Jason Gonsky

The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

Identification of Residues of the Moloney Murine Leukemia Virus Nucleocapsid Critical for Viral DNA Synthesis In Vivo

ABSTRACT The nucleocapsid (NC) protein of retroviruses is a small nucleic acid-binding protein important in virion assembly and in the encapsidation of the viral RNA genome into the virion particle. Multiple single-amino-acid substitutions were introduced into the NC of Moloney murine leukemia virus to examine further its role in viral replication. Two residues were shown to play important roles in the early events of replication. Unlike viruses with previously characterized NC mutations, these viruses showed no impairment in the late events of replication. Viruses containing the substitutions L21A and K30A expressed the normal complement of properly processed viral Gag proteins. Analysis of the RNA content of mutant virions revealed normal levels of unspliced and spliced viral RNA, and the tRNAPro primer was properly annealed to the primer binding site on the viral genome. The virions demonstrated no defect in initiation of reverse transcription using the endogenous tRNA primer or in the synthesis of long viral DNA products in vitro. Nonetheless, viruses possessing these NC mutations demonstrated significant defects in the synthesis and accumulation of viral DNA products in vivo.

Thrombolytic therapy is effective in paroxysmal nocturnal hemoglobinuria: a series of nine patients and a review of the literature

Background Thrombosis is the major risk factor for death in patients with paroxysmal nocturnal hemoglobinuria. Previous case reports indicate that venous thrombosis in patients with paroxysmal nocturnal hemoglobinuria is amenable to thrombolysis. Design and Methods We reviewed the outcome of thrombolytic therapy for patients with paroxysmal nocturnal hemoglobinuria who had thromboses refractory to anticoagulation at our institutions. Results In this study of 41 patients who had at least one thrombotic event, we confirmed a very high incidence of recurrence despite anticoagulation. Nine patients with thrombosis were regarded as eligible for administration of intravenous tissue plasminogen activator, which was effective in reversing thrombi in all of 15 occasions in which it was given. Serious hemorrhagic complications developed in three cases. At last follow-up visit, of the nine patients treated, three had died, and six were in very good to excellent condition in terms of clinical outcome and radiological findings. The only patient in whom thrombolysis may have contributed to a fatal outcome also had complications of “heparin induced thrombocytopenia with thrombosis”, which we diagnosed in three additional patients. In our review of the literature, nine out of 15 patients treated with thrombolysis have had a good outcome. Conclusions Although it is associated with a significant but manageable risk of bleeding, systemic thrombolysis is a highly effective treatment for reversing venous thromboses in patients with paroxysmal nocturnal hemoglobinuria.

Inaccuracies in 24-Hour Urine Testing for Monoclonal Gammopathies Serum Free Light Chain Analysis Provides a More Accurate Measure of Light Chain Burden Than Urine Protein Electrophoresis

Background Urine testing is important to measure monoclonal proteins and to evaluate renal function in patients with monoclonal gammopathies. Methods Creatinine clearance (CrCl), urine protein electrophoresis (UPEP), 24-h urine protein, and serum free light chain results were analyzed during an 11-mo period. Normal ranges for CrCl were 97–137 mL/min (men) and 88–128 mL/min (women). Results Among 623 urine samples from 207 patients with a monoclonal gammopathy, CrCl was abnormally increased (≥150 mL/min) in 119 samples (19%). For CrCl ≥300 mL/min, the median percent increase in CrCl from prior samples was 61%, and the median percent decrease in subsequent samples was 71%. Unexpected increases in urinary M protein and total protein were observed when CrCl was unexpectedly increased, but clonal serum free light chain results were not unexpectedly increased. Conclusions Results requiring 24-hour urine samples were highly susceptible to error. Serum free light chain analysis was unaffected by these errors.

Speaking Up: How Patient and Physician Voices Shaped a Trial to Improve Goals-of-Care Discussions

BackgroundPatients with advanced cancer benefit from early goals-of-care (GoC) conversations, but few facilitators are known.ObjectiveWe describe the process and outcomes of involving patient and physician stakeholders in the design and development of a trial, funded by the Patient-Centered Outcomes Research Institute (PCORI), to enhance oncologists’ communication skills and their propensity to facilitate productive, meaningful GoC discussions with patients with advanced cancer.MethodsWe recruited oncologists, palliative care physicians, and patient stakeholders to participate in proposal development, intervention design and modification, identification of outcome measures, and refinement of study tools. Formats for exchange included 1:1 structured interviews, workshops, and stakeholder meetings.ResultsPatient and physician voices helped craft and implement a study of an intervention to enhance oncologists’ ability to facilitate GoC discussions with patients with advanced cancer. Physician inputs guided the creation of an oncologist and palliative care physician “joint visit” intervention at a turning point in disease management. Patient inputs impacted on the language used, outcome measures assessed, and approaches used to introduce patients to the intervention visit.ConclusionsStakeholder input informed the development of a novel intervention that physicians seemed to find both valuable and in sync with their needs and their practice schedules. Where communication about difficult subjects and shared decision making are involved, including multiple stakeholder groups in study design, implementation, and outcomes measurement may have far-reaching effects.