Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location.

G protein-coupled receptors (GPCRs) are membrane bound signaling molecules that regulate many aspects of human physiology. Recent advances have demonstrated that GPCR signaling can occur both at the cell surface and internal cellular membranes. Our findings suggest that cannabinoid receptor 1 (CB1) signaling is highly dependent on its subcellular location. We find that intracellular CB1 receptors predominantly couple to Gαi while plasma membrane receptors couple to Gαs. Here we show subcellular location of CB1, and its signaling, is contingent on the choice of promoters and receptor tags. Heterologous expression with a strong promoter or N-terminal tag resulted in CB1 predominantly localizing to the plasma membrane and signaling through Gαs. Conversely, CB1 driven by low expressing promoters and lacking N-terminal genetic tags largely localized to internal membranes and signals via Gαi. Lastly, we demonstrate that genetically encodable non-canonical amino acids (ncAA) offer a solution to the problem of non-native N-terminal tags disrupting CB1 signaling. We identified sites in CB1R and CB2R which can be tagged with fluorophores without disrupting CB signaling or trafficking using (trans-cyclooctene attached to lysine (TCO*A)) and copper-free click chemistry to attach fluorophores in live cells. Together, our data demonstrate the origin of location bias in cannabinoid signaling which can be experimentally controlled and tracked in living cells through promoters and novel CBR tagging strategies.

Exploring the role of professional identity in the implementation of clinical decision support systems-a narrative review.

BACKGROUND: Clinical decision support systems (CDSSs) have the potential to improve quality of care, patient safety, and efficiency because of their ability to perform medical tasks in a more data-driven, evidence-based, and semi-autonomous way. However, CDSSs may also affect the professional identity of health professionals. Some professionals might experience these systems as a threat to their professional identity, as CDSSs could partially substitute clinical competencies, autonomy, or control over the care process. Other professionals may experience an empowerment of the role in the medical system. The purpose of this study is to uncover the role of professional identity in CDSS implementation and to identify core human, technological, and organizational factors that may determine the effect of CDSSs on professional identity. METHODS: We conducted a systematic literature review and included peer-reviewed empirical studies from two electronic databases (PubMed, Web of Science) that reported on key factors to CDSS implementation and were published between 2010 and 2023. Our explorative, inductive thematic analysis assessed the antecedents of professional identity-related mechanisms from the perspective of different health care professionals (i.e., physicians, residents, nurse practitioners, pharmacists). RESULTS: One hundred thirty-one qualitative, quantitative, or mixed-method studies from over 60 journals were included in this review. The thematic analysis found three dimensions of professional identity-related mechanisms that influence CDSS implementation success: perceived threat or enhancement of professional control and autonomy, perceived threat or enhancement of professional skills and expertise, and perceived loss or gain of control over patient relationships. At the technological level, the most common issues were the system's ability to fit into existing clinical workflows and organizational structures, and its ability to meet user needs. At the organizational level, time pressure and tension, as well as internal communication and involvement of end users were most frequently reported. At the human level, individual attitudes and emotional responses, as well as familiarity with the system, most often influenced the CDSS implementation. Our results show that professional identity-related mechanisms are driven by these factors and influence CDSS implementation success. The perception of the change of professional identity is influenced by the user's professional status and expertise and is improved over the course of implementation. CONCLUSION: This review highlights the need for health care managers to evaluate perceived professional identity threats to health care professionals across all implementation phases when introducing a CDSS and to consider their varying manifestations among different health care professionals. Moreover, it highlights the importance of innovation and change management approaches, such as involving health professionals in the design and implementation process to mitigate threat perceptions. We provide future areas of research for the evaluation of the professional identity construct within health care.

Trifunctional Sphinganine: A New Tool to Dissect Sphingolipid Function.

Functions and cell biology of the sphingolipids sphingosine and sphinganine in cells are not well understood. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. Here, we prepared multifunctionalized derivatives of the two lipid species that differ only in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distributions within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies of the cellular functions of sphingosine and sphinganine.

Overcoming the not-invented-here syndrome in healthcare: The case of German ambulatory physiotherapists' adoption of digital health innovations.

Healthcare is characterized by professional, organizational, and institutional boundaries. Digital health innovations can help overcome these boundaries by providing information access to all healthcare professionals. Such innovations emerge from inputs from different health professionals at different positions along the entire care process and have the potential to substantially change the way in which interprofessional tasks are performed among the involved professionals. Consequently, as less empowered professionals, physiotherapists may resist the adoption of digital health innovations in particular if the innovation is dominated by physicians, and thus the not-invented-here syndrome may become a major barrier. We aim to examine whether the origin of a digital health innovation affects German physiotherapists' adoption decision and whether the collaboration quality and physiotherapists' proactive job crafting behavior may help overcome adoption barriers. We applied a mixed-method sequential design with a qualitative study one in which we interviewed 20 physiotherapists to provide exploratory insights, and a quantitative study two in which we tested our proposed hypotheses with survey data including an experimental vignette from 165 physiotherapists. Physiotherapists adopt digital health innovations developed by their own professional group more likely than digital health innovations developed by physicians. Our results also confirm that physiotherapists' job crafting behavior and the quality of the collaboration with physicians weaken the resistance against physician-driven innovations. Our study underlines (1) the need to involve allied health professionals as physiotherapists in digital health innovation development, (2) the relevance of interprofessional collaboration in daily practice and, (3) an open mind set of allied health professionals to cope with innovation adoption barriers.

Challenges in the Implementation of Disruptive Innovations in Health Care Organizations.

Health organizations face barriers when seeking to deploy radical innovations, such as innovative telemonitoring approaches or AI based Clinical Decision Support Systems (CDSS) into their clinical workflow. However, these barriers are of various types and rarely known to organizations and their management. This study conducted a systematic literature review of 99 selected studies to identify the implementation barriers and factors encountered in this process. Using a hierarchical framework comprising of strategies, resources and capabilities, and processes, the study examined 16 barriers generated from the analysis of the individual studies. The findings highlight implementation barriers on all three levels of the proposed framework. By addressing these barriers comprehensively, health care organizations can successfully implement radical health innovations and enhance patient care outcomes and health care delivery.

Synaptotagmin 1-triggered lipid signaling facilitates coupling of exo- and endocytosis.

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.

Chemical Tools for Lipid Cell Biology.

Lipids are key components of all organisms. We are well educated in their use as fuel and their essential role to form membranes. We also know much about their biosynthesis and metabolism. We are also aware that most lipids have signaling character meaning that a change in their concentration or location constitutes a signal that helps a living cell to respond to changes in the environment or to fulfill its specific function ranging from secretion to cell division. What is much less understood is how lipids change location in cells over time and what other biomolecules they interact with at each stage of their lifetime. Due to the large number of often quite similar lipid species and the sometimes very short lifetime of signaling lipids, we need highly specific tools to manipulate and visualize lipids and lipid-protein interactions. If successfully applied, these tools provide fabulous opportunities for discovery.In this Account, I summarize the development of synthetic tools from our lab that were designed to address crucial properties that allow them to function as tools in live cell experiments. Techniques to change the concentration of lipids by adding a small molecule or by light are described and complemented by examples of biological findings made when applying the tools. This ranges from chemical dimerizer-based systems to synthetic "caged" lipid derivatives. Furthermore, I discuss the problem of locating a lipid in an intact cell. Synthetic molecular probes are described that help to unravel the lipid location and to determine their binding proteins. These location studies require in-cell lipid tagging by click chemistry, photo-cross-linking to prevent further movement and the "caging" groups to avoid premature metabolism. The combination of these many technical features in a single tool allows for the analysis of not only lipid fluxes through metabolism but also lipid transport from one membrane to another as well as revealing the lipid interactome in a cell-dependent manner. This latter point is crucial because with these multifunctional tools in combination with lipidomics we can now address differences in healthy versus diseased cells and ultimately find the changes that are essential for disease development and new therapeutics that prevent these changes.

Developments in the epidemiology and surgical management of patella fractures in Germany.

BACKGROUND: Patella fractures account for approximately 1% of all skeletal injuries. Treatment options are vast and no definitive conclusion on what option is the most beneficial could be made so far. Plate osteosynthesis appears to gain in importance. We aim to give insight into the more recent trends and developments as well as establish the epidemiology of patella fractures in Germany by analysing treatment and epidemiological data from a national database. METHODS: Anonymised data was retrieved form a national database. In the period of 2006 to 2020, all patients with patella fractures as defined in ICD-10 GM as their main diagnosis, who were treated in a German hospital were included. Patients were divided into subgroups based on gender and age. Age groups were created in 10-year intervals from 20 years old up to 80 years old with one group each encompassing all those above the age of 80 years old and below 20 years old and younger. Linear regression was performed were possible to determine statistical significance of possible trends. RESULTS: A total of 151,435 patellar fractures were reported. 95,221 surgical interventions were performed. Women were about 1.5 times more likely to suffer from patella fracture than men. The relative number of surgical interventions rose from about 50% in 2006 to 75% in 2020. Most surgical interventions are performed in those over the age of 50. The incidence of complex fractures and plate osteosynthesis has significantly increased throughout the analysed period. CONCLUSIONS: We found a clear trend for surgical treatment in Germany with an increase in surgical procedures. We could also show that this ratio is age-related, making it more likely for younger patients in the age groups from 0 to 70 years old to receive surgical treatment for their patella fracture.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Genetic Deletion of Mmp9 Does Not Reduce Airway Inflammation and Structural Lung Damage in Mice with Cystic Fibrosis-like Lung Disease.

