Publications by authors named "David Roberts"

1,170 Publications

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Acceptance procedure for the linear accelerator component of the 1.5 T MRI-linac.

J Appl Clin Med Phys 2021 Jul 17. Epub 2021 Jul 17.

Department of Radiotherapy, University Medical Center Utrecht, Utrecht, The Netherlands.

Purpose: To develop and implement an acceptance procedure for the new Elekta Unity 1.5 T MRI-linac.

Methods: Tests were adopted and, where necessary adapted, from AAPM TG106 and TG142, IEC 60976 and NCS 9 and NCS 22 guidelines. Adaptations were necessary because of the atypical maximum field size (57.4 × 22 cm), FFF beam, the non-rotating collimator, the absence of a light field, the presence of the 1.5 T magnetic field, restricted access to equipment within the bore, fixed vertical and lateral table position, and the need for MR image to MV treatment alignment. The performance specifications were set for stereotactic body radiotherapy (SBRT).

Results: The new procedure was performed similarly to that of a conventional kilovoltage x-ray (kV) image guided radiation therapy (IGRT) linac. Results were acquired for the first Unity system.

Conclusions: A comprehensive set of tests was developed, described and implemented for the MRI-linac. The MRI-linac met safety requirements for patients and operators. The system delivered radiation very accurately with, for example a gantry rotation locus of isocenter of radius 0.38 mm and an average MLC absolute positional error of 0.29 mm, consistent with use for SBRT. Specifications for clinical introduction were met.
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http://dx.doi.org/10.1002/acm2.13068DOI Listing
July 2021

Optimizing Remote Learning: Leveraging Zoom to Develop and Implement Successful Education Sessions.

J Med Educ Curric Dev 2021 Jan-Dec;8:23821205211020760. Epub 2021 Jun 28.

Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Virtual meeting platforms, such as Zoom, have become essential to medical education during the SARS-CoV-2 pandemic. However, many medical educators do not have experience planning or leading these sessions. Despite the prevalence of Zoom learning, there has been little published on best practices. In this article we describe best practices for using Zoom for remote learning, acknowledging technical considerations, and recommending workflows for designing and implementing virtual sessions. Furthermore, we discuss the important role of cognitive learning theory and how to incorporate these key pedagogical insights into a successful virtual session. While eventually in-person classrooms will open, virtual teaching will remain a component of medical education. If we utilize these inventive tools creatively and functionally, then virtual learning can augment and elevate the practice of medical education.
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http://dx.doi.org/10.1177/23821205211020760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243086PMC
June 2021

High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry.

Anal Chem 2021 Jul 14;93(29):10013-10021. Epub 2021 Jul 14.

Department of Chemistry, University of Wisconsin - Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-to-antibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by low-energy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.
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http://dx.doi.org/10.1021/acs.analchem.1c00150DOI Listing
July 2021

Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro.

J Proteome Res 2021 Jul 8. Epub 2021 Jul 8.

Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States.

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample cleanup to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.
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http://dx.doi.org/10.1021/acs.jproteome.1c00446DOI Listing
July 2021

Effects of adiposity on the human plasma proteome: observational and Mendelian randomisation estimates.

Int J Obes (Lond) 2021 Jul 5. Epub 2021 Jul 5.

Medical Research Council (MRC) Integrative Epidemiology Unit at the University of Bristol, Bristol, UK.

Background: Variation in adiposity is associated with cardiometabolic disease outcomes, but mechanisms leading from this exposure to disease are unclear. This study aimed to estimate effects of body mass index (BMI) on an extensive set of circulating proteins.

Methods: We used SomaLogic proteomic data from up to 2737 healthy participants from the INTERVAL study. Associations between self-reported BMI and 3622 unique plasma proteins were explored using linear regression. These were complemented by Mendelian randomisation (MR) analyses using a genetic risk score (GRS) comprised of 654 BMI-associated polymorphisms from a recent genome-wide association study (GWAS) of adult BMI. A disease enrichment analysis was performed using DAVID Bioinformatics 6.8 for proteins which were altered by BMI.

