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1.
Toxicol Appl Pharmacol ; 440: 115913, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1671180

ABSTRACT

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.


Subject(s)
COVID-19 , Influenza, Human , Animals , Cell Communication , Cetylpyridinium/chemistry , Cetylpyridinium/pharmacology , Dinucleoside Phosphates , Humans , Immunity , Mammals , Mice , Microscopy, Fluorescence , Pandemics , Phosphatidylinositols , Rats , SARS-CoV-2 , Zebrafish
2.
Microb Cell Fact ; 21(1): 21, 2022 Feb 05.
Article in English | MEDLINE | ID: covidwho-1666655

ABSTRACT

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Viral/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mass Spectrometry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
3.
Archives of Disease in Childhood ; 106(SUPPL 1):A194, 2021.
Article in English | EMBASE | ID: covidwho-1495063

ABSTRACT

Background The COVID-19 pandemic required doctors to quickly adapt to new infection-control policies, rota restructuring and pathway changes. There was uncertainty on how the pandemic would affect paediatrics, as well as anxiety of the personal COVID-19 effects, frustration with media reporting and potential isolation with social-distancing measures. Our department recognised that both junior and senior doctors needed a platform to come together to address these feelings and reflect on them, ensuring a supportive team at work during this challenging period. Objectives To implement a supportive debrief session within the paediatric unit's teaching programme to improve team morale and reduce anxiety on the uncertainties of the pandemic. Methods During the first wave of the pandemic (April 2020- August 2020) we ran a weekly 'debrief hour' scheduled within the departmental teaching programme. Co-led by the college tutor, clinical director and trainee representatives, it was open to paediatric junior doctors and consultants. These sessions were held face-to-face and virtually. During the second wave (October 2020-February 2021) these sessions were held fortnightly and were focused on wellbeing. One week prior to each session questionnaires were completed anonymously by junior doctors to collate issues they wished to reflect on. Postdebrief surveys were completed by participants. Results We ran a total of 30 sessions. During the first wave 18 junior doctors and 5 consultants on average attended each debrief. Topics of discussion varied from difficult clinical cases and the emotional challenges of the pandemic to learning about individual approaches to mindfulness. Anecdotally junior doctors appreciated this dedicated time to 'offload' in a safe space. It helped forge bonds and personal connections within the team. Overall doctors were grateful their wellbeing was prioritised by these sessions. We took this initiative forward into the second wave where sessions became fortnightly. Our post-debrief surveys revealed that 96% (N=19) of junior doctors valued this time for team reflection and connection. 92% (N=19) found them useful. 94% (N=19) would like to see these sessions continue after the pandemic. Feedback included junior doctors 'feeling supported', 'paediatrics being the best team' and 'bonding during a worrying time'. From March 2021 these sessions will be led by the department's clinical psychologist. Conclusions • We advocate scheduling a supportive debrief session within departmental teaching programmes, especially in times of uncertainty and potential anxiety (such as global pandemics). This encourages team bonding. • Embedding debrief sessions within the teaching programme sends a clear message to junior doctors that the department prioritises and promotes the wellbeing of doctors, seeing it as an important part of their working lives. • Supportive debrief sessions allow doctors a safe space to 'offload' with their peers in a reflective, relaxed environment, thus helping create a sense of community at work and improving morale. • The department will continue to hold scheduled team debriefs, which will carry on after the pandemic. The Trust recognises the importance of championing wellbeing;moving forward time has been allocated for a clinical psychologist to lead these sessions. • We strongly recommend that budget planning includes provision for trainee wellbeing support services.

4.
Preprint in English | bioRxiv | ID: ppbiorxiv-449540

ABSTRACT

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach used the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest intact CBM9 fusion protein incorporates spike protein amino acids 540-588, which is a conserved region immediately C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

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