Publications by authors named "Sebastian Maurer-Stroh"

169 Publications

Clinical and virological features of SARS-CoV-2 variants of concern: a retrospective cohort study comparing B.1.1.7 (Alpha), B.1.315 (Beta), and B.1.617.2 (Delta).

Clin Infect Dis 2021 Aug 23. Epub 2021 Aug 23.

National Centre for Infectious Diseases, Singapore.

Background: he impact of SARS-CoV-2 variants of concern (VOCs) on disease severity is unclear. In this retrospective study, we compared outcomes of patients infected with B.1.1.7, B.1.351, and B.1.617.2 with those with wild-type strains from early 2020.

Methods: National surveillance data from 1-January-2021 to 22-May-2021 were obtained from the Ministry of Health, and outcomes in relation to VOC were explored. Detailed patient level data from all patients with VOC infection admitted to our center between 20-December-2020 and 12-May-2021 were analyzed. Clinical outcomes were compared with a cohort of 846 patients admitted from January-April 2020.

Results: 829 patients in Singapore in the study period were infected with these 3 VOCs. After adjusting for age and sex, B.1.617.2 was associated with higher odds of oxygen requirement, ICU admission, or death (adjusted odds ratio (aOR) 4.90, [95% CI 1.43-30.78]). 157 of these patients were admitted to our center. After adjusting for age, sex, comorbidities, and vaccination, aOR for pneumonia with B.1.617.2 was 1.88 [95% CI 0.95-3.76]) compared with wild-type. These differences were not seen with B.1.1.7 and B.1.351. Vaccination status was associated with decreased severity. B.1.617.2 was associated with significantly lower PCR Ct values and longer duration of Ct value ≤30 (median duration 18 days for B.1.617.2, 13 days for wild-type).

Conclusions: There was a signal toward increased severity associated with B.1.617.2. The association of B.1.617.2 with lower Ct value and longer viral shedding provides a potential mechanism for increased transmissibility. These findings provide an impetus for the rapid implementation of vaccination programs.
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http://dx.doi.org/10.1093/cid/ciab721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8522361PMC
August 2021

Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications.

Front Bioeng Biotechnol 2021 2;9:701120. Epub 2021 Aug 2.

Bioinformatics Institute (BII), Agency for Science Technology and Research (ASTAR), Singapore, Singapore.

Large enzyme families such as the groups of zinc-dependent alcohol dehydrogenases (ADHs), long chain alcohol oxidases (AOxs) or amine dehydrogenases (AmDHs) with, sometimes, more than one million sequences in the non-redundant protein database and hundreds of experimentally characterized enzymes are excellent cases for protein engineering efforts aimed at refining and modifying substrate specificity. Yet, the backside of this wealth of information is that it becomes technically difficult to rationally select optimal sequence targets as well as sequence positions for mutagenesis studies. In all three cases, we approach the problem by starting with a group of experimentally well studied family members (including those with available 3D structures) and creating a structure-guided multiple sequence alignment and a modified phylogenetic tree (aka binding site tree) based just on a selection of potential substrate binding residue positions derived from experimental information (not from the full-length sequence alignment). Hereupon, the remaining, mostly uncharacterized enzyme sequences can be mapped; as a trend, sequence grouping in the tree branches follows substrate specificity. We show that this information can be used in the target selection for protein engineering work to narrow down to single suitable sequences and just a few relevant candidate positions for directed evolution towards activity for desired organic compound substrates. We also demonstrate how to find the closest thermophile example in the dataset if the engineering is aimed at achieving most robust enzymes.
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http://dx.doi.org/10.3389/fbioe.2021.701120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8366029PMC
August 2021

Factors influencing SARS-CoV-2 transmission and outbreak control measures in densely populated settings.

Sci Rep 2021 07 27;11(1):15297. Epub 2021 Jul 27.

Ministry of Health, Singapore, Singapore.

Starting with a handful of SARS-CoV-2 infections in dormitory residents in late March 2020, rapid transmission in their dense living environments ensued and by October 2020, more than 50,000 acute infections were identified across various dormitories in Singapore. The aim of the study is to identify combination of factors facilitating SARS-CoV-2 transmission and the impact of control measures in a dormitory through extensive epidemiological, serological and phylogenetic investigations, supported by simulation models. Our findings showed that asymptomatic cases and symptomatic cases who did not seek medical attention were major drivers of the outbreak. Furthermore, each resident had about 30 close contacts and each infected resident spread to 4.4 (IQR 3.5-5.3) others at the start of the outbreak. The final attack rate of the current outbreak was 76.2% (IQR 70.6-98.0%) and could be reduced by further 10% under a modified dormitory housing condition. These findings are important when designing living environments in a post COVID-19 future to reduce disease spread and facilitate rapid implementation of outbreak control measures.
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http://dx.doi.org/10.1038/s41598-021-94463-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316572PMC
July 2021

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

Nat Microbiol 2021 08 22;6(8):991-999. Epub 2021 Jul 22.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia. P. vivax preferentially invades reticulocytes (immature red blood cells). Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. ) and the reticulocyte-binding proteins (PvRBPs). PvRBP2b binds to the transferrin receptor CD71 (ref. ), which is selectively expressed on immature reticulocytes. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.
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http://dx.doi.org/10.1038/s41564-021-00939-3DOI Listing
August 2021

Structure-based virtual screening of CYP1A1 inhibitors: towards rapid tier-one assessment of potential developmental toxicants.

Arch Toxicol 2021 09 28;95(9):3031-3048. Epub 2021 Jun 28.

Innovations in Food and Chemical Safety Programme and Bioinformatics Institute, Agency for Science, Technology, and Research, Singapore, Singapore.

Cytochrome P450 1A1 (CYP1A1) metabolizes estrogens, melatonin, and other key endogenous signaling molecules critical for embryonic/fetal development. The enzyme has increasing expression during pregnancy, and its inhibition or knockout increases embryonic/fetal lethality and/or developmental problems. Here, we present a virtual screening model for CYP1A1 inhibitors based on the orthosteric and predicted allosteric sites of the enzyme. Using 1001 reference compounds with CYP1A1 activity data, we optimized the decision thresholds of our model and classified the training compounds with 68.3% balanced accuracy (91.0% sensitivity and 45.7% specificity). We applied our final model to 11 known CYP1A1 orthosteric binders and related compounds, and found that our ranking of the known orthosteric binders generally agrees with the relative activity of CYP1A1 in metabolizing these compounds. We also applied the model to 22 new test compounds with unknown/unclear CYP1A1 inhibitory activity, and predicted 16 of them are CYP1A1 inhibitors. The CYP1A1 potency and modes of inhibition of these 22 compounds were experimentally determined. We confirmed that most predicted inhibitors, including drugs contraindicated during pregnancy (amiodarone, bicalutamide, cyproterone acetate, ketoconazole, and tamoxifen) and environmental agents suspected to be endocrine disruptors (bisphenol A, diethyl and dibutyl phthalates, and zearalenone), are indeed potent inhibitors of CYP1A1. Our results suggest that virtual screening may be used as a rapid tier-one method to screen for potential CYP1A1 inhibitors, and flag them out for further experimental evaluations.
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http://dx.doi.org/10.1007/s00204-021-03111-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380238PMC
September 2021

Global spectrum of population-specific common missense variation in cytochrome P450 pharmacogenes.

Hum Mutat 2021 Sep 29;42(9):1107-1123. Epub 2021 Jun 29.

Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Next-generation sequencing technology has afforded the discovery of many novel variants that are of significance to inheritable pharmacogenomics (PGx) traits but a large proportion of them have unknown consequences. These include missense variants resulting in single amino acid substitutions in cytochrome P450 (CYP) proteins that can impair enzyme function, leading to altered drug efficacy and toxicity. While most unknown variants are rare, an overlooked minority are variants that are collectively rare but enriched in specific populations. Here, we analyzed sequence variation data in 141,456 individuals from across eight study populations in gnomAD for 38 CYP genes to identify such variants in addition to common variants. By further comparison with data from two PGx-specific databases (PharmVar and PharmGKB) and ClinVar, we identified 234 missense variants in 35 CYP genes, of which 107 were unknown to these databases. Most unknown variants (n = 83) were population-specific common variants and several (n = 7) were found in important CYP pharmacogenes (CYP2D6, CYP4F2, and CYP2C19). Overall, 29% (n = 31) of 107 unknown variants were predicted to affect CYP enzyme function although further biochemical characterization is necessary. These variants may elucidate part of the unexplained interpopulation differences observed in drug response.
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http://dx.doi.org/10.1002/humu.24243DOI Listing
September 2021

Loss of C2orf69 defines a fatal autoinflammatory syndrome in humans and zebrafish that evokes a glycogen-storage-associated mitochondriopathy.

Am J Hum Genet 2021 07 25;108(7):1301-1317. Epub 2021 May 25.

Institute of Molecular and Cell Biology, A(∗)STAR, Biopolis, Singapore 138673, Singapore.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.
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http://dx.doi.org/10.1016/j.ajhg.2021.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322802PMC
July 2021

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Cell 2021 06 24;184(12):3192-3204.e16. Epub 2021 Apr 24.

QBI COVID-19 Research Group (QCRG), San Francisco, CA, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco (UCSF), San Francisco, CA, USA. Electronic address:

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.
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http://dx.doi.org/10.1016/j.cell.2021.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064868PMC
June 2021

Association of SARS-CoV-2 clades with clinical, inflammatory and virologic outcomes: An observational study.

EBioMedicine 2021 Apr 8;66:103319. Epub 2021 Apr 8.

Singapore Ministry of Health, Singapore; Saw Swee Hock School of Public Health, National University of Singapore, Singapore. Electronic address:

Background: Host determinants of severe coronavirus disease 2019 include advanced age, comorbidities and male sex. Virologic factors may also be important in determining clinical outcome and transmission rates, but limited patient-level data is available.

Methods: We conducted an observational cohort study at seven public hospitals in Singapore. Clinical and laboratory data were collected and compared between individuals infected with different SARS-CoV-2 clades. Firth's logistic regression was used to examine the association between SARS-CoV-2 clade and development of hypoxia, and quasi-Poisson regression to compare transmission rates. Plasma samples were tested for immune mediator levels and the kinetics of viral replication in cell culture were compared.

Findings: 319 patients with PCR-confirmed SARS-CoV-2 infection had clinical and virologic data available for analysis. 29 (9%) were infected with clade S, 90 (28%) with clade L/V, 96 (30%) with clade G (containing D614G variant), and 104 (33%) with other clades 'O' were assigned to lineage B.6. After adjusting for age and other covariates, infections with clade S (adjusted odds ratio (aOR) 0·030 (95% confidence intervals (CI): 0·0002-0·29)) or clade O (B·6) (aOR 0·26 (95% CI 0·064-0·93)) were associated with lower odds of developing hypoxia requiring supplemental oxygen compared with clade L/V. Patients infected with clade L/V had more pronounced systemic inflammation with higher concentrations of pro-inflammatory cytokines, chemokines and growth factors. No significant difference in the severity of clade G infections was observed (aOR 0·95 (95% CI: 0·35-2·52). Though viral loads were significantly higher, there was no evidence of increased transmissibility of clade G, and replicative fitness in cell culture was similar for all clades.

Interpretation: Infection with clades L/V was associated with increased severity and more systemic release of pro-inflammatory cytokines. Infection with clade G was not associated with changes in severity, and despite higher viral loads there was no evidence of increased transmissibility.
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http://dx.doi.org/10.1016/j.ebiom.2021.103319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027908PMC
April 2021

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

Nat Commun 2021 03 19;12(1):1739. Epub 2021 Mar 19.