Results: Observationally, BMI was associated with 1576 proteins (P < 1.4 × 10), with particularly strong evidence for a positive association with leptin and fatty acid-binding protein-4 (FABP4), and a negative association with sex hormone-binding globulin (SHBG). Observational estimates were likely confounded, but the GRS for BMI did not associate with measured confounders. MR analyses provided evidence for a causal relationship between BMI and eight proteins including leptin (0.63 standard deviation (SD) per SD BMI, 95% CI 0.48-0.79, P = 1.6 × 10), FABP4 (0.64 SD per SD BMI, 95% CI 0.46-0.83, P = 6.7 × 10) and SHBG (-0.45 SD per SD BMI, 95% CI -0.65 to -0.25, P = 1.4 × 10). There was agreement in the magnitude of observational and MR estimates (R = 0.33) and evidence that proteins most strongly altered by BMI were enriched for genes involved in cardiovascular disease.

Conclusions: This study provides evidence for a broad impact of adiposity on the human proteome. Proteins strongly altered by BMI include those involved in regulating appetite, sex hormones and inflammation; such proteins are also enriched for cardiovascular disease-related genes. Altogether, results help focus attention onto new proteomic signatures of obesity-related disease.
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http://dx.doi.org/10.1038/s41366-021-00896-1DOI Listing
July 2021

Dating first cases of COVID-19.

PLoS Pathog 2021 06 24;17(6):e1009620. Epub 2021 Jun 24.

Biology Centre of the Czech Academy of Sciences, Institute of Hydrobiology, České Budějovice, Czech Republic.

Questions persist as to the origin of the COVID-19 pandemic. Evidence is building that its origin as a zoonotic spillover occurred prior to the officially accepted timing of early December, 2019. Here we provide novel methods to date the origin of COVID-19 cases. We show that six countries had exceptionally early cases, unlikely to represent part of their main case series. The model suggests a likely timing of the first case of COVID-19 in China as November 17 (95% CI October 4). Origination dates are discussed for the first five countries outside China and each continent. Results infer that SARS-CoV-2 emerged in China in early October to mid-November, and by January, had spread globally. This suggests an earlier and more rapid timeline of spread. Our study provides new approaches for estimating dates of the arrival of infectious diseases based on small samples that can be applied to many epidemiological situations.
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http://dx.doi.org/10.1371/journal.ppat.1009620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224943PMC
June 2021

Nonlethal age estimation of three threatened fish species using DNA methylation: Australian lungfish, Murray cod and Mary River cod.

Mol Ecol Resour 2021 Jun 23. Epub 2021 Jun 23.

School of Biological Sciences, The University of Western Australia, Crawley, WA, Australia.

Age-based demography is fundamental to management of wild fish populations. Age estimates for individuals can determine rates of change in key life-history parameters such as length, maturity, mortality and fecundity. These age-based characteristics are critical for population viability analysis in endangered species and for developing sustainable harvest strategies. For teleost fish, age has traditionally been determined by counting increments formed in calcified structures such as otoliths. However, the collection of otoliths is lethal and therefore undesirable for threatened species. At a molecular level, age can be predicted by measuring DNA methylation. Here, we use previously identified age-associated sites of DNA methylation in zebrafish (Danio rerio) to develop two epigenetic clocks for three threatened freshwater fish species. One epigenetic clock was developed for the Australian lungfish (Neoceratodus forsteri) and the second for the Murray cod (Maccullochella peelii) and Mary River cod (Maccullochella mariensis). Age estimation models were calibrated using either known-age individuals, ages derived from otoliths or bomb radiocarbon dating of scales. We demonstrate a high Pearson's correlation between the chronological and predicted age in both the Lungfish clock (cor = .98) and Maccullochella clock (cor = .92). The median absolute error rate for both epigenetic clocks was also low (Lungfish = 0.86 years; Maccullochella = 0.34 years). This study demonstrates the transferability of DNA methylation sites for age prediction between highly phylogenetically divergent fish species. Given the method is nonlethal and suited to automation, age prediction by DNA methylation has the potential to improve fisheries and other wildlife management settings.
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http://dx.doi.org/10.1111/1755-0998.13440DOI Listing
June 2021

Machine Learning-Enabled Renal Cell Carcinoma Status Prediction Using Multiplatform Urine-Based Metabolomics.

J Proteome Res 2021 Jul 23;20(7):3629-3641. Epub 2021 Jun 23.

Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.

Renal cell carcinoma (RCC) is diagnosed through expensive cross-sectional imaging, frequently followed by renal mass biopsy, which is not only invasive but also prone to sampling errors. Hence, there is a critical need for a noninvasive diagnostic assay. RCC exhibits altered cellular metabolism combined with the close proximity of the tumor(s) to the urine in the kidney, suggesting that urine metabolomic profiling is an excellent choice for assay development. Here, we acquired liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data followed by the use of machine learning (ML) to discover candidate metabolomic panels for RCC. The study cohort consisted of 105 RCC patients and 179 controls separated into two subcohorts: the model cohort and the test cohort. Univariate, wrapper, and embedded methods were used to select discriminatory features using the model cohort. Three ML techniques, each with different induction biases, were used for training and hyperparameter tuning. Assessment of RCC status prediction was evaluated using the test cohort with the selected biomarkers and the optimally tuned ML algorithms. A seven-metabolite panel predicted RCC in the test cohort with 88% accuracy, 94% sensitivity, 85% specificity, and 0.98 AUC. Metabolomics Workbench Study IDs are ST001705 and ST001706.
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http://dx.doi.org/10.1021/acs.jproteome.1c00213DOI Listing
July 2021

Seroprevalence of SARS-CoV-2 among Blood Donors and Changes after Introduction of Public Health and Social Measures, London, UK.

Emerg Infect Dis 2021 07;27(7):1795-1801

We describe results of testing blood donors in London, UK, for severe acute respiratory disease coronavirus 2 (SARS-CoV-2) IgG before and after lockdown measures. Anonymized samples from donors 17-69 years of age were tested using 3 assays: Euroimmun IgG, Abbott IgG, and an immunoglobulin receptor-binding domain assay developed by Public Health England. Seroprevalence increased from 3.0% prelockdown (week 13, beginning March 23, 2020) to 10.4% during lockdown (weeks 15-16) and 12.3% postlockdown (week 18) by the Abbott assay. Estimates were 2.9% prelockdown, 9.9% during lockdown, and 13.0% postlockdown by the Euroimmun assay and 3.5% prelockdown, 11.8% during lockdown, and 14.1% postlockdown by the receptor-binding domain assay. By early May 2020, nearly 1 in 7 donors had evidence of past SARS-CoV-2 infection. Combining results from the Abbott and Euroimmun assays increased seroprevalence by 1.6%, 2.3%, and 0.6% at the 3 timepoints compared with Euroimmun alone, demonstrating the value of using multiple assays.
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http://dx.doi.org/10.3201/eid2707.203167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237903PMC
July 2021

Plasma Proteomics of Renal Function: A Trans-ethnic Meta-analysis and Mendelian Randomization Study.

J Am Soc Nephrol 2021 Jun 16. Epub 2021 Jun 16.

M Prunotto, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

Background: Studies on the relationship between renal function and the human plasma proteome have identified several potential biomarkers. However, investigations have been conducted largely in European populations, and causality of the associations between plasma proteins and kidney function has never been addressed.

Methods: A cross-sectional study of 993 plasma proteins among 2,882 participants in four studies of European and admixed ancestries (KORA, INTERVAL, HUNT, QMDiab) identified trans-ethnic associations between eGFR/CKD and proteomic biomarkers. For the replicated associations, two-sample bidirectional Mendelian randomization (MR) was used to investigate potential causal relationships. Publicly available datasets and transcriptomic data from independent studies were used to examine the association between gene expression in kidney tissue and eGFR .

Results: Fifty-seven plasma proteins were associated with eGFR, including one novel protein. Twenty-three of these were additionally associated with CKD. The strongest inferred causal effect was the positive effect of eGFR on testican-2, in line with the known biological role of this protein and the expression of its protein-coding gene (SPOCK2) in renal tissue. We also observed suggestive evidence of an effect of melanoma inhibitory activity (MIA), carbonic anhydrase III, and cystatin-M on eGFR.

Conclusions: In a discovery-replication setting, we identified 57 proteins trans-ethnically associated with eGFR. The revealed causal relationships are an important stepping-stone in establishing testican-2 as a clinically relevant physiological marker of kidney disease progression, and point to additional proteins warranting further investigation.
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http://dx.doi.org/10.1681/ASN.2020071070DOI Listing
June 2021

Training Future International Clinical Academic Leaders Through a Structured Observership Program: Impact and Outcomes from the Initial Cohort.

J Contin Educ Health Prof 2021 Jun 8. Epub 2021 Jun 8.

