PDZK1 is a four PDZ domain-containing scaffold protein that binds to scavenger receptor class B, type I (SR-BI), the high density lipoprotein receptor, by its first PDZ domain (PDZ1). PDZK1 knock-out mice exhibit a >95% decrease in hepatic SR-BI protein and consequently an ϳ70% increase in plasma cholesterol in abnormally large high density lipoprotein particles. These defects are corrected by hepatic overexpression of full-length PDZK1 but not the PDZ1 domain alone, which partially restores SR-BI protein abundance but not cell surface expression or function. We have generated PDZK1 knock-out mice with hepatic expression of four PDZK1 transgenes encoding proteins with nested C-terminal truncations: pTEM, which lacks the three C-terminal residues (putative PDZ-binding motif), and PDZ1.2, PDZ1.2.3, or PDZ1.2.3.4, which contain only the first two, three, or four N-terminal PDZ domains, respectively, but not the remaining C-terminal sequences. Hepatic overexpression of pTEM restored normal hepatic SR-BI abundance, localization, and function. Hepatic overexpression of PDZ1.2 or PDZ1.2.3 partially restored SR-BI abundance (ϳ12 or ϳ30% of wild type, respectively) but did not (PDZ1.2) or only slightly (PDZ1.2.3) restored hepatic SR-BI cell surface localization and function. Hepatic overexpression of PDZ1.2.3.4 completely restored SR-BI protein abundance, cell surface expression, and function (normalization of plasma cholesterol levels). Thus, all four PDZ domains in PDZK1, but not PDZ1-3 alone, are sufficient for its normal control of the abundance, localization, and therefore function of hepatic SR-BI, whereas the residues C-terminal to the PDZ4 domain, including the C-terminal putative PDZ-binding domain, are not required. SR-BI3 plays a significant role in lipoprotein metabolism as the high density lipoprotein (HDL) receptor (1). Through a mechanism called selective uptake (2-4), SR-BI mediates the uptake of cholesteryl esters from HDL and other lipoproteins into cells (5-10) as well as the bidirectional movement of unesterified cholesterol between cells and lipoproteins (11,12).SR-BI is most highly expressed in the liver and steroidogenic tissues (5), which exhibit the highest levels of HDL selective cholesterol uptake (2, 4). Deficiency of SR-BI in SR-BI knockout (KO) mice leads to elevated (ϳ2.2-fold) plasma cholesterol levels transported in abnormally large HDL particles with an unesterified cholesterol:total cholesterol ratio roughly double that of wild-type (WT) mice (13-16). This dyslipidemia is a consequence of the reduced uptake of cholesterol from plasma HDL by the liver and can be reversed by hepatic SR-BI transgene expression (17).Hepatic SR-BI activity is controlled by the intracellular adaptor protein PDZK1 (18 -20). PDZK1 is a four PDZ domaincontaining scaffold protein (see Fig. 1A, top left) that binds to the C termini of numerous membrane-associated transporter proteins, including ion channels (e.g. cystic fibrosis transmembrane conductance regulator) and cell surface receptors (19,21). Its most N-te...

Objectives Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) was identified in late 2019, spreading to over 200 countries and resulting in almost two million deaths worldwide. The emergence of safe and effective vaccines provides a route out of the pandemic, with vaccination uptake of 75-90% needed to achieve population protection. Vaccine hesitancy is problematic for vaccine rollout; global reports suggest only 73% of the population may agree to being vaccinated. As a result, there is an urgent need to develop equitable and accessible interventions to address vaccine hesitancy at the population level. Study Design & Method: We report the development of a scalable digital intervention seeking to address COVID-19 vaccine hesitancy and enhance uptake of COVID-19 vaccines in the UK. Guided by motivational interviewing (MI) principles, the intervention includes a series of therapeutic dialogues addressing 10 key concerns of vaccine hesitant individuals. Development of the intervention occurred linearly across four stages. During stage 1, we identified common reasons for COVID-19 vaccine hesitancy through analysis of existing survey data, a rapid systematic literature review, and public engagement workshops. Stage 2 comprised qualitative interviews with medical, immunological, and public health experts. Rapid content and thematic analysis of the data provided evidence-based responses to common vaccine concerns. Stage 3 involved the development of therapeutic dialogues through workshops with psychological and digital behaviour change experts. Dialogues were developed to address concerns using MI principles, including embracing resistance and supporting self-efficacy. Finally, stage 4 involved digitisation of the dialogues and pilot testing with members of the public. Discussion The digital intervention provides an evidence-based approach to addressing vaccine hesitancy through MI principles. The dialogues are user-selected, allowing exploration of relevant issues associated with hesitancy in a non-judgmental context. The text-based content and digital format allow for rapid modification to changing information and scalability for wider dissemination.

Background: Human challenge studies involve the deliberate exposure of healthy volunteers to an infectious micro-organism in a highly controlled and monitored way. They are used to understand infectious diseases and have contributed to the development of vaccines. In early 2020, the UK started exploring the feasibility of establishing a human challenge study with SARS-CoV-2. Given the significant public interest and the complexity of the potential risks and benefits, it is vital that public views are considered in the design and approval of any such study and that investigators and ethics boards remain accountable to the public. Methods: Mixed methods study comprising online surveys conducted with 2,441 UK adults and in-depth virtual focus groups with 57 UK adults during October 2020 to explore the public’s attitudes to a human challenge study with SARS-CoV-2 taking place in the UK. Results: There was overall agreement across the surveys and focus groups that a human challenge study with SARS-CoV-2 should take place in the UK. Transparency of information, trust and the necessity to provide clear information on potential risks to study human challenge study participants were important. The perceived risks of taking part included the risk of developing long-term effects from COVID, impact on personal commitments and mental health implications of isolation. There were a number of practical realities to taking part that would influence a volunteer’s ability to participate (e.g. Wi-Fi, access to exercise, outside space and work, family and pet commitments). Conclusions: The results identified practical considerations for teams designing human challenge studies. Recommendations were grouped: 1) messaging to potential study participants, 2) review of the protocol and organisation of the study, and 3) more broadly, making the study more inclusive and relevant. This study highlights the value of public consultation in research, particularly in fields attracting public interest and scrutiny.

Mammary MCF-10A cells seeded on reconstituted basement membrane form spherical structures with a hollow central lumen, termed acini, which are a physiologically relevant model of mammary morphogenesis. Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional protein overexpressed in breast cancer and ductal carcinoma in situ. When present in the nucleus Bag-1 is predictive of clinical outcome in breast cancer. Bag-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA. The long isoform of Bag-1, Bag-1L, contains a nuclear localisation sequence not present in the other isoforms. When present in the nucleus Bag-1L, but not the other Bag-1 isoforms, can interact with and modulate the activities of estrogen-, androgen- and vitamin D-receptors. Overexpression of Bag-1 mRNA in MCF-10A is known to produce acini with luminal filling reminiscent of ductal carcinoma in situ. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1L isoform is sufficient to drive premalignant change by developing a Bag-1L-overexpressing MCF-10A model. Two clones differentially overexpressing Bag-1L were grown in two-dimensional (2D) and three-dimensional (3D) cultures and compared with an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect growth factor responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared with controls. An abnormal branching morphology was also observed which correlated with the level of Bag-1L overexpression, suggesting further malignant change. Treatment with Thio-2, a small-molecule inhibitor of Bag-1, reduced the level of branching. In summary, 3D cultures of MCF-10A mammary epithelial cells overexpressing Bag-1L demonstrate a premalignant phenotype with features of ductal carcinoma in situ. Using this model to test the small-molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini that characterizes this premalignant change.