RESUMEN
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Asunto(s)
Sistemas CRISPR-Cas , Hexosiltransferasas , Lipopolisacáridos , Proteínas de la Membrana , FN-kappa B , Transducción de Señal , Receptor Toll-Like 4 , Hexosiltransferasas/metabolismo , Hexosiltransferasas/genética , FN-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Receptor Toll-Like 4/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Células HEK293 , Inflamación/metabolismo , Inflamación/genética , Glicosilación , Microscopía por Crioelectrón , Dominio Catalítico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
In this article, we present two medicolegal cases illustrating medical and diagnostic pitfalls that can lead to litigation for missed testicular torsion. Testicular torsion (TT) is a urologic emergency with potentially devastating consequences and costs, for providers and patients alike. TT occurs in approximately 4.5 per 100,000 males under the age of 25. While uncommon, TT is the third most common cause of medical malpractice suits in this demographic. As a consequence of varying presentations and physical exam findings, and diagnostic imaging subject to individual interpretation, this time-sensitive diagnosis may be missed by emergency department providers. Delays in diagnosis significantly increases the morbidity associated with TT, and 31.9%-41.9% of such cases result in testicular loss. The average reported settlement for TT malpractice litigation is $60,000. This article discusses two actual malpractice cases involving TT and provides insight and caveats to ensure an optimal evaluation and diagnostic approach to this often-elusive condition.