domesticated P. sativum identified. Lastly, this study provides a framework for defining global Pisum germplasm which will be useful for designing core collections.

Abstract Herbal medicines are used in many countries for maintaining health and treating diseases. Their efficacy depends on the use of the correct materials, and life-threatening poisoning may occur if toxic adulterants or substitutes are administered instead. Identification of a medicinal material at the DNA level provides an objective and powerful tool for quality control. Extraction of high-quality DNA is the first crucial step in DNA authentication, followed by a battery of DNA techniques including whole genome fingerprinting, DNA sequencing and DNA microarray to establish the identity of the material. New or improved technologies have been developed and valuable data have been collected and compiled for DNA authentication. Some of these technologies and data are patentable. This article provides an overview of some recent patents that cover the extraction of DNA from medicinal materials, the amplification of DNA using improved reaction conditions, the generation of DNA sequences and fingerprints, and the development of high-throughput authentication methods. It also briefly explains why these patents have been granted.

Abstract Background Grasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum. Results The DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins. Conclusion The resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.

Abstract Background Vascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS. Methods We have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor. Results No mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS. Conclusion Mechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.

Background: Missing data in confounding variables present a frequent challenge in generating evidence using real-world data, including electronic health records (EHR). Our objective was to apply a recently published toolkit for characterizing missing data patterns and based on the toolkit results about likely missingness mechanisms, illustrate the decision-making process for analyses in an empirical case example., Methods: We utilized the Structural Missing Data Investigations (SMDI) toolkit to characterize missing data patterns in the context of a pharmacoepidemiology study comparing cardiovascular outcomes of initiating sodium-glucose-cotransporter-2 inhibitors (SGLT2i) and dipeptidyl peptidase-4 inhibitors (DPP-4i) among older adults. The study used a linked EHR-Medicare claims dataset from Duke Health patients (2015-2017), focusing on partially observed confounders from EHR data (HbA1c lab and body mass index [BMI] values). Our analysis incorporated SMDI's descriptive functions and diagnostic tests to explore missingness patterns and determine missingness mitigation approaches. We used findings from these investigations to inform estimation of adjusted hazard ratios comparing the two classes of medications., Results: High levels of missingness were noted for important confounding variables including HbA1c (63.6%) and BMI (16.5%). Diagnostic tests resulted in output that described: 1) the distributions of patient characteristics, exposure, and outcome between patients with or without an observed value of the partially observed covariate, 2) the ability to predict missingness based on observed covariates, and 3) estimate if the missingness of a partially observed covariate is differential with respect to the outcome. There was evidence that missingness could be sufficiently described using observed data, which allowed multiple imputation by chained equations using random forests to address missing confounder data in estimating treatment effects. Multiple imputation resulted in improved alignment of effect estimates with previous studies., Conclusions: We were able to demonstrate the practical application of the SMDI toolkit in a real-world setting. Application of the SMDI toolkit and the resulting insights of potential missingness patterns can inform the choice of appropriate analytic methods and increase transparency of research methods in handling missing data. This type of approach can inform analytic decision making and may increase our ability to generate evidence from real-world data., (© 2024. The Author(s).)

Background & Aims: Mailed outreach for colorectal cancer (CRC) screening increases uptake but it is unclear how to offer the choice of testing. We evaluated if the active choice between colonoscopy and fecal immunochemical test (FIT), or FIT alone, increased response compared with colonoscopy alone., Methods: This pragmatic, randomized, controlled trial at a community health center included patients between ages 50 and 74 who were not up to date with CRC screening. Patients were randomized 1:1:1 to the following: (1) colonoscopy only, (2) active choice of colonoscopy or FIT, or (3) FIT only. Patients received an outreach letter with instructions for testing (colonoscopy referral and/or an enclosed FIT kit), a reminder letter at 2 months, and another reminder at 3 to 5 months via text message or automated voice recording. The primary outcome was CRC screening completion within 6 months., Results: Among 738 patients in the final analysis, the mean age was 58.7 years (SD, 6.2 y); 48.6% were insured by Medicaid and 24.3% were insured by Medicare; and 71.7% were White, 16.9% were Black, and 7.3% were Hispanic/Latino. At 6 months, 5.6% (95% CI, 2.8-8.5) completed screening in the colonoscopy-only arm, 12.8% (95% CI, 8.6-17.0) in the active-choice arm, and 11.3% (95% CI, 7.4-15.3) in the FIT-only arm. Compared with colonoscopy only, there was a significant increase in screening in active choice (absolute difference, 7.1%; 95% CI, 2.0-12.2; P = .006) and FIT only (absolute difference, 5.7%; 95% CI, 0.8-10.6; P = .02)., Conclusions: Both choice of testing and FIT alone increased response and may align with patient preferences., Trial Registration: clinicaltrials.gov NCT04711473., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Changes in sleep, physical activity and mental health were observed in older adults during early stages of the COVID-19 pandemic. Here we describe effects of the COVID-19 pandemic on older adult mental health, wellbeing, and lifestyle behaviors and explore predictors of better mid-pandemic mental health and wellbeing., Methods: Participants in the Adult Changes in Thought study completed measures of lifestyle behaviors (e.g., sleep, physical activity) and mental health and wellbeing both pre-pandemic during regular study visits and mid-pandemic via a one-time survey. We used paired t-tests to compare differences in these measures pre- vs. mid-pandemic. Using multivariate linear regression, we further explored demographic, health, and lifestyle predictors of pandemic depressive symptoms, social support, and fatigue. We additionally qualitatively coded free text data from the mid-pandemic survey for related comments., Results: Participants (N = 896) reported significant changes in mental health and lifestyle behaviors at pre-pandemic vs. mid-pandemic measurements (p < 0.0001). Qualitative findings supported these behavioral and wellbeing changes. Being male, never smoking, and lower pre-pandemic computer time and sleep disturbance were significantly associated with lower pandemic depressive symptoms. Being partnered, female, never smoking, and lower pre-pandemic sleep disturbance were associated with higher pandemic social support. Pre-pandemic employment, more walking, less computer time, and less sleep disturbance were associated with less pandemic fatigue. Participant comments supported these quantitative findings, highlighting gender differences in pandemic mental health, changes in computer usage and physical activity during the pandemic, the value of spousal social support, and links between sleep disturbance and mental health and wellbeing. Qualitative findings also revealed additional factors, such as stresses from personal and family health situations and the country's concurrent political environment, that impacted mental health and wellbeing., Conclusions: Several demographic, health, and lifestyle behaviors appeared to buffer the effects of the COVID-19 pandemic and may be key sources of resilience. Interventions and public health measures targeting men and unpartnered individuals could promote social support resilience, and intervening on modifiable behaviors like sleep quality, physical activity and sedentary activities like computer time may promote resilience to fatigue and depressive symptoms during future community stressor events. Further research into these relationships is warranted., (© 2024. The Author(s).)

The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46 + ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity., Competing Interests: Dr. Pamela Ohashi is on Scientific Advisory Boards for Providence Therapeutics, Treadwell Therapeutics, Tikva Allocell and Rondo Therapeutics. Inc. Dr. Ohashi holds a sponsored research agreement with Providence Therapeutics. No potential conflict of interest was reported by the other authors., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Objectives: Partially observed confounder data pose a major challenge in statistical analyses aimed to inform causal inference using electronic health records (EHRs). While analytic approaches such as imputation are available, assumptions on underlying missingness patterns and mechanisms must be verified. We aimed to develop a toolkit to streamline missing data diagnostics to guide choice of analytic approaches based on meeting necessary assumptions., Materials and Methods: We developed the smdi (structural missing data investigations) R package based on results of a previous simulation study which considered structural assumptions of common missing data mechanisms in EHR., Results: smdi enables users to run principled missing data investigations on partially observed confounders and implement functions to visualize, describe, and infer potential missingness patterns and mechanisms based on observed data., Conclusions: The smdi R package is freely available on CRAN and can provide valuable insights into underlying missingness patterns and mechanisms and thereby help improve the robustness of real-world evidence studies., Competing Interests: The FDA approved the study protocol, statistical analysis plan and reviewed and approved this manuscript. Coauthors from the FDA participated in the results interpretation and in the preparation and decision to submit the manuscript for publication. The FDA had no role in data collection, management, or analysis. The views expressed are those of the authors and not necessarily those of the US FDA. J.W. reports prior employment by Hoffmann-La Roche and previously held shares in Hoffmann-La Roche. P.A.S. is a named inventor on a patent licensed to Novartis by the University of Pennsylvania for an unrelated project. S.T. serves as a consultant for Pfizer, Inc. and TriNetX, LLC. R.J.G. has received research funding through his employer from Amarin, Kowa, Novartis, and Pfizer. R.J.D. reports serving as Principal Investigator on investigator-initiated grants to the Brigham and Women’s Hospital from Novartis, Vertex, and Bristol-Myers-Squibb on unrelated projects. All remaining authors report no disclosures or conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

Aircraft noise can disrupt sleep and impair recuperation. The last U.S. investigation into the effects of aircraft noise on sleep dates back more than 20 years. Since then, traffic patterns and the noise levels produced by single aircraft have changed substantially. It is therefore important to acquire current data on sleep disturbance relative to varying degrees of aircraft noise exposure in the U.S. that can be used to check and potentially update the existing noise policy. This manuscript describes the design, procedures, and analytical approaches of the FAA's National Sleep Study. Seventy-seven U.S. airports with relevant nighttime air traffic from 39 states are included in the sampling frame. Based on simulation-based power calculations, the field study aims to recruit 400 participants from four noise strata and record an electrocardiogram (ECG), body movement, and sound pressure levels in the bedroom for five consecutive nights. The primary outcome of the study is an exposure-response function between the instantaneous, maximum A-weighted sound pressure levels (dBA) of individual aircraft measured in the bedroom and awakening probability inferred from changes in heart rate and body movement. Self-reported sleep disturbance due to aircraft noise is the secondary outcome that will be associated with long-term average noise exposure metrics such as the Day-Night Average Sound Level (DNL) and the Nighttime Equivalent Sound Level ( L night ). The effect of aircraft noise on several other physiological and self-report outcomes will also be investigated. This study will provide key insights into the effects of aircraft noise on objectively and subjectively assessed sleep disturbance.

In response to the escalating global obesity crisis and its associated health and financial burdens, this paper presents a novel methodology for analyzing longitudinal weight loss data and assessing the effectiveness of financial incentives. Drawing from the Keep It Off trial-a three-arm randomized controlled study with 189 participants-we examined the potential impact of financial incentives on weight loss maintenance. Given that some participants choose not to weigh themselves because of small weight change or weight gains, which is a common phenomenon in many weight-loss studies, traditional methods, for example, the Generalized Estimating Equations (GEE) method tends to overestimate the effect size due to the assumption that data are missing completely at random. To address this challenge, we proposed a framework which can identify evidence of missing not at random and conduct bias correction using the estimating equation derived from pairwise composite likelihood. By analyzing the Keep It Off data, we found that the data in this trial are most likely characterized by non-random missingness. Notably, we also found that the enrollment time (i.e., duration time) would be positively associated with the weight loss maintenance after adjusting for the baseline participant characteristics (e.g., age, sex). Moreover, the lottery-based intervention was found to be more effective in weight loss maintenance compared with the direct payment intervention, though the difference was non-statistically significant. This framework's significance extends beyond weight loss research, offering a semi-parametric approach to assess missing data mechanisms and robustly explore associations between exposures (e.g., financial incentives) and key outcomes (e.g., weight loss maintenance). In essence, the proposed methodology provides a powerful toolkit for analyzing real-world longitudinal data, particularly in scenarios with data missing not at random, enriching comprehension of intricate dataset dynamics., (© 2023. The Author(s).)

Regression calibration is a popular approach for correcting biases in estimated regression parameters when exposure variables are measured with error. This approach involves building a calibration equation to estimate the value of the unknown true exposure given the error-prone measurement and other covariates. The estimated, or calibrated, exposure is then substituted for the unknown true exposure in the health outcome regression model. When used properly, regression calibration can greatly reduce the bias induced by exposure measurement error. Here, we first provide an overview of the statistical framework for regression calibration, specifically discussing how a special type of error, called Berkson error, arises in the estimated exposure. We then present practical issues to consider when applying regression calibration, including: 1) how to develop the calibration equation and which covariates to include; 2) valid ways to calculate standard errors of estimated regression coefficients; and 3) problems arising if one of the covariates in the calibration model is a mediator of the relationship between the exposure and outcome. Throughout, we provide illustrative examples using data from the Hispanic Community Health Study/Study of Latinos (United States, 2008-2011) and simulations. We conclude with recommendations for how to perform regression calibration., (Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2023. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Measurement error is a major issue in self-reported diet that can distort diet-disease relationships. Use of blood concentration biomarkers has the potential to mitigate the subjective bias inherent in self-reporting. As part of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) baseline visit (2008-2011), self-reported information on diet was collected from all participants (n = 16,415). The HCHS/SOL also included annual telephone follow-up, as well as a second (2014-2017) and third (2020-2023) clinic visit. Blood concentration biomarkers for carotenoids, tocopherols, retinol, vitamin B12, and folate were measured in a subset of participants (n = 476) as part of the Study of Latinos: Nutrition and Physical Activity Assessment Study (SOLNAS) (2010-2012). We examined the relationships among biomarker levels, self-reported intake, Hispanic/Latino background (Central American, Cuban, Dominican, Mexican, Puerto Rican, or South American), and other participant characteristics in this diverse cohort. We built regression calibration-based prediction equations for 10 nutritional biomarkers and used a simulation to study the power of detecting a diet-disease association in a multivariable Cox model using a predicted concentration level. Good statistical power was observed for some nutrients with high prediction model R2 values, but further research is needed to understand how best to realize the potential of these dietary biomarkers. This study provides a comprehensive examination of several nutritional biomarkers within the HCHS/SOL, characterizing their associations with subject characteristics and the influence of the measurement characteristics on the power to detect associations with health outcomes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The 24-h activity cycle (24HAC) is a new paradigm for studying activity behaviors in relation to health outcomes. This approach inherently captures the interrelatedness of the daily time spent in physical activity (PA), sedentary behavior (SB), and sleep. We describe three popular approaches for modeling outcome associations with the 24HAC exposure. We apply these approaches to assess an association with a cognitive outcome in a cohort of older adults, discuss statistical challenges, and provide guidance on interpretation and selecting an appropriate approach. We compare the use of the isotemporal substitution model (ISM), compositional data analysis (CoDA), and latent profile analysis (LPA) to analyze 24HAC. We illustrate each method by exploring cross-sectional associations with cognition in 1,034 older adults (Mean age = 77; Age range = 65-100; 55.8% female; 90% White) who were part of the Adult Changes in Thought (ACT) Activity Monitoring (ACT-AM) sub-study. PA and SB were assessed with thigh-worn activPAL accelerometers for 7-days. For each method, we fit a multivariable regression model to examine the cross-sectional association between the 24HAC and Cognitive Abilities Screening Instrument item response theory (CASI-IRT) score, adjusting for baseline characteristics. We highlight differences in assumptions and the scientific questions addressable by each approach. ISM is easiest to apply and interpret; however, the typical ISM assumes a linear association. CoDA uses an isometric log-ratio transformation to directly model the compositional exposure but can be more challenging to apply and interpret. LPA can serve as an exploratory analysis tool to classify individuals into groups with similar time-use patterns. Inference on associations of latent profiles with health outcomes need to account for the uncertainty of the LPA classifications, which is often ignored. Analyses using the three methods did not suggest that less time spent on SB and more in PA was associated with better cognitive function. The three standard analytical approaches for 24HAC each have advantages and limitations, and selection of the most appropriate method should be guided by the scientific questions of interest and applicability of each model's assumptions. Further research is needed into the health implications of the distinct 24HAC patterns identified in this cohort., Competing Interests: The 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., (Copyright © 2023 Wu, Rosenberg, Greenwood-Hickman, McCurry, Proust-Lima, Nelson, Crane, LaCroix, Larson and Shaw.)

