The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface ...receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8
T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.
CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the ...biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.
Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found ...activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma.
Selective laser melting (SLM), a method used in the nuclear, space, and racing industries, allows the creation of customized titanium alloy scaffolds with highly defined external shape and internal ...structure using rapid prototyping as supporting external structures within which bone tissue can grow. Human osteoblasts were cultured on SLM-produced Ti6Al4V mesh scaffolds to demonstrate biocompatibility using scanning electron microscopy (SEM), fluorescence microscopy after cell vitality staining, and common biocompatibility tests (lactate dihydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-bromo-2-deoxyuridine (BrdU), and water soluble tetrazolium (WST)). Cell occlusion of pores of different widths (0.45-1.2 mm) was evaluated. Scaffolds were tested for resistance to compressive force. SEM investigations showed osteoblasts with well-spread morphology and multiple contact points. Cell vitality staining and biocompatibility tests confirmed osteoblast vitality and proliferation on the scaffolds. Pore overgrowth increased during 6 weeks' culture at pore widths of 0.45 and 0.5 mm, and in the course of 3 weeks for pore widths of 0.55, 0.6, and 0.7 mm. No pore occlusion was observed on pores of width 0.9-1.2 mm. Porosity and maximum compressive load at failure increased and decreased with increasing pore width, respectively. In summary, the scaffolds are biocompatible, and pore width influences pore overgrowth, resistance to compressive force, and porosity.
According to the facial feedback hypothesis, people's affective responses can be influenced by their own facial expression (e.g., smiling, pouting), even when their expression did not result from ...their emotional experiences. For example, Strack, Martin, and Stepper (1988) instructed participants to rate the funniness of cartoons using a pen that they held in their mouth. In line with the facial feedback hypothesis, when participants held the pen with their teeth (inducing a "smile"), they rated the cartoons as funnier than when they held the pen with their lips (inducing a "pout"). This seminal study of the facial feedback hypothesis has not been replicated directly. This Registered Replication Report describes the results of 17 independent direct replications of Study 1 from Strack et al. (1988), all of which followed the same vetted protocol. A meta-analysis of these studies examined the difference in funniness ratings between the "smile" and "pout" conditions. The original Strack et al. (1988) study reported a rating difference of 0.82 units on a 10-point Likert scale. Our meta-analysis revealed a rating difference of 0.03 units with a 95% confidence interval ranging from −0.11 to 0.16.
Abstract Background Accurate initial patient triage in the emergency department (ED) is pivotal in reducing time to effective treatment by the medical team and in expediting patient flow. The ...Manchester Triage System (MTS) is widely implemented for this purpose. Yet the overall effectiveness of its performance remains unclear. Objectives We investigated the ability of MTS to accurately assess high treatment priority and to predict adverse clinical outcomes in a large unselected population of medical ED patients. Methods We prospectively followed consecutive medical patients seeking ED care for 30 days. Triage nurses implemented MTS upon arrival of patients admitted to the ED. The primary endpoint was high initial treatment priority adjudicated by two independent physicians. Secondary endpoints were 30-day all-cause mortality, admission to the intensive care unit (ICU), and length of stay. We used regression models with area under the receiver operating characteristic curve (AUC) as a measure of discrimination. Results Of the 2407 patients, 524 (21.8%) included patients (60.5 years, 55.7% males) who were classified as high treatment priority; 3.9% (n = 93) were transferred to the ICU; and 5.7% (n = 136) died. The initial MTS showed fair prognostic accuracy in predicting treatment priority (AUC 0.71) and ICU admission (AUC 0.68), but not in predicting mortality (AUC 0.55). Results were robust across most predefined subgroups, including patients diagnosed with infections, or cardiovascular or gastrointestinal diseases. In the subgroup of neurological symptoms and disorders, the MTS showed the best performance. Conclusion The MTS showed fair performance in predicting high treatment priority and adverse clinical outcomes across different medical ED patient populations. Future research should focus on further refinement of the MTS so that its performance can be improved. Trial Registration Clinicaltrials.gov : NCT01768494.
Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial ...infarction (MI).
MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using in vitro and in vivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention.
Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) in vitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression in vitro rescues the LCM-mediated down-regulation of PSD-95. In vivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window.
Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI.
Knut and Alice Wallenberg Foundation F 2015/2112; Swedish Research Council VR; 2017-01243; the German Research Foundation DFG; ME 4667/2-1; Hjärnfonden FO2021-0112; The Crafoord Foundation; Åke Wibergs Stiftelse M19-0380, NMMP 2021 V2021-2102; the Albert Påhlsson Research Foundation; STINT MG19-8469, Lund University; Canadian Institutes of Health Research PJT-153269 and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.
On the basis of phase II trials, we considered that transcranial laser therapy could have neuroprotective effects in patients with acute ischemic stroke.
We studied transcranial laser therapy in a ...double-blind, sham-controlled randomized clinical trial intended to enroll 1000 patients with acute ischemic stroke treated ≤24 hours after stroke onset and who did not undergo thrombolytic therapy. The primary efficacy measure was the 90-day functional outcome as assessed by the modified Rankin Scale, with hierarchical Bayesian analysis incorporating relevant previous data. Interim analyses were planned after 300 and 600 patients included.
The study was terminated on recommendation by the Data Monitoring Committee after a futility analysis of 566 completed patients found no difference in the primary end point (transcranial laser therapy 140/282 49.6% versus sham 140/284 49.3% for good functional outcome; modified Rankin Scale, 0-2). The results remained stable after inclusion of all 630 randomized patients (adjusted odds ratio, 1.024; 95% confidence interval, 0.705-1.488).
Once the results of the interim futility analysis became available, all study support was immediately withdrawn by the capital firms behind PhotoThera, and the company was dissolved. Proper termination of the trial was difficult but was finally achieved through special efforts by former employees of PhotoThera, the CRO Parexel and members of the steering and the safety committees. We conclude that transcranial laser therapy does not have a measurable neuroprotective effect in patients with acute ischemic stroke when applied within 24 hours after stroke onset.
http://www.clinicaltrials.gov. Unique identifier: NCT01120301.
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