Elevated levels of matrix metalloprotease 9 (MMP-9) and neutrophil elastase (NE) are associated with bronchiectasis and lung function decline in patients with cystic fibrosis (CF). MMP-9 is a potent extracellular matrix-degrading enzyme which is activated by NE and has been implicated in structural lung damage in CF. However, the role of MMP-9 in the in vivo pathogenesis of CF lung disease is not well understood. Therefore, we used ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice as a model of CF-like lung disease and determined the effect of genetic deletion of Mmp9 (Mmp9-/-) on key aspects of the pulmonary phenotype. We found that MMP-9 levels were elevated in the lungs of ßENaC-Tg mice compared with wild-type littermates. Deletion of Mmp9 had no effect on spontaneous mortality, inflammatory markers in bronchoalveolar lavage, goblet cell metaplasia, mucus hypersecretion and emphysema-like structural lung damage, while it partially reduced mucus obstruction in ßENaC-Tg mice. Further, lack of Mmp9 had no effect on increased inspiratory capacity and increased lung compliance in ßENaC-Tg mice, whereas both lung function parameters were improved with genetic deletion of NE. We conclude that MMP-9 does not play a major role in the in vivo pathogenesis of CF-like lung disease in mice.

Data platforms for open life sciences-A systematic analysis of management instruments.

Open data platforms are interfaces between data demand of and supply from their users. Yet, data platform providers frequently struggle to aggregate data to suit their users' needs and to establish a high intensity of data exchange in a collaborative environment. Here, using open life science data platforms as an example for a diverse data structure, we systematically categorize these platforms based on their technology intermediation and the range of domains they cover to derive general and specific success factors for their management instruments. Our qualitative content analysis is based on 39 in-depth interviews with experts employed by data platforms and external stakeholders. We thus complement peer initiatives which focus solely on data quality, by additionally highlighting the data platforms' role to enable data utilization for innovative output. Based on our analysis, we propose a clearly structured and detailed guideline for seven management instruments. This guideline helps to establish and operationalize data platforms and to best exploit the data provided. Our findings support further exploitation of the open innovation potential in the life sciences and beyond.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response.

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1's kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL's enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

"Flash & Click": Multifunctionalized Lipid Derivatives as Tools To Study Viral Infections.

In this perspective article, we describe the current status of lipid tools for studying host lipid-virus interactions at the cellular level. We discuss the potential lipidomic changes that viral infections impose on host cells and then outline the tools available and the resulting options to investigate the host cell lipid interactome. The future outcome will reveal new targets for treating virus infections.

Recruitment of a splicing factor to the nuclear lamina for its inactivation.

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease.

BACKGROUND: Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. METHODS: Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. RESULTS: Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. CONCLUSION: Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants.

A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. Here, we map alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We find that SARS-CoV-2 rewires host lipid metabolism, significantly altering hundreds of lipid species to effectively establish infection. We correlate these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We find that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We find that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

Changes in Microbiome Dominance Are Associated With Declining Lung Function and Fluctuating Inflammation in People With Cystic Fibrosis.

Airway inflammation and microbiome dysbiosis are hallmarks of cystic fibrosis (CF) lung disease. However, longitudinal studies are needed to decipher which factors contribute to the long-term evolution of these key features of CF. We therefore evaluated the relationship between fluctuation in microbiome and inflammatory parameters in a longitudinal study including a short- (1-year) and a long-term (3+ years) period. We collected 118 sputum samples from 26 CF adult patients and analyzed them by 16S rRNA gene sequencing. We measured the levels of inflammatory cytokines, neutrophil elastase, and anti-proteinases; lung function (FEV1% predicted); and BMI. The longitudinal evolution was analyzed based on (i) the rates of changes; (ii) the intra-patient stability of the variables; and (iii) the dependency of the rates of changes on the baseline values. We observed that the diversity of the microbiome was highly variable over a 1-year period, while the inflammatory markers showed a slower evolution, with significant changes only observed in the 3+ year cohort. Further, the degree of fluctuation of the biomass and the dominance of the microbiome were associated with changes in inflammatory markers, especially IL-1ß and IL-8. This longitudinal study demonstrates for the first time that the long-term establishment and periodical variation of the abundance of a dominant pathogen is associated with a more severe increase in inflammation. This result indicates that a single time point or 1-year study might fail to reveal the correlation between microbial evolution and clinical degradation in cystic fibrosis.

Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission.

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.