Background: Molecular stable isotope ratios are a novel type of dietary biomarker with high sensitivity and specificity for certain foods. Among these, fatty acid carbon isotope ratios (CIRs) have strong potential but have not been investigated as dietary biomarkers., Objectives: We evaluated whether fatty acid CIRs and mass proportions were associated with meat, fish, and sugar-sweetened beverage (SSB) intake., Methods: Thirty-two men [aged 46.2 ± 10.5 y; BMI (kg/m2): 27.2 ± 4.0] underwent a 12-wk inpatient dietary intervention at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona. Men were randomly assigned to 1 of 8 dietary treatments varying the presence/absence of dietary meat, fish, and SSBs in all combinations. Fatty acid CIRs and mass proportions were measured in fasting blood samples and adipose tissue biopsies that were collected pre- and postintervention. Dietary effects were analyzed using multivariable regression and receiver operating characteristic AUCs were calculated using logistic regression., Results: CIRs of the several abundant SFAs, MUFAs and arachidonic acid (20:4n-6) in plasma were strongly associated with meat, as were a subset of these fatty acids in RBCs. Effect sizes in plasma ranged from 1.01‰ to 1.93‰ and were similar but attenuated in RBCs. Mass proportions of those fatty acids were not associated with diet. CIRs of plasma dihomo-γ-linolenic acid (20:3n-6) and adipose palmitic acid (16:0) were weakly associated with SSBs. Mass proportions of plasma odd-chain fatty acids were associated with meat, and mass proportions of plasma EPA and DHA (20:5n-3 and 22:6n-3) were associated with fish., Conclusions: CIRs of plasma and RBC fatty acids show promise as sensitive and specific measures of dietary meat. These provide different information from that provided by fatty acid mass proportions, and are informative where mass proportion is not. This trial is registered at www.clinicaltrials.gov as NCT01237093., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Importance: HIV self-testing (HIVST) is a promising strategy to expand the HIV care continuum, particularly among priority populations at high risk of HIV infection. However, little is known about HIVST uptake among men who have sex with men (MSM) outside of clinical trial settings., Objective: To evaluate HIVST use among urban MSM in the US who reported testing within the past 12 months., Design, Setting, and Participants: A cross-sectional study of adult MSM in the 2017 National HIV Behavioral Surveillance system, which used venue-based sampling methods to collect data related to HIV testing, receipt of prevention services, and risk factors for HIV, was conducted at 588 venues in 23 urban areas in the contiguous US and Puerto Rico. All participants were offered HIV testing. Adult cisgender MSM who reported HIV-negative or unknown HIV status and obtained HIV testing in the past 12 months were included. Data for this study were collected between June 4, 2017, and December 22, 2017, and analyzed between October 23, 2020, and August 20, 2021., Main Outcomes and Measures: Self-reported HIVST in the past year. Adjusted prevalence ratios (aPRs) using survey weights were calculated to assess factors associated with HIVST., Results: A total of 6563 MSM in 23 urban areas met inclusion criteria, of whom 506 (7.7%) individuals reported HIVST in the past year. The median age of self-testers was 29 (IQR, 25-35) years, 52.8% had completed college, and 37.9% reported non-Hispanic White race. One self-tester reported seroconverting in the prior 12 months, and an additional 10 self-testers were diagnosed with HIV during the survey. HIVST was associated with sexual orientation disclosure (aPR, 10.27; 95% CI, 3.45-30.60; P < .001), perceived discrimination against people with HIV (aPR, 1.53; 95% CI, 1.09-2.03; P = .01), younger age (aPR, 0.74; 95% CI, 0.66-0.84; P < .001), higher educational level (aPR, 1.20; 95% CI, 1.04-1.37; P = .01), and higher income levels (aPR, 1.18; 95% CI, 1.04-1.32; P = .009). No association was noted with condomless anal sex (aPR, 0.96; 95% CI, 0.88-1.06, P = .88), sexually transmitted infections (aPR, 0.96; 95% CI, 0.70-1.30; P = .77), or preexposure prophylaxis use (aPR, 0.99; 95% CI, 0.75-1.30; P = .92)., Conclusions and Relevance: In this study, HIVST was relatively uncommon in this sample of urban MSM. HIVST may not be reaching those with lower socioeconomic status or who have not disclosed their sexual identity. The findings of this study suggest that efforts to increase HIVST should focus on engaging underserved and vulnerable subgroups of MSM.

Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP), a passive polyclonal antibody therapeutic agent, has had mixed clinical results. Although antibody neutralization is the predominant approach to benchmarking CCP efficacy, CCP may also influence the evolution of the endogenous antibody response. Using systems serology to comprehensively profile severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) functional antibodies of hospitalized people with COVID-19 enrolled in a randomized controlled trial of CCP (ClinicalTrials.gov: NCT04397757), we find that the clinical benefits of CCP are associated with a shift toward reduced inflammatory Spike (S) responses and enhanced nucleocapsid (N) humoral responses. We find that CCP has the greatest clinical benefit in participants with low pre-existing anti-SARS-CoV-2 antibody function and that CCP-induced immunomodulatory Fc glycan profiles and N immunodominant profiles persist for at least 2 months. We highlight a potential mechanism of action of CCP associated with durable immunomodulation, outline optimal patient characteristics for CCP treatment, and provide guidance for development of a different class of COVID-19 hyperinflammation-targeting antibody therapeutic agents., Competing Interests: Declaration of interests G.A. is a founder of SeromYx Systems, Inc., an equity holder in Leyden Labs, and a member of the scientific advisory board of Sanofi Pasteur., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor T-cell (CAR-T) therapy is an exciting development in the field of cancer immunology and has received a lot of interest in recent years. Many time-to-event (TTE) endpoints related to relapse, disease progression, and remission are analyzed in CAR-T studies to assess treatment efficacy. Definitions of these TTE endpoints are not always consistent, even for the same outcomes (e.g., progression-free survival), which often stems from analysis choices regarding which events to consider as part of the composite endpoint, censoring or competing risk in the analysis. Subsequent therapies such as hematopoietic stem cell transplantation are common but are not treated the same in different studies. Standard survival analysis methods are commonly applied to TTE analyses but often without full consideration of the assumptions inherent in the chosen analysis. We highlight two important issues of TTE analysis that arise in CAR-T studies, as well as in other settings in oncology: the handling of competing risks and assessing the association between a time-varying (post-infusion) exposure and the TTE outcome. We review existing analytical methods, including the cumulative incidence function and regression models for analysis of competing risks, and landmark and time-varying covariate analysis for analysis of post-infusion exposures. We clarify the scientific questions that the different analytical approaches address and illustrate how the application of an inappropriate method could lead to different results using data from multiple published CAR-T studies. Codes for implementing these methods in standard statistical software are provided., (©2022 The Authors; Published by the American Association for Cancer Research.)

