Circadian Rhythms in the light of COVID-19: Formulating optimal time-of-day regimens for antiviral drugs using human 3D models and in silico modelling
Lead Research Organisation:
University of Liverpool
Department Name: Musculoskeletal & Ageing Science
Abstract
A number of antiviral, immunomodulatory and anti-inflammatory drugs are being repurposed for COVID-19. Clinical trials worldwide are testing their use as a treatment and/or prophylactic. At present, no clinically available antiviral drugs have been established for SARS-CoV-2. We have recently prioritized promising drug candidates for COVID-19 based on in vivo pharmacokinetic drug modelling in target tissues. Moreover, a number of new animal models are being tested in SARS CoV-2 infections studies in combination with promising drug candidates. Human immune defence, inflammatory responses as well as efficacy of therapeutics and vaccines follow robust daily circadian rhythms, but their role in SARS-CoV-2 infection has yet to be studied. Moreover, recent systems-level studies have defined many host factors and physiological pathways as potential therapeutic targets, many of them which are under the circadian control. To devise therapeutic strategies to counteract SARS CoV-2 infection and the associated COVID-19 pathology, it is crucial to understand how SARS COV-2 affects the host circadian rhythms during infection, and to apply this knowledge towards improved repurposing of existing drugs and development of new drugs and drug formulations.
Our interdisciplinary study aims to determine optimal time-of-day regimens for currently repurposed antiviral COVID-19 drugs and develop in silico mathematical models for their time-of-day use in vivo. This will provide time-of-day information on when such drugs exert most potent effects (on viral load and/or viral-induced inflammation) without perturbing circadian clock timing (least toxic). Disruption of circadian timing has serious consequences on many physiological processes especially given its essential role in drug metabolism and recovery from bacterial/viral infections with emerging relevance to SARS-CoV-2. This inter-disciplinary project will lead to novel understanding of how SARS-2 viral infection impacts human circadian rhythms and clock-controlled inflammatory pathways. This project will utilise cutting-edge molecular and biochemical techniques (gene/protein expression, 'omic' profiling, real-time bioluminescence imaging) to analyse cellular and organ-level circadian rhythms upon SARS CoV-2 infection using susceptible human cell types and experimental animal models as well as combined in vitro/in vivo pharmacokinetic drug modelling to project optimal times-of-administration of antiviral drugs in a human setting. This new knowledge will provide new insights on time-of-day dosing profiles of current repurposed COVID-19 drugs, which will have help guide ongoing/future clinical trials to obtain improved clinical outcomes and in vivo experimental studies. This study will also uncover novel drug targets at the interface between circadian control and SARS CoV-2 viral infection to aid development of improved drug formulations thus moving towards precision medicine based on chronotherapy for COVID-19.
Our interdisciplinary study aims to determine optimal time-of-day regimens for currently repurposed antiviral COVID-19 drugs and develop in silico mathematical models for their time-of-day use in vivo. This will provide time-of-day information on when such drugs exert most potent effects (on viral load and/or viral-induced inflammation) without perturbing circadian clock timing (least toxic). Disruption of circadian timing has serious consequences on many physiological processes especially given its essential role in drug metabolism and recovery from bacterial/viral infections with emerging relevance to SARS-CoV-2. This inter-disciplinary project will lead to novel understanding of how SARS-2 viral infection impacts human circadian rhythms and clock-controlled inflammatory pathways. This project will utilise cutting-edge molecular and biochemical techniques (gene/protein expression, 'omic' profiling, real-time bioluminescence imaging) to analyse cellular and organ-level circadian rhythms upon SARS CoV-2 infection using susceptible human cell types and experimental animal models as well as combined in vitro/in vivo pharmacokinetic drug modelling to project optimal times-of-administration of antiviral drugs in a human setting. This new knowledge will provide new insights on time-of-day dosing profiles of current repurposed COVID-19 drugs, which will have help guide ongoing/future clinical trials to obtain improved clinical outcomes and in vivo experimental studies. This study will also uncover novel drug targets at the interface between circadian control and SARS CoV-2 viral infection to aid development of improved drug formulations thus moving towards precision medicine based on chronotherapy for COVID-19.