Ms. Kuc: Former Associate Director, Professional Programs, Department of Postgraduate Medical Education, Office for External Education, Harvard Medical School, Boston, MA. Dr. Roberts: Dean for External Education and Associate Professor of Medicine, Harvard Medical School, Boston, MA. Dr. Caballero: Faculty Director, International Innovation Programs, Faculty Director of Diabetes Education in the Office for External Education, Harvard Medical School, Boston, MA, and Endocrinologist and Clinical Investigator, Brigham and Women's Hospital, Boston, MA.

Introduction: Clinical observership programs have existed for many years as both formal initiatives organized at the institutional or professional society level and informal arrangements between individual physicians and prospective mentors. However, few programs longitudinally assess their impact on patient care and require implementation of a postobservership project to demonstrate learning. In 2018, the Harvard Medical School Office for External Education launched the International Clinical Leaders Observership Program (ICLOP) as an opportunity for midcareer physicians to shadow Harvard Medical School faculty in one of three specialty areas: oncology, cardiology, or diabetology. The culminating activity for each participant was presentation of a capstone project and commitment to implement it on returning home.

Methods: We summarize here the initial cohort and results from a follow-up survey measuring the impact of the program on their subsequent patient care, academic activities, and achievements.

Results: At 1 year of follow-up, 90% of survey respondents had implemented their project. Improvements were reported in areas of clinical, surgical, therapeutic, system, patient education, and team innovation, as well as personal benefits to the participants.

Discussion: ICLOP is a robust and reproducible initiative that resulted in meaningful improvements in patient care and physician-related outcomes for the first cohort of participants.
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http://dx.doi.org/10.1097/CEH.0000000000000368DOI Listing
June 2021

CD47 and thrombospondin-1 regulation of mitochondria, metabolism, and diabetes.

Am J Physiol Cell Physiol 2021 Aug 9;321(2):C201-C213. Epub 2021 Jun 9.

Radiation Control Technologies, Inc., Loudonville, New York.

Thrombospondin-1 (TSP1) is the prototypical member of a family of secreted proteins that modulate cell behavior by engaging with molecules in the extracellular matrix and with receptors on the cell surface. CD47 is widely displayed on many, if not all, cell types and is a high-affinity TSP1 receptor. CD47 is a marker of self that limits innate immune cell activities, a feature recently exploited to enhance cancer immunotherapy. Another major role for CD47 in health and disease is to mediate TSP1 signaling. TSP1 acting through CD47 contributes to mitochondrial, metabolic, and endocrine dysfunction. Studies in animal models found that elevated TSP1 expression, acting in part through CD47, causes mitochondrial and metabolic dysfunction. Clinical studies established that abnormal TSP1 expression positively correlates with obesity, fatty liver disease, and diabetes. The unabated increase in these conditions worldwide and the availability of CD47 targeting drugs justify a closer look into how TSP1 and CD47 disrupt metabolic balance and the potential for therapeutic intervention.
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http://dx.doi.org/10.1152/ajpcell.00175.2021DOI Listing
August 2021

Biogeographical patterns and speciation of the genus Pinguicula (Lentibulariaceae) inferred by phylogenetic analyses.

PLoS One 2021 7;16(6):e0252581. Epub 2021 Jun 7.

Florida Museum of Natural History, University of Florida, Gainesville, Florida, United States of America.

Earlier phylogenetic studies in the genus Pinguicua (Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matK and rpl32-trnL), from up to ca. 80% of the taxa in the genus Pinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genus Pinguicula has involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252581PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184156PMC
June 2021

Association between Business Travel, Health-Related Behaviors, and Adiposity.

J Occup Environ Med 2021 Jun 4. Epub 2021 Jun 4.

Department of Medicine; School of Medicine; Emory University, Atlanta, GA, USA (Bergquist, Roberts); Departments of Epidemiology, Environmental Health and Pediatrics; Rollins School of Public Health and School of Medicine; Emory University; Atlanta, GA, USA (Marcus); Department of Biostatistics & Bioinformatics, Rollins School of Public Health; Emory Biostatistics Collaboration Core; Emory University; Atlanta, GA, USA (Meng, Fei, Moore); Department of Biomedical Informatics; School of Medicine; Emory University, Atlanta, GA, USA (Robichaux).

Objective: To evaluate the associations between frequency of business travel and health behaviors and adiposity.

Methods: Retrospective cross-sectional analysis of de-identified electronic medical records from 795 corporate physical exams.