Although several therapeutics are used to treat coronavirus disease 2019 (COVID-19) patients, there is still no definitive metabolic marker to evaluate disease severity and recovery or a quantitative test to end quarantine. Because severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects human cells via the angiotensin-converting-enzyme 2 (ACE2) receptor and COVID-19 is associated with renin-angiotensin system dysregulation, we evaluated soluble ACE2 (sACE2) activity in the plasma/saliva of 80 hospitalized COVID-19 patients and 27 non-COVID-19 volunteers, and levels of ACE2/Ang (1-7) in plasma or membrane (mACE2) in lung autopsy samples. sACE2 activity was markedly reduced (p < 0.0001) in COVID-19 plasma (n = 59) compared with controls (n = 27). Nadir sACE2 activity in early hospitalization was restored during disease recovery, irrespective of patient age, demographic variations, or comorbidity; in convalescent plasma-administered patients (n = 45), restoration was statistically higher than matched controls (n = 22, p = 0.0021). ACE2 activity was also substantially reduced in the saliva of COVID-19 patients compared with controls (p = 0.0065). There is a strong inverse correlation between sACE2 concentration and sACE2 activity and Ang (1-7) levels in participant plasmas. However, there were no difference in membrane ACE2 levels in lungs of autopsy tissues of COVID-19 (n = 800) versus other conditions (n = 300). These clinical observations suggest sACE2 activity as a potential biomarker and therapeutic target for COVID-19., Competing Interests: Authors declare no competing interests., (© 2022 The Authors.)

Background: The natural abundance nitrogen stable isotope ratio (NIR) of whole tissue correlates with animal protein intakes, including meat and fish. Amino acid (AA) NIRs (NIRAAs) are more variable than the whole-tissue NIRs and may thus better differentiate among foods., Objectives: We evaluated whether NIRAAs were associated with intakes of fish and meat and whether these dietary associations were larger than with whole-tissue NIRs., Methods: Men were recruited at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona, and randomly assigned to one of eight 12-wk inpatient dietary interventions, which varied the presence/absence of fish, meat, and sugar-sweetened beverages (SSBs) in all possible combinations. Fasting blood was drawn pre- and postintervention and plasma and RBC NIRAAs (free and protein-bound) were measured as secondary outcomes in 32 participants. Multivariable regression was used to determine responses of postintervention NIRAAs to dietary variables, and logistic regression was used to calculate receiver operating characteristic AUCs., Results: Most plasma NIRAAs increased with fish and meat intakes, but to a greater extent with fish intake. The largest increase in response to fish intake was plasma NIRLeucine (β = 2.19, SE = 0.26). The NIRThreonine decreased with both fish and meat intakes. Fewer RBC NIRAAs increased with fish intake, and only RBC NIRProline increased with meat intake. No plasma or RBC NIRAA responded to SSB intake. We identified fish intake with a high degree of accuracy using plasma NIRLeucine (corrected AUC, cAUC = 0.96) and NIRGlutamic acid/glutamine (cAUC = 0.93), and meat intake to a lower degree using plasma NIRProline (cAUC = 0.80) and RBC NIRProline (cAUC = 0.85)., Conclusions: Plasma and RBC NIRAAs were associated with fish and meat intakes but were not superior to whole-tissue stable isotope biomarkers in identifying these intakes in a US diet. This trial is registered at www.clinicaltrials.gov as NCT01237093., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The way in which photons travel through biological tissues and subsequently become scattered or absorbed is a key limitation for traditional optical medical imaging techniques using visible light. In contrast, near-infrared wavelengths, in particular those above 1000 nm, penetrate deeper in tissues and undergo less scattering and cause less photo-damage, which describes the so-called "second biological transparency window". Unfortunately, current dyes and imaging probes have severely limited absorption profiles at such long wavelengths, and molecular engineering of novel NIR-II dyes can be a tedious and unpredictable process, which limits access to this optical window and impedes further developments. Two-photon (2P) absorption not only provides convenient access to this window by doubling the absorption wavelength of dyes, but also increases the possible resolution. This review aims to provide an update on the available 2P instrumentation and 2P luminescent materials available for optical imaging in the NIR-II window., Competing Interests: The 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., (Copyright © 2022 Shaw, Forsyth, Haseeb, Yang, Bradley and Klausen.)

