Design of fast proteolysis-based signaling and logic circuits in mammalian cells.

Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, we present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chemical or biological signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.

Enhancing LGBT + primary healthcare in Slovenia: A national qualitative study of experiences and expectations of LGBT + people and family doctors.

BACKGROUND: Despite growing acceptance of LGBT + individuals, an underlying stigma persists even in healthcare, resulting in substandard care and worse healthcare outcomes for LGBT + individuals. OBJECTIVES: To examine and compare the experiences and expectations regarding primary healthcare among LGBT + individuals and general practitioners (GPs) in Slovenia. METHODS: We conducted an online national qualitative study using open-ended questions. To reach LGBT + population snowball method of recruitment was employed by sharing the questionnaire through LGBT + organisations, while GPs were invited by email of Association of family doctors in Slovenia. Anonymous data was collected from October to December 2021 and the questionnaires of 25 GPs and 90 LGBT + individuals of various ages, backgrounds, gender identities and sexual orientations were reviewed using thematic analysis. RESULTS: Both LGBT + participants and GPs expressed a desire for equal treatment. However, while all GPs claimed to treat all patients equally, LGBT + participants reported more varied experiences. Specific knowledge, especially on LGBT + terminology and healthcare, was perceived as lacking among GPs, leading LGBT + individuals to seek advice from specialists or community counselling. Systemic barriers, including societal stigmatisation and limited formal education on LGBT + issues, were identified, highlighting the need for designated safe spaces and improved GP training. Safety emerged as a central theme, crucial for fostering trust and disclosure between patients and healthcare providers. CONCLUSION: The study underscores the significance of a sense of safety in the patient-doctor relationship and highlights the need for improved training and attitudes to provide inclusive and affirming healthcare for LGBT + individuals.

Primary care settings must prioritise creating safe and non-judgmental environments for better LGBT + healthcare.Addressing formal GP education on LGBT + terminology, identities, and healthcare needs is crucial to provide inclusive care.Overcoming systemic barriers, such as societal prejudice and homophobia, requires creating visible safe space signals in primary care settings.

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage.

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.

The Onset of Depression in Middle-Aged Presumed Healthy Slovenian Family Practice Attendees and Its Associations with Genetic Risk Assessment, Quality of Life and Health Status: A Contribution for Family Medicine Practitioners' Early Detection.

Despite depression being a major driver of morbidity and mortality, the majority of primary care patients remain undiagnosed, so this study aimed to assess the prevalence of depression and the association with demographic and clinical variables, genetic risk, and quality of life. The participants were presumably healthy model family medicine practice (MFMP) attendees between 30 and 65 years of age and recruited during a preventive check-up in 2019. Each of the 40 pre-selected MFMP pragmatically invited 30 attendees to voluntarily participate. They completed a questionnaire of demographic, clinical, and social determinants, as well as a three-generational family history. The results were analyzed using multivariable modelling to calculate the associations with signs of depression. A modified Scheuner method was used to calculate the level genetic risk level using family history. Of 968 participants, aged 42.8 ± 8.6 years, 627 (64.8%) were women. The prevalence of depression was 4.1%. Signs of depression were negatively associated with health-related quality of life score, in particular in the domains of self-care (p = 0.001) and anxiety/depression (p < 0.001). Depression was also associated with predicted high risk for comorbidities given the family history (p = 0.030). Primary care directed at improving patients' quality of life should implement more widespread screening for mental health disorders. Family history for disease even beyond depression can be used by physicians as an important primary prevention tool.