Results: Business travel frequency demonstrates a curvilinear relationship with body mass index and body composition in men and women, with domestic and international travel. Linear and quadratic term beta coefficients indicate stronger associations between the sum of domestic and international travel and BMI, body fat percentage, and visceral adipose tissue in women than men, after accounting for age, exercise, and sleep. Based on our male sample population, international travel frequency has a greater influence on adiposity than summed (mostly domestic) travel.

Conclusions: Frequent business travel adversely affects body composition, with differences by gender and type of travel.
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http://dx.doi.org/10.1097/JOM.0000000000002278DOI Listing
June 2021

Ethics and governance for internet-based conservation science research.

Conserv Biol 2021 May 31. Epub 2021 May 31.

Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent, UK.

Internet-based research is increasingly important for conservation science and has wide-ranging applications and contexts, including culturomics, illegal wildlife trade, and citizen science. However, online research methods pose a range of ethical and legal challenges. Online data may be protected by copyright, database rights, or contract law. Privacy rights may also restrict the use and access of data, as well as ethical requirements from institutions. Online data have real-world meaning, and the ethical treatment of individuals and communities must not be marginalized when conducting internet-based research. As ethics frameworks originally developed for biomedical applications are inadequate for these methods, we propose that research activities involving the analysis of preexisting online data be treated analogous to offline social science methods, in particular, nondeceptive covert observation. By treating internet users and their data with respect and due consideration, conservationists can uphold the public trust needed to effectively address real-world issues.
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http://dx.doi.org/10.1111/cobi.13778DOI Listing
May 2021

Virological and serological characterization of critically ill patients with COVID-19 in the UK: Interactions of viral load, antibody status and B.1.1.7 variant infection.

J Infect Dis 2021 May 24. Epub 2021 May 24.

Microbiology Services, NHS Blood and Transplant, London, UK.

Background: Convalescent plasma containing neutralising antibody to SARS-CoV-2 is under investigation for COVID-19 treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomised controlled trial that potentially influence treatment outcomes.

Methods: SARS-CoV-2 RNA in nasopharyngeal swabs collected pre-treatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H.

Results: Of 1274 subjects, 90% were PCR-positive with viral loads 118-1.7x10 11 IU/ml. Median viral loads were 40-fold higher in those seronegative for IgG antibodies (n=354; 28%) compared to seropositives (n=939; 72%). Frequencies of B.1.1.7 increased from <1% in early November, 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8x10 6 and 2.0 x10 5 IU/ml respectively; p=2x10 -15). However, viral load distributions were elevated in both seronegative and seropositive subjects infected with B.1.1.7 (4.0x10 6 and 1.6x10 6 IU/ml respectively).

Conclusions: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads and antibody status define subgroups for analysis of treatment efficacy.
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http://dx.doi.org/10.1093/infdis/jiab283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241475PMC
May 2021

Development of a Dissemination Platform for Spatiotemporal and Phylogenetic Analysis of Avian Infectious Bronchitis Virus.

Front Vet Sci 2021 4;8:624233. Epub 2021 May 4.

Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, United States.

Infecting large portions of the global poultry populations, the avian infectious bronchitis virus (IBV) remains a major economic burden in North America. With more than 30 serotypes globally distributed, Arkansas, Connecticut, Delaware, Georgia, and Massachusetts are among the most predominant serotypes in the United States. Even though vaccination is widely used, the high mutation rate exhibited by IBV is continuously triggering the emergence of new viral strains and hindering control and prevention measures. For that reason, targeted strategies based on constantly updated information on the IBV circulation are necessary. Here, we sampled IBV-infected farms from one US state and collected and analyzed 65 genetic sequences coming from three different lineages along with the immunization information of each sampled farm. Phylodynamic analyses showed that IBV dispersal velocity was 12.3 km/year. The majority of IBV infections appeared to have derived from the introduction of the Arkansas DPI serotype, and the Arkansas DPI and Georgia 13 were the predominant serotypes. When analyzed against IBV sequences collected across the United States and deposited in the GenBank database, the most likely viral origin of our sequences was from the states of Alabama, Georgia, and Delaware. Information about vaccination showed that the MILDVAC-MASS+ARK vaccine was applied on 26% of the farms. Using a publicly accessible open-source tool for real-time interactive tracking of pathogen spread and evolution, we analyzed the spatiotemporal spread of IBV and developed an online reporting dashboard. Overall, our work demonstrates how the combination of genetic and spatial information could be used to track the spread and evolution of poultry diseases, providing timely information to the industry. Our results could allow producers and veterinarians to monitor in near-real time the current IBV strain circulating, making it more informative, for example, in vaccination-related decisions.
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http://dx.doi.org/10.3389/fvets.2021.624233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129014PMC
May 2021

Convalescent plasma or hyperimmune immunoglobulin for people with COVID-19: a living systematic review.

Cochrane Database Syst Rev 2021 05 20;5:CD013600. Epub 2021 May 20.

Cochrane Cancer, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.

Background: Convalescent plasma and hyperimmune immunoglobulin may reduce mortality in patients with viral respiratory diseases, and are being investigated as potential therapies for coronavirus disease 2019 (COVID-19). A thorough understanding of the current body of evidence regarding benefits and risks of these interventions is required.  OBJECTIVES: Using a living systematic review approach, to assess whether convalescent plasma or hyperimmune immunoglobulin transfusion is effective and safe in the treatment of people with COVID-19; and to maintain the currency of the evidence.

Search Methods: To identify completed and ongoing studies, we searched the World Health Organization (WHO) COVID-19 Global literature on coronavirus disease Research Database, MEDLINE, Embase, the Cochrane COVID-19 Study Register, the Epistemonikos COVID-19 L*OVE Platform, and trial registries. Searches were done on 17 March 2021.

Selection Criteria: We included randomised controlled trials (RCTs) evaluating convalescent plasma or hyperimmune immunoglobulin for COVID-19, irrespective of disease severity, age, gender or ethnicity. For safety assessments, we also included non-controlled non-randomised studies of interventions (NRSIs) if 500 or more participants were included. We excluded studies that included populations with other coronavirus diseases (severe acute respiratory syndrome (SARS) or Middle East respiratory syndrome (MERS)), as well as studies evaluating standard immunoglobulin.

Data Collection And Analysis: We followed standard Cochrane methodology. To assess bias in included studies, we used the Cochrane 'Risk of Bias 2' tool for RCTs, and for NRSIs, the assessment criteria for observational studies, provided by Cochrane Childhood Cancer. We rated the certainty of evidence, using the GRADE approach, for the following outcomes: all-cause mortality, improvement and worsening of clinical status (for individuals with moderate to severe disease), development of severe clinical COVID-19 symptoms (for individuals with asymptomatic or mild disease), quality of life (including fatigue and functional independence), grade 3 or 4 adverse events, and serious adverse events.

Main Results: We included 13 studies (12 RCTs, 1 NRSI) with 48,509 participants, of whom 41,880 received convalescent plasma. We did not identify any completed studies evaluating hyperimmune immunoglobulin. We identified a further 100 ongoing studies evaluating convalescent plasma or hyperimmune immunoglobulin, and 33 studies reporting as being completed or terminated. Individuals with a confirmed diagnosis of COVID-19 and moderate to severe disease Eleven RCTs and one NRSI investigated the use of convalescent plasma for 48,349 participants with moderate to severe disease. Nine RCTs compared convalescent plasma to placebo treatment or standard care alone, and two compared convalescent plasma to standard plasma (results not included in abstract). Effectiveness of convalescent plasma We included data on nine RCTs (12,875 participants) to assess the effectiveness of convalescent plasma compared to placebo or standard care alone.  Convalescent plasma does not reduce all-cause mortality at up to day 28 (risk ratio (RR) 0.98, 95% confidence interval (CI) 0.92 to 1.05; 7 RCTs, 12,646 participants; high-certainty evidence). It has little to no impact on clinical improvement for all participants when assessed by liberation from respiratory support (RR not estimable; 8 RCTs, 12,682 participants; high-certainty evidence). It has little to no impact on the chance of being weaned or liberated from invasive mechanical ventilation for the subgroup of participants requiring invasive mechanical ventilation at baseline (RR 1.04, 95% CI 0.57 to 1.93; 2 RCTs, 630 participants; low-certainty evidence). It does not reduce the need for invasive mechanical ventilation (RR 0.98, 95% CI 0.89 to 1.08; 4 RCTs, 11,765 participants; high-certainty evidence). We did not identify any subgroup differences.  We did not identify any studies reporting quality of life, and therefore, do not know whether convalescent plasma has any impact on quality of life. One RCT assessed resolution of fatigue on day 7, but we are very uncertain about the effect (RR 1.21, 95% CI 1.02 to 1.42; 309 participants; very low-certainty evidence).  Safety of convalescent plasma We included results from eight RCTs, and one NRSI, to assess the safety of convalescent plasma. Some of the RCTs reported on safety data only for the convalescent plasma group.  We are uncertain whether convalescent plasma increases or reduces the risk of grade 3 and 4 adverse events (RR 0.90, 95% CI 0.58 to 1.41; 4 RCTs, 905 participants; low-certainty evidence), and serious adverse events (RR 1.24, 95% CI 0.81 to 1.90; 2 RCTs, 414 participants; low-certainty evidence).  A summary of reported events of the NRSI (reporting safety data for 20,000 of 35,322 transfused participants), and four RCTs reporting safety data only for transfused participants (6125 participants) are included in the full text. Individuals with a confirmed diagnosis of SARS-CoV-2 infection and asymptomatic or mild disease We identified one RCT reporting on 160 participants, comparing convalescent plasma to placebo treatment (saline).  Effectiveness of convalescent plasma We are very uncertain about the effect of convalescent plasma on all-cause mortality (RR 0.50, 95% CI 0.09 to 2.65; very low-certainty evidence). We are uncertain about the effect of convalescent plasma on developing severe clinical COVID-19 symptoms (RR not estimable; low-certainty evidence).  We identified no study reporting quality of life.  Safety of convalescent plasma We do not know whether convalescent plasma is associated with a higher risk of grade 3 or 4 adverse events (very low-certainty evidence), or serious adverse events (very low-certainty evidence). This is a living systematic review. We search weekly for new evidence and update the review when we identify relevant new evidence. Please refer to the Cochrane Database of Systematic Reviews for the current status of this review.

Authors' Conclusions: We have high certainty in the evidence that convalescent plasma for the treatment of individuals with moderate to severe disease does not reduce mortality and has little to no impact on measures of clinical improvement. We are uncertain about the adverse effects of convalescent plasma. While major efforts to conduct research on COVID-19 are being made, heterogeneous reporting of outcomes is still problematic. There are 100 ongoing studies and 33 studies reporting in a study registry as being completed or terminated. Publication of ongoing studies might resolve some of the uncertainties around hyperimmune immunoglobulin therapy for people with any disease severity, and convalescent plasma therapy for people with asymptomatic or mild disease.
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http://dx.doi.org/10.1002/14651858.CD013600.pub4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135693PMC
May 2021

Novel Strategies to Address the Challenges in Top-Down Proteomics.

J Am Soc Mass Spectrom 2021 Jun 13;32(6):1278-1294. Epub 2021 May 13.

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Top-down mass spectrometry (MS)-based proteomics is a powerful technology for comprehensively characterizing proteoforms to decipher post-translational modifications (PTMs) together with genetic variations and alternative splicing isoforms toward a proteome-wide understanding of protein functions. In the past decade, top-down proteomics has experienced rapid growth benefiting from groundbreaking technological advances, which have begun to reveal the potential of top-down proteomics for understanding basic biological functions, unraveling disease mechanisms, and discovering new biomarkers. However, many challenges remain to be comprehensively addressed. In this Account & Perspective, we discuss the major challenges currently facing the top-down proteomics field, particularly in protein solubility, proteome dynamic range, proteome complexity, data analysis, proteoform-function relationship, and analytical throughput for precision medicine. We specifically review the major technology developments addressing these challenges with an emphasis on our research group's efforts, including the development of top-down MS-compatible surfactants for protein solubilization, functionalized nanoparticles for the enrichment of low-abundance proteoforms, strategies for multidimensional chromatography separation of proteins, and a new comprehensive user-friendly software package for top-down proteomics. We have also made efforts to connect proteoforms with biological functions and provide our visions on what the future holds for top-down proteomics.
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http://dx.doi.org/10.1021/jasms.1c00099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310706PMC
June 2021

Functions of Thrombospondin-1 in the Tumor Microenvironment.