Importance: COVID-19 vaccine uptake among urban populations remains low., Objective: To evaluate whether text messaging with outbound or inbound scheduling and behaviorally informed content might increase COVID-19 vaccine uptake., Design, Setting, and Participants: This randomized clinical trial with a factorial design was conducted from April 29 to July 6, 2021, in an urban academic health system. The trial comprised 16 045 patients at least 18 years of age in Philadelphia, Pennsylvania, with at least 1 primary care visit in the past 5 years, or a future scheduled primary care visit within the next 3 months, who were unresponsive to prior outreach. The study was prespecified in the trial protocol, and data were obtained from the intent-to-treat population., Interventions: Eligible patients were randomly assigned in a 1:20:20 ratio to (1) outbound telephone call only by call center, (2) text message and outbound telephone call by call center to those who respond, or (3) text message, with patients instructed to make an inbound telephone call to a hotline. Patients in groups 2 and 3 were concurrently randomly assigned in a 1:1:1:1 ratio to receive different content: standard messaging, clinician endorsement (eg, "Dr. XXX recommends"), scarcity ("limited supply available"), or endowment framing ("We have reserved a COVID-19 vaccine appointment for you")., Main Outcomes and Measures: The primary outcome was the proportion of patients who completed the first dose of the COVID-19 vaccine within 1 month, according to the electronic health record. Secondary outcomes were the completion of the first dose within 2 months and completion of the vaccination series within 2 months of initial outreach. Additional outcomes included the percentage of patients with invalid cell phone numbers (wrong number or nontextable), no response to text messaging, the percentage of patients scheduled for the vaccine, text message responses, and the number of telephone calls made by the access center. Analysis was on an intention-to-treat basis., Results: Among the 16 045 patients included, the mean (SD) age was 36.9 (11.1) years; 9418 (58.7%) were women; 12 869 (80.2%) had commercial insurance, and 2283 (14.2%) were insured by Medicaid; 8345 (52.0%) were White, 4706 (29.3%) were Black, and 967 (6.0%) were Hispanic or Latino. At 1 month, 14 of 390 patients (3.6% [95% CI, 1.7%-5.4%]) in the outbound telephone call-only group completed 1 vaccine dose, as did 243 of 7890 patients (3.1% [95% CI, 2.7%-3.5%]) in the text plus outbound call group (absolute difference, -0.5% [95% CI, -2.4% to 1.4%]; P = .57) and 253 of 7765 patients (3.3% [95% CI, 2.9%-3.7%]) in the text plus inbound call group (absolute difference, -0.3% [95% CI, -2.2% to 1.6%]; P = .72). Among the 15 655 patients receiving text messaging, 118 of 3889 patients (3.0% [95% CI, 2.5%-3.6%]) in the standard messaging group completed 1 vaccine dose, as did 135 of 3920 patients (3.4% [95% CI, 2.9%-4.0%]) in the clinician endorsement group (absolute difference, 0.4% [95% CI, -0.4% to 1.2%]; P = .31), 100 of 3911 patients (2.6% [95% CI, 2.1%-3.1%]) in the scarcity group (absolute difference, -0.5% [95% CI, -1.2% to 0.3%]; P = .20), and 143 of 3935 patients (3.6% [95% CI, 3.0%-4.2%]) in the endowment group (absolute difference, 0.6% [95% CI, -0.2% to 1.4%]; P = .14)., Conclusions and Relevance: There was no detectable increase in vaccination uptake among patients receiving text messaging compared with telephone calls only or behaviorally informed message content., Trial Registration: ClinicalTrials.gov Identifier: NCT04834726.

There is growing evidence that racial and ethnic minorities bear a disproportionate burden from COVID-19. Temporal changes in the pandemic epidemiology and diversity in the clinical course require careful study to identify determinants of poor outcomes. We analyzed 6255 hospitalized individuals with PCR-confirmed SARS-CoV-2 infection from one of 5 hospitals in the University of Pennsylvania Health System between March 2020 and March 2021, using electronic health records to assess risk factors and outcomes through 8 weeks post-admission. Discharge, readmission and mortality outcomes were analyzed in a multi-state model with multivariable Cox models for each transition. Mortality varied markedly over time, with cumulative incidence (95% CI) 30 days post-admission of 19.1% (16.9, 21.3) in March-April 2020, 5.7% (4.2, 7.5) in July-October 2020 and 10.5% (9.1,12.0) in January-March 2021; 26% of deaths occurred after discharge. Average age (SD) at admission varied from 62.7 (17.6) to 54.8 (19.9) to 60.5 (18.1); mechanical ventilation use declined from 21.3% to 9-11%. Compared to Caucasian, Black race was associated with more severe disease at admission, higher rates of co-morbidities and residing in a low-income zip code. Between-race risk differences in mortality risk diminished in multivariable models; while admitting hospital, increasing age, admission early in the pandemic, and severe disease and low blood pressure at admission were associated with increased mortality hazard. Hispanic ethnicity was associated with fewer baseline co-morbidities and lower mortality hazard (0.57, 95% CI: 0.37, .087). Multi-state modeling allows for a unified framework to analyze multiple outcomes throughout the disease course. Morbidity and mortality for hospitalized COVID-19 patients varied over time but post-discharge mortality remained non-trivial. Black race was associated with more risk factors for morbidity and with treatment at hospitals with lower mortality. Multivariable models suggest there are not between-race differences in outcomes. Future work is needed to better understand the identified between-hospital differences in mortality., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Youth account for a disproportionate number of new HIV infections; however, pre-exposure prophylaxis (PrEP) use is limited. We evaluated PrEP counselling rates among non-Hispanic Black youth in the United States after a bacterial sexually transmitted infection (STI) diagnosis., Methods: We conducted a retrospective cohort study of Black youth receiving care at two academically affiliated clinics in Philadelphia between June 2014 and June 2019. We compared PrEP counselling for youth who received primary care services versus those who did not receive primary care services, all of whom met PrEP eligibility criteria due to STI diagnosis per U.S. Centers for Disease Control and Prevention clinical practice guidelines. Two logistic regression models for receipt of PrEP counselling were fit: Model 1 focused on sexual and gender minority (SGM) status and Model 2 on rectal STIs with both models adjusted for patient- and healthcare-level factors., Results: Four hundred and sixteen patients met PrEP eligibility criteria due to STI based on sex assigned at birth and sexual partners. Thirty patients (7%) had documentation of PrEP counselling. Receipt of primary care services was not significantly associated with receipt of PrEP counselling in either Model 1 (adjusted OR (aOR) 0.10 [95% CI 0.01, 0.99]) or Model 2 (aOR 0.52 [95% CI 0.10, 2.77]). Receipt of PrEP counselling was significantly associated with later calendar years of STI diagnosis (aOR 6.80 [95% CI 1.64, 29.3]), assigned male sex at birth (aOR 26.2 [95% CI 3.46, 198]) and SGM identity (aOR 317 [95% CI 39.9, 2521]) in Model 1 and later calendar years of diagnosis (aOR 3.46 [95% CI 1.25, 9.58]), assigned male sex at birth (aOR 18.6 [95% CI 3.88, 89.3]) and rectal STI diagnosis (aOR 28.0 [95% CI 8.07, 97.5]) in Model 2. Fourteen patients (3%) started PrEP during the observation period; 12/14 (86%) were SGM primary care patients assigned male sex at birth., Conclusions: PrEP counselling and uptake among U.S. non-Hispanic Black youth remain disproportionately low despite recent STI diagnosis. These findings support the need for robust investment in PrEP-inclusive sexual health services that are widely implemented and culturally tailored to Black youth, particularly cisgender heterosexual females., (© 2022 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

BackgroundAntibody-based strategies for COVID-19 have shown promise in prevention and treatment of early disease. COVID-19 convalescent plasma (CCP) has been widely used but results from randomized trials supporting its benefit in hospitalized patients with pneumonia are limited. Here, we assess the efficacy of CCP in severely ill, hospitalized adults with COVID-19 pneumonia.MethodsWe performed a randomized control trial (PennCCP2), with 80 adults hospitalized with COVID-19 pneumonia, comparing up to 2 units of locally sourced CCP plus standard care versus standard care alone. The primary efficacy endpoint was comparison of a clinical severity score. Key secondary outcomes include 14- and 28-day mortality, 14- and 28-day maximum 8-point WHO ordinal score (WHO8) score, duration of supplemental oxygenation or mechanical ventilation, respiratory SARS-CoV-2 RNA, and anti-SARS-CoV-2 antibodies.ResultsEighty hospitalized adults with confirmed COVID-19 pneumonia were enrolled at median day 6 of symptoms and day 1 of hospitalization; 60% were anti-SARS-CoV-2 antibody seronegative. Participants had a median of 3 comorbidities, including risk factors for severe COVID-19 and immunosuppression. CCP treatment was safe and conferred significant benefit by clinical severity score (median [MED] and interquartile range [IQR] 10 [5.5-30] vs. 7 [2.75-12.25], P = 0.037) and 28-day mortality (n = 10, 26% vs. n = 2, 5%; P = 0.013). All other prespecified outcome measures showed weak evidence toward benefit of CCP.ConclusionTwo units of locally sourced CCP administered early in hospitalization to majority seronegative participants conferred a significant benefit in clinical severity score and 28-day mortality. Results suggest CCP may benefit select populations, especially those with comorbidities who are treated early.Trial RegistrationClinicalTrials.gov NCT04397757.FundingUniversity of Pennsylvania.