Organisations
- University of Liverpool (Lead Research Organisation)
- University of Manchester (Collaboration)
- University of Sheffield (Collaboration)
- UNIVERSITY OF NOTTINGHAM (Collaboration)
- DURHAM UNIVERSITY (Collaboration)
- UNIVERSITY OF GLASGOW (Collaboration)
- University of Belgrade (Collaboration)
- Newcastle University (Collaboration)
- UNIVERSITY OF LIVERPOOL (Collaboration)
Publications
Giannos P
(2023)
Shorter sleep duration is associated with greater visceral fat mass in US adults: Findings from NHANES, 2011-2014.
in Sleep medicine
Pekovic-Vaughan V
(2023)
Antioxidant transcription factor NRF2 regulates skeletal muscle homeostasis through a circadian mechanism that is disrupted with ageing
in Free Radical Biology and Medicine
Sutton E
(2021)
NRF2/KEAP1 pathway is required to fine-tune circadian oscillations as part of the negative feedback loop of the molecular clock: implications for tissue homeostasis and therapeutic interventions
in Free Radical Biology and Medicine
Sutton E
(2021)
A novel role for an antioxidant transcription factor Nrf2 as a transcriptional repressor of the circadian molecular clock
in Free Radical Biology and Medicine
Zakic T
(2023)
Redox-metabolic reprogramming of skin in mice lacking functional Nrf2 under basal conditions and cold acclimation.
in BioFactors (Oxford, England)
Zakic T
(2023)
Redox and metabolic reprogramming in breast cancer and cancer-associated adipose tissue.
in FEBS letters
Zakic T
(2023)
The discrepancy between morphological and functional activation of brown adipose tissue in the absence of functional Nrf2
in Free Radical Biology and Medicine
Zakic T
(2022)
Structural and redox-metabolic remodelling of brown adipose tissue in mice lacking nuclear factor erythroid 2-related factor 2 under basal conditions and cold acclimation
in Free Radical Biology and Medicine
| Title | Cellular Vessel |
| Description | Through this film, it is depicted the desynchronisation between the rhythms of the expanding port of Liverpool, and the biological rhythms of local residents |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2022 |
| Impact | The film was shown to the two local communities in Liverpool, leading to discussions with local residents and their participation in the video making. It led to raising awareness of social determinants of ageing that impact on their daily lives and healthspan, including the highest risk of chronic lung disease in the UK. |
| URL | https://www.liverpool.ac.uk/sociology-social-policy-and-criminology/research/research-projects/dysrh... |
| Description | We are developing human reporter cell line susceptible to SARS-COV2 infection which can be used to replace animals in research an provide a drug screening platform for antiviral candidates. |
| Exploitation Route | By using in vitro molecular tools to screen for promising drug candidates and model pharmacological interactions |
| Sectors | Healthcare |
| Description | Our human reporter lines are being used for testing novel synthetic drugs developed by our industrial collaborator. |
| First Year Of Impact | 2022 |
| Sector | Pharmaceuticals and Medical Biotechnology |
| Description | training of Mres Clinical Sciences researchers including majority of final year Medical Degree students |
| Geographic Reach | National |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Description | training of practitioners or researchers -MRes Clinical Sciences |
| Geographic Reach | National |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Impact | Influenced training of practitioners or researchers - training of MRes Clinical Sciences researchers including majority of intercalating Medical Degree students |
| Description | Biotechnology and Biological Sciences Research Council (BBSRC): - The role of circadian rhythms and redox signalling in musculoskeletal stem cell ageing |
| Amount | £100,000 (GBP) |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2021 |
| End | 09/2025 |
| Description | Circadian disruption and fibrosis progression in Systemic Sclerosis: towards improved prognosis and chrono-based therapeutic stratification |
| Amount | £129,781 (GBP) |
| Funding ID | CF-2021-2 133 |
| Organisation | Rosetrees Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 12/2022 |
| End | 12/2025 |
| Description | Circadian rhythms in neuromuscular function: the effects of age and exercise |
| Amount | £180,000 (GBP) |
| Organisation | Government of Libya |
| Sector | Public |
| Country | Libya |
| Start | 09/2022 |
| End | 09/2025 |
| Description | EcMagine': Extracellular Matrix ageing across the life course interdisciplinary research network |
| Amount | £176,276 (GBP) |
| Funding ID | BB/W018314/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2022 |
| End | 02/2024 |
| Description | Exploring new avenues in breast cancer research: Redox and metabolic reprogramming of cancer and associated adipose tissue |
| Amount | € 300,000 (EUR) |
| Organisation | University of Belgrade |
| Sector | Academic/University |
| Country | Serbia |
| Start | 02/2022 |
| End | 02/2025 |
| Description | Investigating the mechanisms and therapeutic potential of targeting circadian clocks in collagen VI myopathies |
| Amount | £70,688 (GBP) |
| Funding ID | 21GRO-PG12-0532 |
| Organisation | Muscular Dystrophy UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 07/2022 |
| End | 07/2023 |
| Description | Time to heal: circadian regulation of chronic wound repair |
| Amount | £80,000 (GBP) |
| Organisation | Liverpool John Moores University |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 02/2022 |
| End | 02/2025 |
| Description | Understanding the role of physical activity in the entrainment mechanisms of circadian biological cycles' |
| Amount | £20,000 (GBP) |
| Organisation | The Royal Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2021 |
| End | 09/2022 |
| Title | Circadian tissue and cell timecourses |
| Description | We have harvested and preserved precious tissue and cell samples for both biochemistry and histology from circadian time-courses from different environmental conditions, ages, genotypes and gender. |
| Type Of Material | Biological samples |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | This will expand our outputs and collaborations and enable multiple forms of scientific investigation. |
| Title | Clock reporter cell line for drug screening |
| Description | This is essential tool for reporting biological oscillations in real time and using it for drug screening |
| Type Of Material | Cell line |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | It will expand our ability to test time-of-day drugs and treatments in mammalian cells ex vivo |
| Title | Human tissue biopsies |
| Description | We have developed a method for measuring biological cycles in real time from human biopsies. This will help progress research in examining disruption of biological cycles as a result of disease, ageing or injury and can be used as a outcome measure. |
| Type Of Material | Physiological assessment or outcome measure |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | We have developed a method for measuring biological cycles in real time from tissue biopsies. This will help progress research in examining disruption of biological cycles as a result of disease, ageing or injury and can be used as a outcome measure. |
| Title | Plasmid tools for assessing circadian oscillations |
| Description | Essential tool for reporting biological oscillations in real time |
| Type Of Material | Model of mechanisms or symptoms - in vitro |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | It will expand our ability to test time-of-day drugs and treatments in mammalian cells in vitro |
| Title | Reporter cell line for antiviral drug screening |
| Description | Our team has created a reporter plasmid which can be used as an in vitro readout of clock gene activation during SARS-COV2 infection. We have hence generated a stable cell line having this reporter which can be used for real-time bioluminescence imaging of pharmacological drugs and stimuli involved in activation of this reporter. |
| Type Of Material | Cell line |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | This will enable reporting clock gene activity in response to SARS-COV2 infection in susceptible human lung cell lines. |
| Title | Mathematical model of clock oscillations |
| Description | The model in question is addressing a mathematical challenge that will provide a first ever model of these interactions. The identification of a suitable detailed clock model will be made was made following reviews of most recent models. A detailed model of different molecular pathways was combined to the clock model to generate a more complete model which we can then use to generate further experimental predictions. Understanding of these network interactions between transcription factors and the circadian timing system will aid future development of novel chrono-based drug therapies for a number of age-related diseases. |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | It will expand our ability to predict outcomes of time-of-day drug treatments, interventions and molecular pathway interactions. |
| Description | CIMA ageing network |
| Organisation | Newcastle University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, resources and data. |
| Collaborator Contribution | We share expertise, resources and data. |
| Impact | This is a multi-disciplinary collaboration of basic scientists, clinicians and industrial partners. We have published joint publications and obtained joint research funding including industrial pump-priming award as well as an omics profiling award. |
| Start Year | 2022 |
| Description | CIMA ageing network |
| Organisation | University of Liverpool |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, resources and data. |
| Collaborator Contribution | We share expertise, resources and data. |
| Impact | This is a multi-disciplinary collaboration of basic scientists, clinicians and industrial partners. We have published joint publications and obtained joint research funding including industrial pump-priming award as well as an omics profiling award. |
| Start Year | 2022 |
| Description | CIMA ageing network |
| Organisation | University of Sheffield |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, resources and data. |