Int J Mol Sci 2021 Apr 27;22(9). Epub 2021 Apr 27.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The identification of thrombospondin-1 as an angiogenesis inhibitor in 1990 prompted interest in its role in cancer biology and potential as a therapeutic target. Decreased thrombospondin-1 mRNA and protein expression are associated with progression in several cancers, while expression by nonmalignant cells in the tumor microenvironment and circulating levels in cancer patients can be elevated. is not a tumor suppressor gene, but the regulation of its expression in malignant cells by oncogenes and tumor suppressor genes mediates some of their effects on carcinogenesis, tumor progression, and metastasis. In addition to regulating angiogenesis and perfusion of the tumor vasculature, thrombospondin-1 limits antitumor immunity by CD47-dependent regulation of innate and adaptive immune cells. Conversely, thrombospondin-1 is a component of particles released by immune cells that mediate tumor cell killing. Thrombospondin-1 differentially regulates the sensitivity of malignant and nonmalignant cells to genotoxic stress caused by radiotherapy and chemotherapy. The diverse activities of thrombospondin-1 to regulate autophagy, senescence, stem cell maintenance, extracellular vesicle function, and metabolic responses to ischemic and genotoxic stress are mediated by several cell surface receptors and by regulating the functions of several secreted proteins. This review highlights progress in understanding thrombospondin-1 functions in cancer and the challenges that remain in harnessing its therapeutic potential.
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http://dx.doi.org/10.3390/ijms22094570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123789PMC
April 2021

Discovery of RSV-Induced BRD4 Protein Interactions Using Native Immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) Mass Spectrometry.

Viruses 2021 03 11;13(3). Epub 2021 Mar 11.

Institute for Clinical and Translational Research (ICTR), University of Wisconsin-Madison, Madison, WI 53705, USA.

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4's acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.
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http://dx.doi.org/10.3390/v13030454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000986PMC
March 2021

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2.

Nat Commun 2021 03 29;12(1):1951. Epub 2021 Mar 29.

Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.
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http://dx.doi.org/10.1038/s41467-021-22045-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007702PMC
March 2021

Statistical inference of earlier origins for the first flaked stone technologies.

J Hum Evol 2021 05 24;154:102976. Epub 2021 Mar 24.

Biology Centre of the Czech Academy of Sciences, Institute of Hydrobiology, České Budějovice, Czech Republic; University of South Bohemia, Faculty of Science, Department of Ecosystem Biology, České Budějovice, Czech Republic.

Identifying when hominins first produced Lomekwian, Oldowan, and Acheulean technologies is vital to multiple avenues of human origins research. Yet, like most archaeological endeavors, our understanding is currently only as accurate as the artifacts recovered and the sites identified. Here we use optimal linear estimation (OLE) modelling to identify the portion of the archaeological record not yet discovered, and statistically infer the date of origin of the earliest flaked stone technologies. These models provide the most accurate framework yet for understanding when hominins first produced these tool types. Our results estimate the Oldowan to have originated 2.617 to 2.644 Ma, 36,000 to 63,000 years earlier than current evidence. The Acheulean's origin is pushed back further through OLE, by at least 55,000 years to 1.815 to 1.823 Ma. We were unable to infer the Lomekwian's date of origin using OLE, but an upper bound of 5.1 million years can be inferred using alternative nonparametric techniques. These dates provide a new chronological foundation from which to understand the emergence of the first flaked stone technologies, alongside their behavioral and evolutionary implications. Moreover, they suggest there to be substantial portions of the artifact record yet to be discovered.
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http://dx.doi.org/10.1016/j.jhevol.2021.102976DOI Listing
May 2021

Antibody testing for COVID-19: A report from the National COVID Scientific Advisory Panel.

Wellcome Open Res 2020 11;5:139. Epub 2020 Jun 11.

Oxford University Hospitals NHS Foundation Trust, Oxford, OX3 9DU, UK.

The COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. We tested plasma for COVID (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). ELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Currently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.
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http://dx.doi.org/10.12688/wellcomeopenres.15927.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941096PMC
June 2020

Prolonged Symptoms After COVID-19 Infection in Outpatients.

Open Forum Infect Dis 2021 Mar 2;8(3):ofab060. Epub 2021 Feb 2.

Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

We review 127 encounters for polymerase chain reaction-confirmed coronavirus disease 2019 (COVID-19) infection at a multidisciplinary outpatient clinic. We describe the symptomatology, time course, exam, and radiographic findings in this population. Patients with COVID-19 can experience persistent symptoms, primarily respiratory in nature, which can be severe enough to warrant hospitalization.
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http://dx.doi.org/10.1093/ofid/ofab060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928621PMC
March 2021

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry.

bioRxiv 2021 Mar 1. Epub 2021 Mar 1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.
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http://dx.doi.org/10.1101/2021.02.28.433291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941619PMC
March 2021
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