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.
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http://dx.doi.org/10.1038/s41467-021-21996-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979722PMC
March 2021

How the lessons of previous epidemics helped successful countries fight covid-19.

BMJ 2021 03 11;372:n486. Epub 2021 Mar 11.

Bioinformatics Institute, Agency for Science, Technology and Research, Singapore.

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http://dx.doi.org/10.1136/bmj.n486DOI Listing
March 2021

Bioinformatics-aided identification, characterization and applications of mushroom linalool synthases.

Commun Biol 2021 02 17;4(1):223. Epub 2021 Feb 17.

Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Giessen, Germany.

Enzymes empower chemical industries and are the keystone for metabolic engineering. For example, linalool synthases are indispensable for the biosynthesis of linalool, an important fragrance used in 60-80% cosmetic and personal care products. However, plant linalool synthases have low activities while expressed in microbes. Aided by bioinformatics analysis, four linalool/nerolidol synthases (LNSs) from various Agaricomycetes were accurately predicted and validated experimentally. Furthermore, we discovered a linalool synthase (Ap.LS) with exceptionally high levels of selectivity and activity from Agrocybe pediades, ideal for linalool bioproduction. It effectively converted glucose into enantiopure (R)-linalool in Escherichia coli, 44-fold and 287-fold more efficient than its bacterial and plant counterparts, respectively. Phylogenetic analysis indicated the divergent evolution paths for plant, bacterial and fungal linalool synthases. More critically, structural comparison provided catalytic insights into Ap.LS superior specificity and activity, and mutational experiments validated the key residues responsible for the specificity.
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http://dx.doi.org/10.1038/s42003-021-01715-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890063PMC
February 2021

An epidemiological surveillance of hand foot and mouth disease in paediatric patients and in community: A Singapore retrospective cohort study, 2013-2018.

PLoS Negl Trop Dis 2021 02 10;15(2):e0008885. Epub 2021 Feb 10.

Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Background: While hand, foot and mouth disease (HFMD) is primarily self-resolving-soaring incidence rate of symptomatic HFMD effectuates economic burden in the Asia-Pacific region. Singapore has seen a conspicuous rise in the number of HFMD cases from 2010s. Here, we aims to identify the serology and genotypes responsible for such outbreaks in hospitals and childcare facilities.

Methods: We studied symptomatic paediatric HFMD cases from 2013 to 2018 in Singapore. Surveillance for subclinical enterovirus infections was also performed in childcares at the same time period.

Results: Genotyping 101 symptomatic HFMD samples revealed CV-A6 as the major etiological agent for recent outbreaks. We detected infections with CV-A6 (41.0%), EV-A71 (7%), CV-A16 (3.0%), coxsackievirus A2, CV-A2 (1.0%) and coxsackievirus A10, CV-A10 (1.0%). Phylogenetic analysis of local CV-A6 strains revealed a high level of heterogeneity compared against others worldwide, dissimilar to other HFMD causative enteroviruses for which the dominant strains and genotypes are highly region specific. We detected sub-clinical enterovirus infections in childcare centres; 17.1% (n = 245) tested positive for enterovirus in saliva, without HFMD indicative symptoms at the point of sample collection.

Conclusions: CV-A6 remained as the dominant HFMD causative strain in Singapore. Silent subclinical enteroviral infections were detected and warrant further investigations.
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http://dx.doi.org/10.1371/journal.pntd.0008885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901731PMC
February 2021

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Food Chem 2021 Jun 19;348:129110. Epub 2021 Jan 19.

CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia, QLD 4067, Australia; Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, School of Science, Edith Cowan University, Joondalup, WA 6027, Australia. Electronic address:

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.
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http://dx.doi.org/10.1016/j.foodchem.2021.129110DOI Listing
June 2021

A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells.

Cells 2020 10 30;9(11). Epub 2020 Oct 30.

Center for Brain Research, Department of Pathobiology of the Nervous System, Medical University of Vienna, 1090 Vienna, Austria.

Measuring Förster-resonance-energy-transfer (FRET) efficiency allows the investigation of protein-protein interactions (PPI), but extracting quantitative measures of affinity necessitates highly advanced technical equipment or isolated proteins. We demonstrate the validity of a recently suggested novel approach to quantitatively analyze FRET-based experiments in living mammalian cells using standard equipment using the interaction between different type-1 peroxisomal targeting signals (PTS1) and their soluble receptor peroxin 5 (PEX5) as a model system. Large data sets were obtained by flow cytometry coupled FRET measurements of cells expressing PTS1-tagged EGFP together with mCherry fused to the PTS1-binding domain of PEX5, and were subjected to a fitting algorithm extracting a quantitative measure of the interaction strength. This measure correlates with results obtained by in vitro techniques and a two-hybrid assay, but is unaffected by the distance between the fluorophores. Moreover, we introduce a live cell competition assay based on this approach, capable of depicting dose- and affinity-dependent modulation of the PPI. Using this system, we demonstrate the relevance of a sequence element next to the core tripeptide in PTS1 motifs for the interaction strength between PTS1 and PEX5, which is supported by a structure-based computational prediction of the binding energy indicating a direct involvement of this sequence in the interaction.
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http://dx.doi.org/10.3390/cells9112381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693011PMC
October 2020

Interoperable medical data: The missing link for understanding COVID-19.

Transbound Emerg Dis 2021 Jul 29;68(4):1753-1760. Epub 2021 Jan 29.

Australian Centre for Disease Preparedness, Commonwealth Scientific and Industrial Research Organisation, Geelong, VIC, Australia.

Being able to link clinical outcomes to SARS-CoV-2 virus strains is a critical component of understanding COVID-19. Here, we discuss how current processes hamper sustainable data collection to enable meaningful analysis and insights. Following the 'Fast Healthcare Interoperable Resource' (FHIR) implementation guide, we introduce an ontology-based standard questionnaire to overcome these shortcomings and describe patient 'journeys' in coordination with the World Health Organization's recommendations. We identify steps in the clinical health data acquisition cycle and workflows that likely have the biggest impact in the data-driven understanding of this virus. Specifically, we recommend detailed symptoms and medical history using the FHIR standards. We have taken the first steps towards this by making patient status mandatory in GISAID ('Global Initiative on Sharing All Influenza Data'), immediately resulting in a measurable increase in the fraction of cases with useful patient information. The main remaining limitation is the lack of controlled vocabulary or a medical ontology.
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http://dx.doi.org/10.1111/tbed.13892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8359419PMC
July 2021

SVEP1 as a Genetic Modifier of TEK-Related Primary Congenital Glaucoma.

Invest Ophthalmol Vis Sci 2020 10;61(12)

Genetics Research Center, Molecular and Clinical Sciences Institute, St George's, University of London, Cranmer Terrace, London, United Kingdom.

Purpose: Affecting children by age 3, primary congenital glaucoma (PCG) can cause debilitating vision loss by the developmental impairment of aqueous drainage resulting in high intraocular pressure (IOP), globe enlargement, and optic neuropathy. TEK haploinsufficiency accounts for 5% of PCG in diverse populations, with low penetrance explained by variable dysgenesis of Schlemm's canal (SC) in mice. We report eight families with TEK-related PCG, and provide evidence for SVEP1 as a disease modifier in family 8 with a higher penetrance and severity.

Methods: Exome sequencing identified coding/splice site variants with an allele frequency less than 0.0001 (gnomAD). TEK variant effects were assayed in construct-transfected HEK293 cells via detection of autophosphorylated (active) TEK protein. An enucleated eye from an affected member of family 8 was examined via histology. SVEP1 expression in developing outflow tissues was detected by immunofluorescent staining of 7-day mouse anterior segments. SVEP1 stimulation of TEK expression in human umbilical vascular endothelial cells (HUVECs) was measured by TaqMan quantitative PCR.

Results: Heterozygous TEK loss-of-function alleles were identified in eight PCG families, with parent-child disease transmission observed in two pedigrees. Family 8 exhibited greater disease penetrance and severity, histology revealed absence of SC in one eye, and SVEP1:p.R997C was identified in four of the five affected individuals. During SC development, SVEP1 is secreted by surrounding tissues. SVEP1:p.R997C abrogates stimulation of TEK expression by HUVECs.

Conclusions: We provide further evidence for PCG caused by TEK haploinsufficiency, affirm autosomal dominant inheritance in two pedigrees, and propose SVEP1 as a modifier of TEK expression during SC development, affecting disease penetrance and severity.
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http://dx.doi.org/10.1167/iovs.61.12.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545080PMC
October 2020

Virtual screening of potentially endocrine-disrupting chemicals against nuclear receptors and its application to identify PPARγ-bound fatty acids.

Arch Toxicol 2021 01 9;95(1):355-374. Epub 2020 Sep 9.

Bioinformatics Institute (BII), Agency for Science, Technology, and Research (A*STAR), 30 Biopolis Street, Matrix No. 07-01, Singapore, 138671, Singapore.

Nuclear receptors (NRs) are key regulators of energy homeostasis, body development, and sexual reproduction. Xenobiotics binding to NRs may disrupt natural hormonal systems and induce undesired adverse effects in the body. However, many chemicals of concerns have limited or no experimental data on their potential or lack-of-potential endocrine-disrupting effects. Here, we propose a virtual screening method based on molecular docking for predicting potential endocrine-disrupting chemicals (EDCs) that bind to NRs. For 12 NRs, we systematically analyzed how multiple crystal structures can be used to distinguish actives and inactives found in previous high-throughput experiments. Our method is based on (i) consensus docking scores from multiple structures at a single functional state (agonist-bound or antagonist-bound), (ii) multiple functional states (agonist-bound and antagonist-bound), and (iii) multiple pockets (orthosteric site and alternative sites) of these NRs. We found that the consensus enrichment from multiple structures is better than or comparable to the best enrichment from a single structure. The discriminating power of this consensus strategy was further enhanced by a chemical similarity-weighted scoring scheme, yielding better or comparable enrichment for all studied NRs. Applying this optimized method, we screened 252 fatty acids against peroxisome proliferator-activated receptor gamma (PPARγ) and successfully identified 3 previously unknown fatty acids with Kd = 100-250 μM including two furan fatty acids: furannonanoic acid (FNA) and furanundecanoic acid (FUA), and one cyclopropane fatty acid: phytomonic acid (PTA). These results suggested that the proposed method can be used to rapidly screen and prioritize potential EDCs for further experimental evaluations.
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http://dx.doi.org/10.1007/s00204-020-02897-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811525PMC
January 2021

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study.

Lancet 2020 08 18;396(10251):603-611. Epub 2020 Aug 18.

Infectious Diseases Horizontal Technology Centre, Agency for Science, Technology, and Research, Singapore; Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore. Electronic address:

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection.

Methods: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed.

Findings: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only.

Interpretation: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines.

Funding: National Medical Research Council Singapore.
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http://dx.doi.org/10.1016/S0140-6736(20)31757-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434477PMC
August 2020

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020.

Euro Surveill 2020 Aug;25(32)

The members of the working group are listed in the Investigator tab.

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.32.2001410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427299PMC
August 2020

Determination of the relative allergenic potency of proteins: hurdles and opportunities.

Crit Rev Toxicol 2020 07 30;50(6):521-530. Epub 2020 Jul 30.

GF3 Consultancy LLC, West Chester, OH, USA.

The use of proteins and protein-containing materials in a variety of industrial and commercial products is increasing, with applications in pharmaceuticals, agrochemicals and consumer and personal care products. As a consequence there is a need to ensure potential and environmental risks are understood. One important requirement is an appreciation of the ability of proteins to induce allergic sensitization and allergic disease. However, there is currently no clear guidance for determination of whether or not to accept a new protein in a product based on potential allergenicity. A key requirement for effective risk assessment in this respect is an understanding of sensitizing potency. Here we describe issues and challenges associated with measurement of allergenic potency and explore emerging opportunities and possible ways forward. Effective assessment of the risk of allergy demands not only information about the likely conditions of exposure, but also an understanding of the sensitizing potency of protein allergens. For the purposes of this article sensitizing potency can be viewed as being the ease with which, and the concentration at which, proteins will induce sensitization in a previously non-sensitized subject. The immunological bases of protein allergy are summarized, and the properties that confer on proteins the ability to induce allergic sensitization are considered prior to a detailed exploration of the issues that have to be addressed for evaluation of sensitizing potency. Included among the important considerations are: the impact of route of exposure, identification of relevant dose metrics, and the requirement for reference standards. Finally, new and emerging opportunities to evaluate the sensitizing potency of allergenic proteins are reviewed, including the use of modeling.
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http://dx.doi.org/10.1080/10408444.2020.1793895DOI Listing
July 2020

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

Euro Surveill 2020 07;25(28)

Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.28.2000291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376845PMC
July 2020

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization.

bioRxiv 2020 Jul 15. Epub 2020 Jul 15.

antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection . Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction . Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.
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http://dx.doi.org/10.1101/2020.07.14.203414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373131PMC
July 2020

Discovery and Genomic Characterization of a 382-Nucleotide Deletion in ORF7b and ORF8 during the Early Evolution of SARS-CoV-2.

mBio 2020 07 21;11(4). Epub 2020 Jul 21.

Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore

To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans. During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.
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http://dx.doi.org/10.1128/mBio.01610-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374062PMC
July 2020

Utilising animal models to evaluate oseltamivir efficacy against influenza A and B viruses with reduced in vitro susceptibility.

PLoS Pathog 2020 06 18;16(6):e1008592. Epub 2020 Jun 18.

WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.
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http://dx.doi.org/10.1371/journal.ppat.1008592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326275PMC
June 2020

New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop.

Regul Toxicol Pharmacol 2020 Jul 23;114:104668. Epub 2020 Apr 23.

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK. Electronic address:

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a 'Blue Sky Workshop' on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. "Disruptive thinking" is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity.
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http://dx.doi.org/10.1016/j.yrtph.2020.104668DOI Listing
July 2020

Investigation of three clusters of COVID-19 in Singapore: implications for surveillance and response measures.

Lancet 2020 03 17;395(10229):1039-1046. Epub 2020 Mar 17.

Ministry of Health, Singapore; Saw Swee Hock School of Public Health, Singapore. Electronic address:

Background: Three clusters of coronavirus disease 2019 (COVID-19) linked to a tour group from China, a company conference, and a church were identified in Singapore in February, 2020.

Methods: We gathered epidemiological and clinical data from individuals with confirmed COVID-19, via interviews and inpatient medical records, and we did field investigations to assess interactions and possible modes of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Open source reports were obtained for overseas cases. We reported the median (IQR) incubation period of SARS-CoV-2.

Findings: As of Feb 15, 2020, 36 cases of COVID-19 were linked epidemiologically to the first three clusters of circumscribed local transmission in Singapore. 425 close contacts were quarantined. Direct or prolonged close contact was reported among affected individuals, although indirect transmission (eg, via fomites and shared food) could not be excluded. The median incubation period of SARS-CoV-2 was 4 days (IQR 3-6). The serial interval between transmission pairs ranged between 3 days and 8 days.

Interpretation: SARS-CoV-2 is transmissible in community settings, and local clusters of COVID-19 are expected in countries with high travel volume from China before the lockdown of Wuhan and institution of travel restrictions. Enhanced surveillance and contact tracing is essential to minimise the risk of widespread transmission in the community.

Funding: None.
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http://dx.doi.org/10.1016/S0140-6736(20)30528-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269710PMC
March 2020
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