| Collaborator Contribution | We share expertise, resources and data. |
| Impact | This is a multi-disciplinary collaboration of basic scientists, clinicians and industrial partners. We have published joint publications and obtained joint research funding including industrial pump-priming award as well as an omics profiling award. |
| Start Year | 2022 |
| Description | Nevragenics-led retinoid network UK |
| Organisation | Durham University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have shared resources, data and expertise. It is a multi-disciplinary collaboration involving chemistry, maths, molecular biology, pharmacology, chronobiology. We have obtained industrial pump-priming funding from UKRI and are in the process of applying for a KTP through Innovate UK. |
| Collaborator Contribution | We have shared resources, data and expertise. It is a multi-disciplinary collaboration involving chemistry, maths, molecular biology, pharmacology, chronobiology. We have obtained industrial pump-priming funding from UKRI and are in the process of applying for a KTP through Innovate UK. |
| Impact | We have shared resources, data and expertise. It is a multi-disciplinary collaboration involving chemistry, maths, molecular biology, pharmacology, chronobiology. We have obtained industrial pump-priming funding from UKRI and are in the process of applying for a KTP through Innovate UK. |
| Start Year | 2022 |
| Description | REFRAME redox-metabolic network |
| Organisation | University of Belgrade |
| Department | First Surgical Clinic, Clinical Center of Serbia, University of Belgrade, Serbia |
| Country | Serbia |
| Sector | Academic/University |
| PI Contribution | We have shared expertise, resources and data. |
| Collaborator Contribution | We have shared expertise, resources and data. |
| Impact | We are a multidisciplinary network involving redox biology, biochemistry, chronobiology, imaging and metabolic science. We have published articles together and disseminated our work at scientific conferences as invited speakers, symposium chairs and round table panelists. |
| Start Year | 2022 |
| Description | UKRI-funded ECMAge ageing network |
| Organisation | Newcastle University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Collaborator Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Impact | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. We are planning to contribute a joint manuscript on the current information in the field of ECMageing. We have already contributed to scientific dissemination activities on several national and international conferences and learned societies. We are a multi-disciplinary team involving chronobiology, maths, systems biology, mechano biology, biochemistry and tissue engineering. |
| Start Year | 2022 |
| Description | UKRI-funded ECMAge ageing network |
| Organisation | University of Glasgow |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Collaborator Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Impact | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. We are planning to contribute a joint manuscript on the current information in the field of ECMageing. We have already contributed to scientific dissemination activities on several national and international conferences and learned societies. We are a multi-disciplinary team involving chronobiology, maths, systems biology, mechano biology, biochemistry and tissue engineering. |
| Start Year | 2022 |
| Description | UKRI-funded ECMAge ageing network |
| Organisation | University of Liverpool |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Collaborator Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Impact | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. We are planning to contribute a joint manuscript on the current information in the field of ECMageing. We have already contributed to scientific dissemination activities on several national and international conferences and learned societies. We are a multi-disciplinary team involving chronobiology, maths, systems biology, mechano biology, biochemistry and tissue engineering. |
| Start Year | 2022 |
| Description | UKRI-funded ECMAge ageing network |
| Organisation | University of Manchester |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Collaborator Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Impact | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. We are planning to contribute a joint manuscript on the current information in the field of ECMageing. We have already contributed to scientific dissemination activities on several national and international conferences and learned societies. We are a multi-disciplinary team involving chronobiology, maths, systems biology, mechano biology, biochemistry and tissue engineering. |
| Start Year | 2022 |
| Description | UKRI-funded ECMAge ageing network |
| Organisation | University of Nottingham |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Collaborator Contribution | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. |
| Impact | We share expertise, data, resources and get involved in outreach events. I am on the network operational group and contribute to regular meeting updates with set agenda, including pump-priming opportunities and training for ECRs. We are planning to contribute a joint manuscript on the current information in the field of ECMageing. We have already contributed to scientific dissemination activities on several national and international conferences and learned societies. We are a multi-disciplinary team involving chronobiology, maths, systems biology, mechano biology, biochemistry and tissue engineering. |
| Start Year | 2022 |
| Description | the circadian clock and covid-19 drugs |
| Organisation | University of Liverpool |
| Department | Department of Molecular and Clinical Pharmacology |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Our team has provided transgenic animal models, intellectual input and technical expertise. |
| Collaborator Contribution | The collaborating team has provided transgenic animals, intellectual input and technical expertise. |
| Impact | We have a joint paper in submission and have submitted a grant application. Three disciplines are involved: pharmacology, biology and mathematics. |
| Start Year | 2020 |
| Description | the circadian in silico modelling |
| Organisation | University of Liverpool |
| Department | School of Engineering |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Our team has provided transgenic animal models, intellectual input and technical expertise. |
| Collaborator Contribution | The collaborating team has provided intellectual input and technical expertise. |
| Impact | We have three papers in submission. We have applied for joint grant applications and some have been successfully awarded. This is a multidisciplinary collaboration between biology, maths and chemistry. |
| Start Year | 2020 |
| Title | Chrono-therapy for smarter use of retinoids |
| Description | Novel synthetic retinoids currently tested in preclinical models using circadian medicine approaches. It is expected that this will inform the clinical trial in 2024 on neuromuscular degeneration as well as clinical studies testing their use in other diseases. |
| Type | Therapeutic Intervention - Drug |
| Current Stage Of Development | Refinement. Non-clinical |
| Year Development Stage Completed | 2022 |
| Development Status | Under active development/distribution |
| Impact | This is a lead Nevrargenics drug for the treatment of neurodegenerative and neuromuscular disorders-it will improve the company's delivery of the product for many people with said disorders. It is also expected to have wider uses in connective tissue diseases. |
| Description | Mechanisms and interventions in ageing |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | The purpose was to stimulate thinking and improve understanding. I led one of the four round table discussions as part of the UKRI-funded ECM Age network group. The round table discussion was on the mechanisms and interventions in ageing, which stimulated participants to share their insights and experiences and inform further research. This activity will be repeated in 2023. It led to some joint pump-priming research applications with feedback from public and participants. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Meet the Scientist event at the World Museum, Liverpool |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | The purpose was to to stimulate thinking and share information. The event engaged lots of kids, parents and grandparents, measured their grip strength and heart rate in the morning and afternoon, which sparked their interest in body clocks. The outcome was to an invite to repeat the activity at the Museum in 2023. Also, this activity was carried out at a smaller scale in a local school as a follow-up. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Network Press Release |
| Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Media (as a channel to the public) |
| Results and Impact | Press release on the new network BBSRC/MRC funded grant to tackle ageing. The project grant focuses on interdisciplionary approaches to tackle ageing of extracellular matrix, which is an important structure involved in cellular physiology of many tissues from pulmonary, cutaneous to musculoskeletal systems. Its implications in ageing populations, age-related conditions and Covid-related pathophysiology will also be covered. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://www.liverpool.ac.uk/research/news/articles/liverpool-to-lead-on-new-research-network-to-tack... |
| Description | Utilising chronobiology in assessing sleep quality in chronic disease |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Patients, carers and/or patient groups |
| Results and Impact | The purpose was to share information and inform decision making. I presented a talk on sleep quality to a patient group as part of a workshop on lifestyle interventions in chronic disease; this led to patient interest in the research carried out, discussion on patient experiences and the impact of medication and/or condition on sleep quality; we then formulated important research questions for further research. |
| Year(s) Of Engagement Activity | 2022 |