Screening of NCI-DTP Library to Identify New Drug Candidates for \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e

Lyme disease is the most rapidly growing tick borne zoonotic disease of the Northern Hemisphere and is among the 10 most commonly reported nationally notifiable diseases in the United States.1 Clinical presentations include erythema migrans, fever, chills, muscle and joint pain.2, 3 Though these symptoms tend to fade away even without therapeutic intervention, a significant number of untreated patients develop arthritis and persistent myalgia following exposure to Borrelia burgdorferi.4 Furthermore, 10–20% of patients treated for Lyme disease develop symptoms considered typical, or even exaggerated, including muscle, joint pain and generalized fatigue5, 6. This condition is referred as post-treatment lyme disease syndrome (PTLDS)

Varicella Zoster Virus ORF25 Gene Product: An Essential Hub Protein Linking Encapsidation Proteins and the Nuclear Egress Complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Pothineni, Venkata R.. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Haase, Rudolf. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Woidy, Mathias. Ludwig Maximilians Universitat; AlemaniaFil: Lotz Havla, Amelie. Ludwig Maximilians Universitat; AlemaniaFil: Gersting, Soren W.. Ludwig Maximilians Universitat; AlemaniaFil: Muntau, Ania C.. Ludwig Maximilians Universitat; AlemaniaFil: Haas, Jurgen. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Sommer, Marvin. University of Stanford; Estados UnidosFil: Arvin, Ann M.. University of Stanford; Estados UnidosFil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemani

Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome

Abstract Background Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. Results We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. Conclusions Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.</p

Implantable cardioverter-defibrillator-related procedures and associated complications in continuous flow left ventricular assist device recipients: A multicenter experience

Background: Limited data exist regarding complication rates of implantable cardioverter-defibrillators (ICD) and cardiac resynchronization therapy devices (CRT-D) in patients with left ventricular assist devices (LVAD). Objective: We describe the incidence and characteristics of ICD- and CRT-D-related procedures and complications in a multicenter LVAD cohort. Methods: A total of 537 LVAD patients with a pre-existing ICD or CRT-D from 5 centers were included. Details on device type, device therapies, procedural complications, and long-term survival were analyzed. Results: Of 537 patients, 280 had a CRT-D and 257 had ICD only. During a median follow-up of 538 days, 126 patients underwent generator replacement with significantly higher rate in the CRT group (79 [28.2%] vs 47 [18.3%], P = .0006). Device-related complications occurred in 36 (13%) CRT-D and 20 (8%) ICD patients (P = .06). Incidence of pocket hematoma (3.2% vs 2.7%), infection (4.3% vs 1.6%), and lead malfunction (3.1% vs 2.8%) was similar in both groups, with no effect of device complication on long-term survival (log-rank P = .7). There was a higher incidence of post-LVAD antitachycardia pacing for ventricular arrhythmias in the CRT-D group compared to the ICD group (35% vs 26%, P = .03). Conclusion: Cardiac implantable electronic device-related procedures are common in LVAD patients. Compared to ICD only, continued CRT-D therapy post-LVAD results in a significantly higher number of generator changes and a trend towards higher device- or lead-related complications. Device-related complications were not associated with reduced survival

Amiodarone use and all-cause mortality in patients with a continuous-flow left ventricular assist device

Background Atrial and ventricular arrhythmias are commonly encountered in patients with advanced heart failure, with amiodarone being the most commonly used antiarrhythmic drug in continuous-flow left ventricular assist device (CF-LVAD) recipients. The purpose of this study was to assess the impact of amiodarone use on long-term all-cause mortality in ptients with a CF-LVAD. Methods and Results A retrospective multicenter study of CF-LVAD was conducted at 5 centers including all CF-LVAD implants from 2007 to 2015. Patients were stratified based on pre-CF-LVAD implant amiodarone use. Additional use of amiodarone after CF-LVAD implantation was also evaluated. Primary outcome was all-cause mortality during long-term follow-up. Kaplan-Meier curves were used to assess survival outcomes. Multivariable Cox regression was used to identify predictors of outcomes. Propensity matching was done to address baseline differences. A total of 480 patients with a CF-LVAD (aged 58±13 years, 81% men) were included. Of these, 170 (35.4%) were on chronic amiodarone therapy at the time of CF-LVAD implant, and 310 (64.6%) were not on amiodarone. Rate of all-cause mortality over the follow-up period was 32.9% in the amiodarone group compared with 29.6% in those not on amiodarone (=0.008). Similar results were noted in the propensity-matched group (log-rank, =0.04). On multivariable Cox regression analysis, amiodarone use at baseline was independently associated with all-cause mortality (hazard ratio, 1.68 [95% CI, 1.1-2.5]; =0.01). Conclusions Amiodarone use was associated with significantly increased rates of all-cause mortality in CF-LVAD recipients. Earlier interventions for arrhythmias to avoid long-term amiodarone exposure may improve long-term outcomes in CF-LVAD recipients and needs further study

Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation

Invasive pulmonary disease due to the mold Aspergillus fumigatus can be life-threatening in lung transplant recipients, but the risk factors remain poorly understood. To study this process, we used a tracheal allograft mouse model that recapitulates large airway changes observed in patients undergoing lung transplantation. We report that microhemorrhage-related iron content may be a major determinant of A. fumigatus invasion and, consequently, its virulence. Invasive growth was increased during progressive alloimmune-mediated graft rejection associated with high concentrations of ferric iron in the graft. The role of iron in A. fumigatus invasive growth was further confirmed by showing that this invasive phenotype was increased in tracheal transplants from donor mice lacking the hemochromatosis gene (Hfe(-/-)). The invasive phenotype was also increased in mouse syngrafts treated with topical iron solution and in allograft recipients receiving deferoxamine, a chelator that increases iron bioavailability to the mold. The invasive growth of the iron-intolerant A. fumigatus double-knockout mutant (Delta sreA/Delta cccA) was lower than that of the wild-type mold. Alloimmune-mediated microvascular damage and iron overload did not appear to impair the host's immune response. In human lung transplant recipients, positive staining for iron in lung transplant tissue was more commonly seen in endobronchial biopsy sections from transplanted airways than in biopsies from the patients' own airways. Collectively, these data identify iron as a major determinant of A. fumigatus invasive growth and a potential target to treat or prevent A. fumigatus infections in lung transplant patients

Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell typetropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV proteinexpression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirectimmunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteinswas analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments andshowed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbstested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection.Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thusenabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight intothe potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis