Publications by authors named "Yvonne Jones"

187 Publications

Comparison of characteristics and outcomes of patients admitted to hospital with COVID-19 during wave 1 and wave 2 of the current pandemic.

Intern Emerg Med 2021 Oct 12. Epub 2021 Oct 12.

Department of Endocrinology, Ashford and St Peter's Hospitals NHS Foundation Trust, Guildford Road, Chertsey, Surrey, KT16 0PZ, UK.

In this study of patients admitted with COVID-19, we examined differences between the two waves in patient characteristics and outcomes. Data were collected from the first COVID-19 admission to the end of study (01/03/2020-31/03/2021). Data were adjusted for age and sex and presented as odds ratios (OR) with 95% confidence intervals (CI). Among 12,471 admissions, 1452 (11.6%) patients were diagnosed with COVID-19. On admission, the mean (± SD) age of patients with other causes was 68.3 years (± 19.8) and those with COVID-19 in wave 1 was 69.4 years (± 18.0) and wave 2 was 66.2 years (± 18.4). Corresponding ages at discharge were 67.5 years (± 19.7), 63.9 years (± 18.0) and 62.4 years (± 18.0). The highest proportion of total admissions was among the oldest group (≥ 80 years) in wave 1 (35.0%). When compared with patients admitted with other causes, those admitted with COVID-19 in wave 1 and in wave 2 were more frequent in the 40-59 year band: 20.8, 24.6 and 30.0%; consisted of more male patients: 47.5, 57.6 and 58.8%; and a high LACE (Length of stay, Acuity of admission, Comorbidity and Emergency department visits) index (score ≥ 10): 39.4, 61.3 and 50.3%. Compared to wave-2 patients, those admitted in wave 1 had greater risk of death in hospital: OR = 1.58 (1.18-2.12) and within 30 days of discharge: OR = 2.91 (1.40-6.04). Survivors of COVID-19 in wave 1 stayed longer in hospital (median = 6.5 days; interquartile range = 2.9-12.0) as compared to survivors from wave 2 (4.5 days; interquartile range = 1.9-8.7). Patient characteristics differed significantly between the two waves of COVID-19 pandemic. There was an improvement in outcomes in wave 2, including shorter length of stay in hospital and reduction of mortality.
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http://dx.doi.org/10.1007/s11739-021-02842-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8505475PMC
October 2021

Structural Insights into Notum Covalent Inhibition.

J Med Chem 2021 08 22;64(15):11354-11363. Epub 2021 Jul 22.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, U.K.

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365597PMC
August 2021

Antibody responses to SARS-CoV-2 vaccines in 45,965 adults from the general population of the United Kingdom.

Nat Microbiol 2021 09 21;6(9):1140-1149. Epub 2021 Jul 21.

Nuffield Department of Medicine, University of Oxford, Oxford, UK.

We report that in a cohort of 45,965 adults, who were receiving either the ChAdOx1 or the BNT162b2 SARS-CoV-2 vaccines, in those who had no prior infection with SARS-CoV-2, seroconversion rates and quantitative antibody levels after a single dose were lower in older individuals, especially in those aged >60 years. Two vaccine doses achieved high responses across all ages. Antibody levels increased more slowly and to lower levels with a single dose of ChAdOx1 compared with a single dose of BNT162b2, but waned following a single dose of BNT162b2 in older individuals. In descriptive latent class models, we identified four responder subgroups, including a 'low responder' group that more commonly consisted of people aged >75 years, males and individuals with long-term health conditions. Given our findings, we propose that available vaccines should be prioritized for those not previously infected and that second doses should be prioritized for individuals aged >60 years. Further data are needed to better understand the extent to which quantitative antibody responses are associated with vaccine-mediated protection.
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http://dx.doi.org/10.1038/s41564-021-00947-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294260PMC
September 2021

An observational cohort study on the incidence of SARS-CoV-2 infection and B.1.1.7 variant infection in healthcare workers by antibody and vaccination status.

Clin Infect Dis 2021 Jul 3. Epub 2021 Jul 3.

Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Background: Natural and vaccine-induced immunity will play a key role in controlling the SARS-CoV-2 pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity.

Methods: In a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, UK, we investigated the protection from symptomatic and asymptomatic PCR-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after one versus two vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing.

Results: 13,109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses) and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and two vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95%CI <0.01-0.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [0.02-0.38]) and 85% (0.15 [0.08-0.26]) respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [0.21-0.52]) and any PCR-positive result by 64% (0.36 [0.26-0.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7.

Conclusion: Natural infection resulting in detectable anti-spike antibodies and two vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.
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http://dx.doi.org/10.1093/cid/ciab608DOI Listing
July 2021

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse.

Bone Res 2020 Jun 11;8(1):24. Epub 2020 Jun 11.

Translational Gastroenterology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.
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http://dx.doi.org/10.1038/s41413-020-0098-zDOI Listing
June 2020

Quantitative SARS-CoV-2 anti-spike responses to Pfizer-BioNTech and Oxford-AstraZeneca vaccines by previous infection status.

Clin Microbiol Infect 2021 Oct 7;27(10):1516.e7-1516.e14. Epub 2021 Jun 7.

Oxford University Hospitals NHS Foundation Trust, Oxford, UK.

Objectives: We investigated determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer-BioNTech or Oxford-AstraZeneca vaccines.

Methods: HCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/mL). We used multivariable logistic regression to identify predictors of seropositivity and generalized additive models to track antibody responses over time.

Results: 3570/3610 HCWs (98.9%) were seropositive >14 days post first vaccination and prior to second vaccination: 2706/2720 (99.5%) were seropositive after the Pfizer-BioNTech and 864/890 (97.1%) following the Oxford-AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after the second vaccination were seropositive. Quantitative antibody responses were higher after previous infection: median (IQR) >21 days post first Pfizer-BioNTech 14 604 (7644-22 291) AU/mL versus 1028 (564-1985) AU/mL without prior infection (p < 0.001). Oxford-AstraZeneca vaccine recipients had lower readings post first dose than Pfizer-BioNTech recipients, with and without previous infection, 10 095 (5354-17 096) and 435 (203-962) AU/mL respectively (both p < 0.001 versus Pfizer-BioNTech). Antibody responses >21 days post second Pfizer vaccination in those not previously infected, 10 058 (6408-15 582) AU/mL, were similar to those after prior infection followed by one vaccine dose.

Conclusions: SARS-CoV-2 vaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.
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http://dx.doi.org/10.1016/j.cmi.2021.05.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8180449PMC
October 2021

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

Nature 2021 06 2;594(7863):430-435. Epub 2021 Jun 2.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.
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http://dx.doi.org/10.1038/s41586-021-03525-zDOI Listing
June 2021

Small-molecule inhibitors of carboxylesterase Notum.

Future Med Chem 2021 06 22;13(11):1001-1015. Epub 2021 Apr 22.

Alzheimer's Research UK UCL Drug Discovery Institute, University College London, Cruciform Building, Gower Street, London, WC1E 6BT, UK.

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.
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http://dx.doi.org/10.4155/fmc-2021-0036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130783PMC
June 2021

Notum deacylates octanoylated ghrelin.

Mol Metab 2021 Jul 27;49:101201. Epub 2021 Feb 27.

Division of Structural Biology, Wellcome Centre for Human Genetics, Oxford University, Oxford, OX3 7BN, UK. Electronic address:

Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined.

Methods: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation.

Results: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet.

Conclusions: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.
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http://dx.doi.org/10.1016/j.molmet.2021.101201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010218PMC
July 2021

The Duration, Dynamics, and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Responses in Individual Healthcare Workers.

Clin Infect Dis 2021 08;73(3):e699-e709

Big Data Institute, University of Oxford, Oxford, United Kingdom.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary.

Methods: We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning.

Results: Anti-spike IgG levels remained stably detected after a positive result, for example, in 94% (95% credibility interval [CrI] 91-96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% CrI 19-31) days post first polymerase chain reaction (PCR)-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titer, the mean estimated antibody half-life was 85 (95% CrI 81-90) days. Higher maximum observed anti-nucleocapsid titers were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity, and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives.

Conclusions: SARS-CoV-2 anti-nucleocapsid antibodies wane within months and fall faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.
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http://dx.doi.org/10.1093/cid/ciab004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929225PMC
August 2021

Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers.

N Engl J Med 2021 02 23;384(6):533-540. Epub 2020 Dec 23.

From Oxford University Hospitals NHS Foundation Trust (S.F.L., N.E.S., P.C.M., S.C., T.J., F.W., L.W., D.A., A.-M.O., K.J.), Nuffield Department of Medicine (S.F.L., D.O., N.E.S., P.C.M., A.H., S.B.H., B.D.M., R.J.C., E.Y.J., D.I.S., G.S., D.E., S. Hoosdally, D.W.C., C.P.C., A.S.W., T.E.A.P., T.M.W.), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (N.E.S., P.C.M., S. Hoosdally, D.W.C., A.S.W., T.E.A.P., D.W.E.), the Kennedy Institute of Rheumatology Research (B.D.M.), the Medical School, University of Oxford (L.J.P., T.G.R., Z.T.), Target Discovery Institute (D.E.), Nuffield Department of Population Health (A.-M.O., K.B.P., D.W.E.), and the Big Data Institute (D.W.E.), University of Oxford, and the NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford in partnership with Public Health England (N.E.S., P.C.M., S. Hoosdally, D.W.C., K.B.P., A.S.W., T.E.A.P., D.W.E.), Oxford, and the National Infection Service, Public Health England at Colindale, London (M.C., S. Hopkins) - all in the United Kingdom; and the Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam (T.M.W.).

Background: The relationship between the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the risk of subsequent reinfection remains unclear.

Methods: We investigated the incidence of SARS-CoV-2 infection confirmed by polymerase chain reaction (PCR) in seropositive and seronegative health care workers attending testing of asymptomatic and symptomatic staff at Oxford University Hospitals in the United Kingdom. Baseline antibody status was determined by anti-spike (primary analysis) and anti-nucleocapsid IgG assays, and staff members were followed for up to 31 weeks. We estimated the relative incidence of PCR-positive test results and new symptomatic infection according to antibody status, adjusting for age, participant-reported gender, and changes in incidence over time.

Results: A total of 12,541 health care workers participated and had anti-spike IgG measured; 11,364 were followed up after negative antibody results and 1265 after positive results, including 88 in whom seroconversion occurred during follow-up. A total of 223 anti-spike-seronegative health care workers had a positive PCR test (1.09 per 10,000 days at risk), 100 during screening while they were asymptomatic and 123 while symptomatic, whereas 2 anti-spike-seropositive health care workers had a positive PCR test (0.13 per 10,000 days at risk), and both workers were asymptomatic when tested (adjusted incidence rate ratio, 0.11; 95% confidence interval, 0.03 to 0.44; P = 0.002). There were no symptomatic infections in workers with anti-spike antibodies. Rate ratios were similar when the anti-nucleocapsid IgG assay was used alone or in combination with the anti-spike IgG assay to determine baseline status.

Conclusions: The presence of anti-spike or anti-nucleocapsid IgG antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months. (Funded by the U.K. Government Department of Health and Social Care and others.).
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http://dx.doi.org/10.1056/NEJMoa2034545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781098PMC
February 2021

5-Phenyl-1,3,4-oxadiazol-2(3)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit.

J Med Chem 2020 11 30;63(21):12942-12956. Epub 2020 Oct 30.

Alzheimer's Research UK UCL Drug Discovery Institute, University College London, Cruciform Building, Gower Street, London WC1E 6BT, U.K.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of identified oxadiazol-2-one as a preferred example with properties consistent with drug-like chemical space. Screening in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit into lead (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01391DOI Listing
November 2020

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7-11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9.

Structure 2020 12 1;28(12):1300-1312.e5. Epub 2020 Sep 1.

School of Chemistry, Cantock's Close, University of Bristol, Bristol BS8 1TS, UK; BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol BS8 1TQ, UK. Electronic address:

The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7-10 within two multi-domain constructs, D9-10 and D7-11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7-11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7-11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.
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http://dx.doi.org/10.1016/j.str.2020.08.002DOI Listing
December 2020

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity.

J Med Chem 2020 09 18;63(17):9464-9483. Epub 2020 Aug 18.

Alzheimer's Research UK UCL Drug Discovery Institute, University College London, Cruciform Building, Gower Street, London WC1E 6BT, United Kingdom.

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole , guided by structure-based drug design, identified as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine gave acid . This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable ADME profiles.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00660DOI Listing
September 2020

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.

Nature 2020 09 22;585(7823):85-90. Epub 2020 Jul 22.

The Francis Crick Institute, London, UK.

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.
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http://dx.doi.org/10.1038/s41586-020-2498-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610841PMC
September 2020

A variant in with a selective IL-11 signaling defect in human and mouse.

Bone Res 2020 11;8:24. Epub 2020 Jun 11.

Translational Gastroenterology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK.

The GP130 cytokine receptor subunit encoded by is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.
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http://dx.doi.org/10.1038/s41413-020-0098-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289831PMC
June 2020

Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility.

Structure 2020 05 17;28(5):507-515.e5. Epub 2020 Mar 17.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. Electronic address:

The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.
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http://dx.doi.org/10.1016/j.str.2020.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203548PMC
May 2020

Antiepileptic Drug Carbamazepine Binds to a Novel Pocket on the Wnt Receptor Frizzled-8.

J Med Chem 2020 03 25;63(6):3252-3260. Epub 2020 Feb 25.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.

Misregulation of Wnt signaling is common in human cancer. The development of small molecule inhibitors against the Wnt receptor, frizzled (FZD), may have potential in cancer therapy. During small molecule screens, we observed binding of carbamazepine to the cysteine-rich domain (CRD) of the Wnt receptor FZD8 using surface plasmon resonance (SPR). Cellular functional assays demonstrated that carbamazepine can suppress FZD8-mediated Wnt/β-catenin signaling. We determined the crystal structure of the complex at 1.7 Å resolution, which reveals that carbamazepine binds at a novel pocket on the FZD8 CRD. The unique residue Tyr52 discriminates FZD8 from the closely related FZD5 and other FZDs for carbamazepine binding. The first small molecule-bound FZD structure provides a basis for anti-FZD drug development. Furthermore, the observed carbamazepine-mediated Wnt signaling inhibition may help to explain the phenomenon of bone loss and increased adipogenesis in some patients during long-term carbamazepine treatment.
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http://dx.doi.org/10.1021/acs.jmedchem.9b02020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104226PMC
March 2020

The guidance receptor plexin D1 is a mechanosensor in endothelial cells.

Nature 2020 02 5;578(7794):290-295. Epub 2020 Feb 5.

Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.
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http://dx.doi.org/10.1038/s41586-020-1979-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025890PMC
February 2020

Hand-foot-and-mouth disease virus receptor KREMEN1 binds the canyon of Coxsackie Virus A10.

Nat Commun 2020 01 7;11(1):38. Epub 2020 Jan 7.

Division of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, Headington, Oxford, OX3 7BN, UK.

Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
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http://dx.doi.org/10.1038/s41467-019-13936-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946704PMC
January 2020

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors.

Bioorg Med Chem Lett 2020 02 28;30(3):126751. Epub 2019 Oct 28.

Alzheimer's Research UK UCL Drug Discovery Institute, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK; The Francis Crick Institute, 1 Midland Road, Kings Cross, London NW1 1AT, UK. Electronic address:

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.
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http://dx.doi.org/10.1016/j.bmcl.2019.126751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961116PMC
February 2020

Discovery of 2-phenoxyacetamides as inhibitors of the Wnt-depalmitoleating enzyme NOTUM from an X-ray fragment screen.

Medchemcomm 2019 Aug 29;10(8):1361-1369. Epub 2019 Apr 29.

Alzheimer's Research UK UCL Drug Discovery Institute , University College London , Cruciform Building, Gower Street , London , WC1E 6BT , UK . Email: ; Tel: +44 (0)20 7679 6971.

NOTUM is a carboxylesterase that has been shown to act by mediating the -depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide as binding in the palmitoleate pocket with modest inhibition activity (IC 33 μM). Optimization of hit by SAR studies guided by SBDD identified indazole (IC 0.032 μM) and isoquinoline (IC 0.085 μM) as potent inhibitors of NOTUM. The binding of to NOTUM was rationalized through an X-ray co-crystal structure determination which showed a flipped binding orientation compared to . However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.
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http://dx.doi.org/10.1039/c9md00096hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727465PMC
August 2019

Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning.

Nat Commun 2019 08 15;10(1):3691. Epub 2019 Aug 15.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.

Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.
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http://dx.doi.org/10.1038/s41467-019-11683-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695400PMC
August 2019

iASPP mediates p53 selectivity through a modular mechanism fine-tuning DNA recognition.

Proc Natl Acad Sci U S A 2019 08 8;116(35):17470-17479. Epub 2019 Aug 8.

Ludwig Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom;

The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53-iASPP crystal structure reveals that iASPP displaces the p53 L1 loop-which mediates sequence-specific interactions with the signature-corresponding base-without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF-DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain-except the oncogenic E6 from human papillomaviruses (HPVs)-structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.
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http://dx.doi.org/10.1073/pnas.1909393116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717262PMC
August 2019

An Alternative In Vitro Method for Examining Nanoparticle-Induced Cytotoxicity.

Int J Toxicol 2019 Sep/Oct;38(5):385-394. Epub 2019 Jun 24.

Nanotechnology Core Facility, Office of Scientific Coordination, NCTR, FDA, Jefferson, AR, USA.

Conventional in vitro assays are often used as initial screens to identify potential toxic effects of nanoparticles (NPs). However, many NPs have shown interference with conventional in vitro assays, resulting in either false-positive or -negative outcomes. Here, we report an alternative method for the in vitro assessment of NP-induced cytotoxicity utilizing Fluoro-Jade C (FJ-C). To provide proof of concept and initial validation data, Ag-NPs and Au-NPs were tested in 3 different cell cultures including rat brain microvessel endothelial cells, mouse neural stem cells, and the human SH-SY5Y cell line. Conventional 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase (LDH) assays were run in parallel with the new method and served as references. The results demonstrate for the first time that FJ-C labeling can be a useful tool for assessing NP-induced cytotoxicity in vitro. Using these approaches, it was also demonstrated that removal of Ag-NPs-while keeping the Ag-ions that were released from the Ag-NPs in culture media-abolished the measured cytotoxicity, indicating that Ag-NPs rather than Ag-ions in solution contributed to the observed cytotoxic effects. Further, co-treatment of Ag-NPs with N-acetyl cysteine (NAC) prevented the observed cytotoxicity, suggesting a protective role of NAC in Ag-NP-induced cytotoxicity. Thus, this alternative in vitro assay is well suited for identify potential cytotoxicity associated with exposure to NPs.
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http://dx.doi.org/10.1177/1091581819859267DOI Listing
February 2020

Changes in antibiotic use following implementation of a telehealth stewardship pilot program.

Infect Control Hosp Epidemiol 2019 07;40(7):810-814

Division of Infectious Diseases & HIV Medicine,Case Western Reserve University School of Medicine,Cleveland, Ohio.

Starting in 2016, we initiated a pilot tele-antibiotic stewardship program at 2 rural Veterans Affairs medical centers (VAMCs). Antibiotic days of therapy decreased significantly (P < .05) in the acute and long-term care units at both intervention sites, suggesting that tele-stewardship can effectively support antibiotic stewardship practices in rural VAMCs.
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http://dx.doi.org/10.1017/ice.2019.128DOI Listing
July 2019

Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance.

Cell 2019 02 17;176(4):729-742.e18. Epub 2019 Jan 17.

University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK. Electronic address:

Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.
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http://dx.doi.org/10.1016/j.cell.2018.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370916PMC
February 2019

Designer protein delivers signal of choice.

Authors:
E Yvonne Jones

Nature 2019 01;565(7738):165-166

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http://dx.doi.org/10.1038/d41586-018-07883-zDOI Listing
January 2019

A randomized controlled laboratory study on the long-term effects of methylphenidate on cardiovascular function and structure in rhesus monkeys.

Pediatr Res 2019 02 17;85(3):398-404. Epub 2018 Dec 17.

Department of Pediatrics, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Oishei Children's Hospital, Roswell Park Comprehensive Cancer Center, Buffalo, NY, 14203, USA.

Background: Whether long-term methylphenidate (MPH) results in any changes in cardiovascular function or structure can only be properly addressed through a randomized trial using an animal model which permits elevated dosing over an extended period of time.

Methods: We studied 28 male rhesus monkeys (Macaca mulatta) approximately 7 years of age that had been randomly assigned to one of three MPH dosages: vehicle control (0 mg/kg, b.i.d., n = 9), low dose (2.5 mg/kg, b.i.d., n = 9), or high dose (12.5 mg/kg, b.i.d., n = 10). Dosage groups were compared on serum cardiovascular and inflammatory biomarkers, electrocardiograms (ECGs), echocardiograms, myocardial biopsies, and clinical pathology parameters following 5 years of uninterrupted dosing.

Results: With the exception of serum myoglobin, there were no statistical differences or apparent dose-response trends in clinical pathology, cardiac inflammatory biomarkers, ECGs, echocardiograms, or myocardial biopsies. The high-dose MPH group had a lower serum myoglobin concentration (979 ng/mL) than either the low-dose group (1882 ng/mL) or the control group (2182 ng/mL). The dose response was inversely proportional to dosage (P = .0006).

Conclusions: Although the findings cannot be directly generalized to humans, chronic MPH exposure is unlikely to be associated with increased cardiovascular risk in healthy children.
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http://dx.doi.org/10.1038/s41390-018-0256-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779032PMC
February 2019

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Nat Protoc 2018 12;13(12):2991-3017

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.
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http://dx.doi.org/10.1038/s41596-018-0075-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364805PMC
December 2018
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