Search

Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.

Background: Interventricular septal dissection is a critical disease characterized by the separation of the intraventricular septum into two layers, forming an intermediate layer with a cystic cavity that communicates with the root of the aorta or ventricle. It has low morbidity and high mortality rates. Case presentation: Case 1: A 58-year-old male with a history of hypertension and smoking presented to a local hospital due to chest tightness and pain for 4 days. Coronary angiography revealed diffuse lesions from the proximal to the middle segment of the left circumflex branch, with 80% stenosis at its most severe point, and complete occlusion of the proximal segment of the right coronary artery. A stent was implanted in the middle of the right coronary artery. Three months later, the patient was misdiagnosed with an aneurysm of the membranous ventricular septum with defect via echocardiography at the local hospital. After the implantation of a stent in the left circumflex branch, the patient came to our hospital for further diagnosis and treatment. The first ultrasound of our hospital misdiagnosed it as ventricular septal rupture, and a senior ultrasound doctor diagnosed the patient with interventricular septal dissection secondary to myocardial infarction. The patient underwent follow-up echocardiography every 1–2 months for 6 months. The patient remains asymptomatic with stable hemodynamics. The original treatment regimen and follow-up continues. Case 2: A 70-year-old male was admitted to a local hospital due to repeated chest distress for more than 20 years that worsened over several hours. Coronary angiography revealed complete occlusion of the right coronary artery. Cardiogenic shock occurred after percutaneous coronary intervention. The initial several echocardiography of the local hospital and our hospital misdiagnosed it as interventricular septal rupture secondary to myocardial infarction. The later echocardiography diagnosed it as interventricular septal dissection with rupture secondary to myocardial infarction. The patient underwent interventricular septal repair and mitral valvuloplasty after 25 days of medical treatment and died of multiple organ failure on the fourth day after the operation. Conclusions: These two cases illustrate a complication of acute myocardial infarction and highlight the importance of echocardiography in its diagnosis. By exploring the etiology, pathogenesis, and key diagnostic points of IVSD, this study aims to provide valuable insights for clinical practice. [ABSTRACT FROM AUTHOR]

Conservative treatments for tricuspid regurgitation (TR) frequently yield suboptimal outcomes. Transcatheter interventions provide a new therapeutic avenue, with ongoing assessments of safety and effectiveness. The TriStar (Confirmatory Clinical Study of Treating Tricuspid Regurgitation With K-Clip™ Transcatheter Annuloplasty System) study investigated 1-year outcomes of the K-Clip transcatheter tricuspid annuloplasty system in treating patients with severe functional TR. Patients with TR grade ≥4, resistant to medical management, were enrolled and selected by core laboratory evaluation. Oversight by a data and safety monitoring board and event adjudication by a clinical events committee occurred. Assessments at 30 days, 6 months, and 1 year included echocardiographic data, clinical symptoms, quality of life, study endpoints, and major adverse events. A total of 96 patients (mean age 72.6 years, 59.4% women) were included, with a 97.9% implantation success rate. One-year outcomes showed 5.2% all-cause mortality, a 19.8% composite major adverse event rate, and no device-related reinterventions. TR decreased by ≥1 grade in 94.2%, and by ≥2 grades in 87.2% of patients. Kaplan-Meier estimates demonstrated 94.7% ± 2.3% survival and 90.4% ± 3.0% freedom from heart failure rehospitalization. NYHA functional class significantly improved (P < 0.001), with 97.7% reaching functional class I or II. The 6-minute walk distance improved by 42.8 m (P < 0.001), and Kansas City Cardiomyopathy Questionnaire score increased by 7.0 points (P < 0.001). The K-Clip transcatheter tricuspid annuloplasty system showed high procedural success, a favorable safety profile, sustained TR reduction, and marked improvements in clinical outcomes and quality of life at 1 year. (Confirmatory Clinical Study of Treating Tricuspid Regurgitation With K-Clip™ Transcatheter Annuloplasty System [TriStar]; NCT05173233) [Display omitted] [ABSTRACT FROM AUTHOR]

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.