COVID-19 Related Shifts in Social Interaction, Connection, and Cohesion Impact Psychosocial Health: Longitudinal Qualitative Findings from COVID-19 Treatment Trial Engaged Participants.

While effective for slowing the transmission of SARS-CoV-2, public health measures, such as physical distancing and stay-at-home orders, have significantly shifted the way people interact and maintain social connections. To better understand how people sought social and psychological support amid the pandemic, we conducted a longitudinal qualitative evaluation of participants enrolled in a COVID-19 treatment trial (N = 30). All participants from the parent trial who consented to being contacted for future research studies were recruited electronically via email, and first-round virtual interviews were conducted between December 2020 and March 2021. Participants who participated in first-round interviews were contacted again, and follow-up interviews were conducted in January-February 2022. The results reported significant shifts in how participants connected to social support, including changes from physical to virtual modalities, and using different social networks for distinct purposes (i.e., Reddit/Facebook for information, WhatsApp for community connection). While having COVID-19, profound loneliness during isolation was described; yet, to mitigate effects, virtual support (i.e., emotional, knowledge-seeking) as well as in-person material support (e.g., groceries, snow-shoveling), were key. Public health efforts are needed to develop interventions that will improve the narratives about mental health challenges related to COVID-19 isolation, and to provide opportunities to share challenges in a supportive manner among social networks. Supporting social cohesion, despite the everchanging nature of COVID-19, will necessitate innovative multimodal strategies that learn from lived experiences across various stages of the pandemic.

Design and characterization of a salicylic acid-inducible gene expression system for Jurkat cells.

With continued progress in cell and gene therapies, there is an immediate need for exogenously tunable gene expression systems with safe and predictable behavior in specific human cell types. Here, we demonstrate the ability of the salicylic acid (SA)-inducible MarR repressor protein from Escherichia coli to regulate target gene expression in a human T lymphocyte cell line. Two lentiviral vectors, one encoding an enhanced green fluorescent protein (EGFP) reporter cassette and the other a repressor cassette, were sequentially transduced into Jurkat cells, using fluorescence-activated cell sorting (FACS) to isolate stable Jurkat progeny. As a result, EGFP expression was repressed by MarR and was inducible upon the addition of SA (~1.3 fold). This represents the first example of functional expression of bacterial MarR in mammalian cells, and opens the possibility for further development of regulated, SA-tunable gene expression system for T-cells.

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid.

We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Small-molecule inducible transcriptional control in mammalian cells.

Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies.